SINGLE-MOLECULE MAGNETIC TWEEZERS DEVELOPMENT AND APPLICATION IN STUDIES OF ENZYME DYNAMICS AND CELL MANIPULATION
Meiling Wu
A Dissertation
Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
May 2020
Committee:
Hong Peter Lu, Advisor
Hanfeng Chen Graduate Faculty Representative
John R. Cable
Mikhail A. Zamkov © 2020
Meiling Wu
All Rights Reserved iii
ABSTRACT
Hong Peter Lu, Advisor
Single-molecule Magnetic tweezers have been developed as a powerful approach that
provides detailed insight into single-molecule studies by mechanically manipulating and
simultaneously monitoring the change of the photophysical properties. We have developed a few
generations of magnetic tweezers and further utilized them for the studies of the mechanism of
the product release during an enzymatic reaction, correlated studies of single-molecule
enzymatic reaction dynamics and conformational dynamics, and live-cell motion manipulation. It
is highly informative to manipulate the enzyme under the enzymatic conditions and
simultaneously real-time monitor the conformational fluctuations of the enzymatic active site as
well as the reaction activity change. Understanding how the enzyme dynamics of the
conformational fluctuation relates to the enzyme activity can shed light on the field of
enzymology. Motion manipulation of living cells and other biological entities is also important in the fields of biological research and tissue engineering.
Particularly, in this dissertation, we have employed a Horseradish Peroxidase (HRP
catalyzed fluorogenic enzymatic reaction as a powerful probe to study the reaction and
conformational dynamics of the enzymatic active site under mechanical force manipulation. The
typical fluorogenic feature of this reaction makes the nascent-formed product molecule at its
perfect fitted position at the enzymatic active site, serving as an in-situ probe to report the real-
time active-site configuration and its fluctuations. Interestingly, the product releasing dynamics
ofRP H show the complex conformational behavior with multiple product-releasing pathways. iv
However, under constant magnetic force manipulation, the complex nature of the multiple
product- releasing pathways disappears, and more simplistic conformations of the active site are
populated. Under oscillation force manipulation at different frequencies, we have observed that
conformational dynamics can be significantly perturbed or altered.
We have developed an integrated double-ring magnetic tweezer imaging microscope to
actively manipulate the live-cell motions and selectively pick up the cell with an internalized
paramagnetic bead. We have also demonstrated a lock-in amplifier coupled rotating magnetic tweezers approach, allowing synchronization of magnetic force response with the oscillation force. v
TO MY BELOVED MOM vi
ACKNOWLEDGMENTS
During the journey of pursuing my Ph.D. for five and a half years, I have received immeasurable help, support and guidance from many wonderful people. At this special moment,
I would like to express my deep gratitude to them.
First and foremost, I would like to express my sincere gratitude to my advisor, Dr. H
Peter Lu for his guidance, support, inspired thoughts, and understanding. Being a professional knowledgeable chemist and scientist, Dr. Lu not only provides insightful scientific thoughts and stimulating discussions but also influences me a lot to be a rigorous person with quantitative thinking. His valuable and patient guidance to my academic research has made this dissertation possible.
I would also like to thank my committee members: Dr. John Cable, Dr. Mikhail Zamkov,
Dr. Hanfeng Chen and Dr. Ksenija Glusac for their precious time and valuable comments and suggestions.
I am also grateful to all the group members in Lu’s group, a wonderful family: Dr. Yufan
He, Dr. Nibedita Pal, Dr. Jin Cao, Dr. Qing Guo, Dr. Rajeev Yadav, Dr. Bharat Dhital, Dr.
Dibeyndu K. Sasmal, Dr. Vishal Govind Rao, Dr. Zijian Wang, Dr. Maolin Lu, and my fellow friends Dr. Achuyt Silwal, Lorena Alvarez, Sunidhi Jaiswal, Susovan Roy Chowdhury, Amal
Aburahma, and Uhdari Deshapriya for their friendship, support, stimulating discussions and sharing this wonderful journey with me.
I am thankful to all the faculty and staff in the Department of Chemistry, especially
Charles Codding and Doug Martin for their kind help and technical support for building magnetic tweezers. vii
Last but not least, I would like to thank my family: my parents and my younger sister and brother for their love and support in all these years. Special thanks should also go to my husband,
Dr. Wentao Ge for his endless support and love. I believe that this would become a wonderful experience and precious memory to complete a Ph.D. with him together at Bowling Green State
University. viii
TABLE OF CONTENTS
Page
CHAPTER 1. INTRODUCTION ...... 1
1.1 Protein Conformational Dynamics ...... 1
1.1.1 Introduction of the Enzyme Dynamics ...... 1
1.1.2 Horseradish Peroxide (HRP)-Catalyzed Fluorogenic Enzymatic
Reactions ...... 4
1.2 Single-Molecule Spectroscopy and Imaging ...... 7
1.2.1 Principle of Single-Molecule Spectroscopy...... 7
1.2.2 Single-Molecule Fluorescence Spectroscopy ...... 9
1.3 Magnetic Tweezers ...... 11
1.3.1 Principles of the Magnetic Tweezers Techniques ...... 11
1.3.2 Selection of the Magnetic Bead ...... 15
1.3.3 Force Calibration of the Magnetic Tweezers ...... 17
1.3.4 Magnetic Tweezers in Biological Systems ...... 19
1.4 Research Objectives ...... 22
1.5 References ...... 23
CHAPTER 2. EXPERIMENTAL SECTION ...... 32
2.1 Single-Molecule Techniques ...... 32
2.1.1 Principles of Total Internal Reflection Microscopy ...... 32
2.1.2 Principles of Confocal Microscopy ...... 35
2.1.3 Time Correlated Single Photon Counting ...... 37
2.2 Fluorescence Anisotropy ...... 41 ix
2.3 Force Calibration of the Magnetic Tweezers ...... 44
2.4 Finite Element Method Magnetics (FEMM) Simulation ...... 46
2.5 Principle of Lock-In Amplifier ...... 48
2.6 Experimental Setups of Total Internal Reflection Imaging Guided Confocal
Microscopy ...... 51
2.7 Materials and Sample Preparation ...... 56
2.7.1 Immobilization of Single HRP Enzyme ...... 56
2.7.2 Cell Cultures ...... 58
2.7.3 Rhodamine 6G Stained Paramagnetic Beads ...... 58
2.8 References ...... 59
CHAPTER 3. PROBING CONFORMATIONAL DYNAMICS OF AN ENZYMATIC
ACTIVE SITE BY AN IN SITU SINGLE FLUOROGENIC PROBE UNDER
PICONEWTON FORCE MANIPULATION...... 64
3.1 Introduction ...... 64
3.1.1 Significance of the Protein Conformational Dynamics ...... 64
3.1.2 Product Releasing in an Enzymatic Reaction ...... 66
3.1.3 Force Manipulation on Enzyme Dynamics...... 67
3.2 Experimental Sections ...... 69
3.2.1 Sample Preparation ...... 69
3.2.2 Magnetic Tweezers Coupled Total Internal Reflection Imaging
Guided Confocal Microscopy ...... 72
3.2.3 Data Analysis ...... 73
3.2.4 Force Calibration for Magnetic Tweezers ...... 75 x
3.3 Results and Discussions ...... 77
3.3.1 Time-Correlated Single Photon Time Stamping Spectroscopy ...... 77
3.3.2 Conformational Dynamics Without Force Manipulation ...... 80
3.3.3 Conformational Dynamics Under Force Manipulation ...... 85
3.3.4 Product Release Mechanism ...... 88
3.4 Conclusions ...... 91
3.5 References ...... 92
CHAPTER 4. OSCILLATING PICONEWTON FORCE MANIPULATION ON SINGLE-
MOLECULE CONFORMATIONAL AND REACTION DYNAMICS ...... 98
4.1 Introduction ...... 99
4.1.1 Conformational Dynamics in Enzyme Catalysis ...... 99
4.1.2 Oscillating Force Manipulation ...... 100
4.2 Experimental Sections ...... 102
4.2.1 Sample Preparation ...... 102
4.2.2 Data Analysis ...... 103
4.3 Results and Discussions ...... 107
4.3.1 Home-Developed Rotating Magnetic Tweezers ...... 107
4.3.2 Single Enzyme Activity Analysis ...... 112
4.3.3 Enzymatic Conformational Dynamics in Product Release ...... 119
4.4 Conclusions ...... 127
4.5 References ...... 128
xi
CHAPTER 5. MANIPULATING MOTIONS OF TARGETED SINGLE CELLS IN
SOLUTION BY AN INTEGRATED DOUBLE-RING MAGNETIC TWEEZERS
IMAGING MICROSCOPY ...... 132
5.1 Introduction ...... 133
5.2 Experimental Sections ...... 135
5.2.1 Sample Preparation ...... 135
5.2.2 Integrated Double Ring Magnetic Tweezers ...... 136
5.2.3 Force Calibration ...... 138
5.3 Results and Discussions ...... 141
5.3.1 Finite Element Magnetics Method Simulation ...... 141
5.3.2 Bidirectional Manipulation on Single Paramagnetic Beads ...... 144
5.3.3 Targeted Single Cell Motion Manipulation in Solution ...... 145
5.4 Conclusions ...... 150
5.5 References ...... 151
CHAPTER 6. SYNCHRONIZING MAGNETIC RESPONSE BY A LOCK-IN
AMPLIFIER COUPLED ROTATING MAGNETIC TWEEZERS...... 155
6.1 Introduction ...... 155
6.2 Experimental Sections ...... 158
6.2.1 Sample Preparation ...... 158
6.2.2 Lock-In Amplifier Coupled Rotating Magnetic Tweezers ...... 158
6.3 Results and Discussions ...... 162
6.4 Conclusions ...... 167
6.5 References ...... 168 xii
LIST OF FIGURES
Figure Page
1.1 Commonly used fluorogenic substrates of HRP ...... 5
1.2 (A) Conceptual scheme of HRP as an enzyme to catalyze the non-fluorescent
Amplex Red to fluorescent product Resorufin in presence of H2O2. (B) Crystal
structure of the HRP (PDB ID 1HCH) and its active site configuration ...... 6
1.3 A schematic diagram of the magnetic tweezers ...... 12
1.4 (A) Experimental setup and (B) A schematic representation of a magnetic bead
undergoing Brownian fluctuation ...... 20
1.5 Representation of the device used to measure the binding events ...... 21
2.1 Conceptual representation of Total Internal Reflection ...... 33
2.2 Conceptual scheme of objective-based total internal reflection microscopy...... 34
2.3 Conceptual representation of confocal microscopy ...... 36
2.4 Comparison between wide-field scanning of epifluorescence microscopy and point
scanning of confocal microscopy...... 37
2.5 (A) Time-correlated single-photon counting module detects the photon with two
parameters in the time domain: chronic arrival time and delay time between the laser
excitation and photon detection. (B) The typical raw data of single-molecule photon
time-stamping spectroscopy. (C) Fluorescence intensity trajectory derived from (B) 39
2.6 Histogram of delay time to give fluorescence lifetime in time-resolved fluorescence
measurement with TCSPC ...... 40
2.7 Conceptual diagram of the polarized emission and rotational diffusion ...... 43
2.8 Magnetic Field-distance curve of the magnetic tweezers ...... 45 xiii
2.9 (A) A typical example of model construction and finite mesh generation of a
permanent magnet. (B) Simulated magnetic field distribution by FEMM ...... 47
2.10 Typical experimental set-up of lock-in amplifier ...... 48
2.11 Typical raw data from the lock-in detection ...... 50
2.12 Schematic representation of the Total Reflection Fluorescence Microscopy imaging-
guided Confocal Fluorescence Spectroscopy ...... 52
2.13 Calibration of TIRFM imaging-guided confocal single-molecule fluorescence
spectroscopy ...... 54
2.14 Conceptual scheme for single HRP sample preparation ...... 56
2.15 Protocol for single HRP sample preparation ...... 57
3.1 Conceptual scheme of Horseradish peroxidase (HRP, PDB 1HCH) as an enzyme to
catalyze the non-fluorescent Amplex Red to fluorescent product Resorufin in
presence of H2O2 ...... 70
3.2 (A) Typical snapshot of an image from an electron-multiplying charge-coupled
device (EMCCD) in Total-internal reflection mode under an applied magnetic field.
(B) Fluorescence confocal images of two polarization components from the single-
photon avalanche photodiodes (SAPD1 and SAPD2) in the confocal mode under an
applied magnetic field ...... 72
3.3 Magnetic force calibration curve of the magnetic tweezers ...... 76
3.4 A typical raw data of single-molecule photon time-stamping spectroscopy in the
parallel channel (A) and perpendicular channel (B) ...... 78
xiv
3.5 (A) Single molecule fluorescence intensity and anisotropy trajectories of HRP-
catalyzed oxidation of Amplex Red, binning with 20 ms. (B) Identification of the
rising edge, ON state, and the falling edge of a catalytic event ...... 80
3.6 Two-dimensional joint distribution of anisotropy and lifetime (Upper) and rotational
correlation time and lifetime (Lower) calculated from 281 catalytic turnovers ...... 81
3.7 Two-dimensional joint distribution of anisotropy and lifetime (Upper) and rotational
correlation time and lifetime (Lower) calculated from 356 catalytic turnovers under
∼2 pN of magnetic pulling force ...... 85
3.8 Schematic representation of the product-releasing pathways ...... 90
4.1 Fluorescence detection of HRP enzyme catalysis ...... 105
4.2 A typical fluorescent trajectory of a single molecule in parallel and perpendicular
polarization, as well as the corresponding anisotropy trajectory ...... 106
4.3 Conceptual scheme of our experimental system ...... 108
4.4 Magnetic force calibration curve for the cylinder-permanent magnet (D4C-N52)
used in the magnetic tweezers ...... 109
4.5 Conceptual representation of our developed Magnetic Tweezers system and its
characterization using FEMM simulation ...... 111
4.6 Enzyme activity analysis based on turnover rate mean waiting time, product burst
photon counts and dissociation constant ...... 113
4.7 Distribution of product burst counts of HRP under no force manipulation ...... 114
4.8 Distribution of turnover event counts of HRP from 60s fluorescence trajectory
under no force manipulation ...... 117
xv
4.9 Distribution of turnover event counts of the no paramagnetic bead tethered
HRP (control experiment) from 60s fluorescence trajectory under 0Hz, @15 Hz
and @40 Hz ...... 119
4.10 2D joint distribution of anisotropy and lifetime at the rising edge under different
circumstances ...... 120
4.11 2D joint distribution of anisotropy and lifetime during on-time period under
periodical force manipulation at a frequency of 0Hz (A), 10Hz (B), 15Hz (C),
25Hz (D), 40Hz (E) and 50Hz (F) ...... 121
4.12 2D joint distribution of anisotropy and lifetime at the falling edge under the
periodical force manipulation at a frequency of 0Hz (A), 10Hz (B), 15Hz (C),
25Hz (D), 40Hz (E) and 50Hz (F) ...... 123
5.1 Conceptual scheme of the experimental set-up ...... 137
5.2 The experimental measured magnetic field gradients (dB/dz) when the upper
magnet at different distances away from the sample plane ...... 140
5.3 (A) Model construction and 3D finite mesh generation of the up-down configured
magnetic tweezers using FEMM. FEMM simulation results when upper magnet β
is (B) 2mm, (C) 12mm, and (D) 30mm away from the sample plane ...... 142
5.4 (A) FEMM simulated vector representation of magnetic flux density B when upper
magnet β is 12mm away from the sample plane where a zero magnetic field/force is
achieved. (B) FEMM simulated and experimental measured magnetic field at the
sample plane when moving the upper magnet β up and down ...... 143
xvi
5.5 Representative bright field images of paramagnetic beads in solution under force
manipulation (A1) and its correlated calibration images (A2). Intensity profiles of
the images are plotted in (B). Displacements of paramagnetic beads in z-direction
under force manipulation recorded from ~2000 frames (50ms exposure time) (C) .. 144
5.6 Representative bidirectional manipulated DIC images of HEK-293 with internalized
paramagnetic beads (2.8 µm diameter) by tuning the upward pulling force ...... 147
5.7 Representative DIC images of HT22 with internalized paramagnetic beads (2.8 µm
diameter) by continuously increasing the upward pulling force ...... 149
6.1 Schematic diagrams of the home-developed lock-in amplifier coupled rotating
magnetic tweezers ...... 161
6.2 Typical raw data from the lock-in detection ...... 163
6.3 Surface plot of fluorescence response counts, fluorescence response (Voltage) and
applied oscillation force frequency ...... 164
6.4 Lock-in fluorescence signal response as a function of applied oscillation force
frequency...... 166
xvii
LIST OF TABLES
Table Page
1.1 Rupture forces of typical single chemical bonds as well as forces involved in a
physiological environment ...... 14
1.2 Saturated magnetization of commonly used paramagnetic beads ...... 16
3.1 Summarized Rotational Correlation time of all the domains present in the catalytic
event with and without force manipulation (τf: Fluorescence Lifetime; r: Anisotropy;
τr: Correlation Rotational time.) ...... 83
4.1 Rotational Correlation time of the three domains present at the falling Edge under
force oscillation frequency of 0Hz (Figure. 4.12A), 40Hz (Figure. 4.12E) and 50Hz
(Figure. 4.12F) ...... 126
1
CHAPTER 1. INTRODUCTION
This chapter is dedicated to the biological significance of the protein conformational
dynamics and the introduction of the magnetic tweezers as well as the single-molecule
spectroscopy.
1.1 Protein Conformational Dynamics
1.1.1 Introduction of the Enzyme Dynamics
Enzymes are macromolecular catalysts, catalyzing the biochemical reactions with
associated structural motions, which are often intrinsically stochastic and dynamic.1-7 It has been extensively proven that the 3-dimensional structure, especially the conformational dynamics plays a crucial role in protein functioning, including protein folding/unfolding, misfolding, aggregation processes, as well as in enzyme catalysis and cell signaling functioning.8-13 Over the
years, the enzymatic conformational dynamics have been receiving ever-increasing attention due to their significant roles in regulating enzymatic reactions and biological functioning.5,14-17 To
acquire a comprehensive understanding of the critical role of the enzyme played during the
catalysis is significant in designing and engineering the catalyst with high efficiency. However, it
has been a long journey to obtain a comprehensive understanding of the enzymatic catalysis and
extensive efforts have been made to unravel the detailed mechanism.
In the early stage, the “lock and key” model was proposed to explain the selectivity of the
enzyme in 1894 by Emil Fischer, which believes that the perfect match of the geometry is critical
for the catalysis and the catalysis occurs when the shape of the active site of the enzyme fits with
the substrate.18 However, the “lock and key” model provides a great explanation for the
selectivity of the enzyme but fails to explain the importance of the transition state of the enzyme 2
during the enzymatic reaction.2 Later, the induced fit model was proposed by Daniel Koshland in
1958, which includes the flexibility of the enzyme structure, particularly how the active site of
the enzyme plays an essential role during the process of the enzymatic reaction.19 In fact, despite
the significant role of the active site and its involved amino acids, the overall enzyme
conformational matrix and the associated dynamics are vital for initiating and completing the
enzymatic cycles.20 In 1901, Michaelis-Menten kinetics were proposed to provide
comprehensive understanding on the mechanism of the enzymatic reactions in which multiple
steps are involved.21 As shown in Equation 1.1, Enzyme (E) binds with a substrate (S) to form
enzyme-substrate complex [E·S] and then gets converted to enzyme-product complex [E·P],
which releases the product (P) and regenerates the original enzyme (E) for the following
catalytical cycle.1,21 Moreover, during the processes of the catalytical cycle, such as substrate
binding, catalysis, and product release, enzyme (En) generally possesses different conformational
states for different functionalities to ultimately achieve a productive event (Equation 1.2).
+ [ ] [ ] + Equation 1.1
𝑬𝑬 𝑺𝑺 ⇌ 𝑬𝑬 ∙𝑺𝑺 ⇌ 𝑬𝑬kE ∙𝑷𝑷 ⇌ 𝑬𝑬 𝑷𝑷 E En kE' Equation 1.2
Where E represents enzyme, S represents substrate, [E·S] represents enzyme-substrate complex, [E·P] represents enzyme-product complex and P represents product; En represents
different conformational states of the enzyme, kE and kE represent the rate constants of the interconversion between enzyme conformational states.
Enzymes catalyze the biological chemical reactions with enhanced rates by changing the energy landscape, i.e. lowering the energy barrier between the ground state and transition state through stabilizing the transition state or destabilizing the ground state, as well as providing 3 alternative reaction pathways that have a lower energy barrier.22-24 These functionalities of the enzyme are accomplished by the conformational fluctuation dynamics of the enzyme, i.e. changing the surface charge distribution or the reaction entropy and so on.25 Therefore, instead of being a static structure, a large scale of structural motions are associated during the catalysis and even under normal thermal fluctuations, including the movement of a particular amino acid, domain motions or the entire protein matrix. Due to all these associated structural motions, enzymes fluctuate/interconvert between different conformational states.26
Therefore, unraveling the protein conformational dynamics is critical to understand the intimate relationship between the structure and function. Both experimental and theoretical studies have been developed to probe the protein conformational dynamics, such as NMR studies,27,28 molecular simulations,15,29 and theoretical modeling,30-35. Due to the heterogeneous and fluctuating nature of the conformational dynamics, single-molecule spectroscopy has shown its unique advantages on revealing the dynamics by studying one molecule at a time.28,36-38
Particularly, single-molecule fluorescence measurement has been developed to characterize the enzymatic conformational dynamics, presumably through analyzing the photophysical properties of the fluorophores, such as fluorescence intensity, lifetime, anisotropy and rotational correlation time, which serve as key parameters to directly probe the conformational dynamics of the enzyme.39-45
Generally, three approaches have been developed to probe the enzymatic dynamics for single-molecule fluorescent measurement: intrinsic fluorophores such as Tryptophan, Tyrosine residues or Phenylalanine; site-specific dye labeling; and fluorogenic enzymatic reaction.38,46-48
However, not all the biological macromolecules contain the intrinsic fluorophores, which limits the application only in the fluorophore-containing systems. Besides, the dye labeling has the 4
nonnegligible drawbacks that it could induce significant perturbations on the enzyme active site,
which may further alter the enzymatic activities, or it can not accurately read out the active site conformational dynamics if the dye labeling is far away from the active site to avoid the situation above. Fluorogenic reactions are enzymatic reactions that convert the non-fluorescent substrates
to a nascent formed fluorescent product that emits light. Fluorogenic reaction-based fluorescent
products enable the fluorescence imaging, which is used to probe any structural motion-induced
conformational dynamics with high sensitivity in the progress of the enzymatic reaction.16,46,49-51
Compared to previous two approaches, fluorogenic reactions have several unique advantages.
Firstly, the system is free of photobleaching due to the fluorescent product constantly generated
from the catalytical active site. Besides, it allows real-time observation and recording of the
catalytical events in hours if the enzyme is still active. Moreover, sufficient data collection
facilitates a statistical analysis of all the conformational states in the course of the enzymatic
reaction.
1.1.2 Horseradish Peroxide (HRP)-Catalyzed Fluorogenic Enzymatic Reactions
Horseradish Peroxide (HRP) is a monomeric 44K Dalton enzyme, containing a heme
prosthetic group, which has been studied for more than a century due to its significant role
played in the biological systems. Particularly, HRP isoenzymes have been found to be important
in metabolism, lignification, crosslinking of cell wall polymers, suberin formation and resistance
to infection.52 HRP is found in roots of horseradish and has many isoforms, among which the
most abundant and studied form is type C. HRP isoenzyme C is composed of a single
53 polypeptide chain of 308 amino acid residues. In the presence of hydrogen peroxide (H2O2) as
an oxidizing reagent, HRP catalyzes the oxidation of numerous substrates, including aromatic
amines, indoles, phenols and sulfonates.52,54 5
O HO O OH H N NH HO 2 2 O NH NH 2 N NH NH2 O CH3 O NH 2 O OH TMB Liminol OPD Amplex Red Homovanillic acid NH2 NH2 O HO N S N S O NH2 O N S S N N O OH H2N NH2 ABTS AEC DAB
Figure 1.1 Commonly used fluorogenic substrates of HRP. Amplex Red is used as the
fluorogenic substrate in this dissertation.
Figure 1.1 provides a list of commonly used chromogenic substrates, which are
nonfluorescent by themselves but can be converted into fluorescent products. We have used
Amplex Red as the fluorogenic substrate in this dissertation. Figure 1.2A shows a typical
fluorogenic enzymatic reaction in which Horseradish peroxidase (HRP) enzyme catalyzes the
conversion of non-fluorescent Amplex Red to fluorescent Resorufin, a model system we used for
studying the enzymatic conformational dynamics.55,56 Nascent-formed fluorescent product was generated right in the active site at the perfectly fitted position without perturbing the active site while involving critical molecular interactions, which holds high promise to serve as a real-time in-situ probe for reporting any conformational change due to the structural motions in the process of the enzymatic reaction. 6
Figure 1.2 (A) Conceptual scheme of HRP as an enzyme to catalyze the non-fluorescent Amplex
Red to fluorescent product Resorufin in presence of H2O2. (B) Crystal structure of the HRP
(PDB ID 1HCH) and its active site configuration. The active site involved amino acids are
highlighted.
The crystal structure of HRP and the enzyme-substrate intermediate have been reported
by X-ray crystallography, which reveals the catalytical mechanism of HRP.57,58 Figure 1.2B
shows the crystal structure and the active site configuration as well as the active site-involved amino acids. Particularly, amino acid residues Arginine 38 (Arg38) and Histidine 42 (His42) play essential roles in stabilizing and binding with aromatic substrates. The enzymatic turnover rate and the dynamic disorder have been investigated using single-molecule fluorescence spectroscopy in previous studies.14,55,56,59,60 To obtain deeper understanding on the ultimate
relationship between the structure and function, we have introduced a noninvasive force
perturbation on the protein structure by coupling with the single-molecule magnetic tweezers
technique. By controlling the amplitude of the applied force, we have the capability of imposing 7
a protein-structure perturbation under enzymatic conditions, which hold higher promise and
potential than the conventional protein denaturing assay. A protein denature assay generally
involves a chemical interfering, such as pH, electrolytes and chemical denaturants, which are not
physiologically friendly.
1.2 Single-Molecule Spectroscopy and Imaging
1.2.1 Principle of Single-Molecule Spectroscopy
Single-molecule spectroscopy has been developed to be a powerful and advanced method
within 30 years since its invention by W.E Moerner.61 Moreover, in 2014, the Nobel prize was
awarded to three scientists (W. E Moerner, Eric Betzig and Stefan W Hell) for their contributions
of establishing single-molecule spectroscopy and achieving super-resolution imaging. The
development of single-molecule spectroscopy dates back to 1989, when W.E Moerner, for the
first time, achieved optical detection of single molecules in condensed matter using double
frequency modulation.62 In 1990, Oritt and Bern developed the detection of single molecules by fluorescence-excitation spectroscopy as a proof of absorption for the molecules that emit fluorescence photos when they have absorbed light.63 These two works have been recognized as
the foundation of single-molecule spectroscopy. Later, Eric Betzig achieved sub-wavelength
spatial resolution of single molecule detection using near-field scanning optical microscopy
breaking the Abbe limit.64-66 Stefan Hell invented stimulated-emission-depletion fluorescence microscopy (STEM) to overcome the diffraction limit and achieve super-resolution imaging.67
Since then, single-molecule spectroscopy has opened up many opportunities for studies in
the interface of biology, physics and chemistry. Particularly, since the biological processes are
essentially dynamic and heterogenous, studying single biological molecules has become one 8 significant application in single-molecule spectroscopy. For instance, under thermal fluctuation, a large scale of conformational dynamics is involved for proteins to achieve different functionalities. The main characteristic of single-molecule study is the ability to study the properties of one single molecule at a time, which provides much more rich information beyond the averaged results from the conventional ensemble measurements. The intrinsic dynamics of biological molecules occur in a range from ns to s and 10-2 Å to Å in time and space, respectively. For instance, the macro-biological molecules, such as enzymes, possess a range of conformations to initiate, catalyze and complete a productive enzymatic turnover. Due to the averaging effect, ensemble measurements could possibly provide one global minimum in the reaction coordinate, which fails to give the detailed dynamics of individual steps. A key feature of the biological processes is the fast interconversion between internal states, including the low- energy states and high-energy states. More than often, the transient high-energy conformational states, even with low population or a short lifetime, are important to the function. Single molecule spectroscopy has the capability to capture the transient conformations in the absence of the averaging effect, providing both the number and the population of the conformational states as well as the underlying mechanism that gives rise to the conformational heterogeneity.
Moreover, single molecule spectroscopy can not only provide the detailed conformational fluctuations, but also determine the processive or sequential dynamics in a complicated biological process by monitoring one single molecule throughout the course of the entire process.
On the other hand, with the development and advancement of force spectroscopy, it extended single-molecule spectroscopy to another dimension, which is to study the mechanical properties of single-polymer molecules or proteins, as well as chemical bonds. Over the years, force spectroscopy has developed as a significant single-molecule technique, studying the 9
interactions and binding force between individual molecules. A large variety of force
spectroscopies have been developed, which includes atomic force microscopy (AFM),68-70
optical tweezers (OT),71-74 magnetic tweezers (MT),75,76 acoustic force spectroscopy,77
microneedles,78 and bio-membranes.79 Aided by these force spectroscopies, single-molecule
spectroscopy can simultaneously monitor the distance or the orientation changes as well as the
mechanical impact on the chemical activity of macromolecules, which correlates to the
relationship between conformational changes and function.
In this dissertation, we focused on the development of the magnetic tweezers and applied
the advancement of magnetic tweezers in protein conformational dynamics and cell
manipulation. The detailed introduction of the magnetic tweezers will be discussed later. It is
highly informative to manipulate single molecules while simultaneously monitoring the change
of the photophysical properties using a laser-focused confocal microscopy, which opens a new
window to unravel the ultimate relationship between the structure-dynamics function.
1.2.2 Single-Molecule Fluorescence Spectroscopy
Fluorescence detection of single biological molecules in solutions became an important
application for exploring individual behavior in a complex physiological environment.80-85 As
has been discussed earlier, single-molecule detection eliminates the ensemble average,
measuring the detailed conformational fluctuation and unraveling the heterogeneity that is
usually buried in conventional ensemble measurement.86,87 To accomplish single-molecule
detection, one of the most commonly used strategies is to have only one molecule in the detected
volume, which is achieved by having an ultra-small concentration of the fluorophore. For example, in a typical confocal microscopy, around a concentration of 10-10 M is often used in a 10
probed volume of 10 µm3 to ensure only one molecule present in the focal volume. Optimizing a good signal to noise ratio is the ultimate goal when performing single-molecule fluorescence detection, which requires one to simultaneously maximize the fluorescence photons but minimize the background photons and unwanted noise. Over the years, there are several successful microscopic configurations developed for single-molecule fluorescence detection, including confocal microscopy, total internal microscopy, epifluorescence and near-field optical scanning.88-90 In this dissertation, we have employed total internal microscopy and confocal
microscopy to probe the fluorescence of nascent-formed fluorescent product from single
enzymes, which will be described in detail in chapter 2.
Fluorescence signals are usually collected by the single-photon avalanche diode (SPAD)
or charged coupled device (CCD) camera, measuring fluorescence intensity as a function of time
and fluorescence lifetime. To extract more information regarding photophysical properties of the
fluorescent probe, several techniques have been developed, including polarization microscopy,
fluorescence resonant energy transfer and two-photon excitation, which provide information regarding molecular orientations, conformational changes and energy transfer processes.91 We
have also incorporated polarization microscopy in our dissertation for probing the
conformational change of the enzymatic active site. Polarization microscopy is one of the most
powerful techniques, which is achieved by a polarization excitation beam, and anisotropy is used
to analyze the polarization of the emission.92 Anisotropy measurement by polarization
microscopy describes the exact angular orientation of a molecular transition diploe, which
provides information on its attached protein dynamics and steric constraints, such as the
conformational change and the enzyme flexibility.39-41,46,93 Besides, rotational correlation time
can be derived from the fluorescence lifetime and anisotropy by the Perri Equation, probing the 11
rotational dynamics of a single fluorophore, which serves as a great probe for the local
environment.40
1.3 Magnetic Tweezers
1.3.1 Principles of the Magnetic Tweezers Techniques
Single-molecule magnetic tweezers have been developed as one of the most powerful
force spectroscopic techniques, which provide detailed insight of biological macromolecules by
mechanically manipulating and simultaneously monitoring the change of the photophysical
property. Magnetic tweezers are scientific instruments that generally can be used to study the
mechanical properties of single molecules by applying a magnetic force or torque to the studied
system through the magnetic particles. The basic physical principle underlying the magnetic
tweezers is a gradient of the magnetic field, exerted by the permanent magnet or the electromagnetic devices, under which the magnetic particles experience magnetic force and move towards the magnetic field. In a typical design of the magnetic tweezers (Figure 1.3), the reaction chamber that contains the surface-immobilized single molecule with attached paramagnetic bead is placed on top of the objective while the permanent magnet or the electromagnetic device is above it. 12
Figure 1.3 A schematic diagram of the magnetic tweezers. The studied single molecule is immobilized on the cover glass and tethered to a paramagnetic bead. A permanent magnet is placed above the sample plane, producing a magnetic field under which a magnetic force is applied. 13
Although a few approaches have been employed for single-molecule manipulation, such
as atomic force microscopy (AFM) or optical tweezers, magnetic tweezers feature a number of
unique advantages: 1) force is applied in noncontact mode, meaning no physical or chemical
contact is required; 2) no photon damage, thermal damage and optical background is induced; 3)
force is adjustable in magnitude and direction, and it can be static or oscillatory; 4) a large
amount of targets can be manipulated under the same magnetic field simultaneously.94-96
Besides, magnetic tweezers are low cost and easy to handle. Moreover, magnetic tweezers are ideal to apply a constant force in comparison to the other techniques, which often require
sophisticated feedback systems to impose a constant force.16,51,97,98 Particularly, the constant
force manipulation is significant in studies of protein folding/unfolding and DNA.
These unique characteristics make the magnetic tweezers approach popular and powerful in single-molecule measurements. Over the years, magnetic tweezers have been demonstrated as a capable approach to manipulate biological systems, such as DNA,95,99-101 proteins,16,51,102,103
and biomolecular complexes,104,105 through the micron-sized paramagnetic beads.106 The
dynamic information during the biological processes obtained from single-molecule force
spectroscopy will be served as a complement of the structural aspect from traditional approaches,
such as X-ray crystallography and NMR techniques.
To evaluate the force manipulation on a studied system, force impact can be understood
as the energy divided by a distance. For example, a single carbon-carbon bond has a bond energy
of 5.7×10-19 J (~90 kcal/mol) and a bond length of 1.54 Å (1.54×10-10 m), leading to a rupture
force of ~3.7 nN. Extensive efforts have been made to measure the rupture force of the chemical
bonds, including covalent bonds, noncovalent bonds, and the force in the physiological
environment.107-112 Particularly, H.E Gaub has measured the rupture force of single covalent 14 bonds and intermolecular forces between ligands and receptors.107-109 Table 1.1 summarizes the rupture forces of the important chemical bonds and forces involved in the biological environment.
Table 1.1 Rupture forces of typical single chemical bonds as well as forces involved in a physiological environment.
Covalent bond Hydrogen bond Biotin-streptavidin Langevin force bond Rupture force >1 nN 6-9 pN ~160 pN 0.01pN
Force required to break a typical covalent bond is in the order of a few nanonewtons, while a force of 6-9 pN has the capability to break a single hydrogen bond. The force involved in the thermal motion is ~ 0.01 pN, which serves as the lower limit of the measurable force. Biotin- streptavidin binding is known to be one of the strongest non-covalent bonds in nature, which is commonly used as a linker in the single molecule magnetic tweezers experiment. We have also used the Biotin-streptavidin interaction in the attachment between a single protein and a paramagnetic bead. The force required to break the Biotin-streptavidin binding is ~160 pN. The small constant force applied in single HRP force manipulation in chapter 3 and 4 is ~2 pN, which is not comparable to the biotin-streptavidin binding and the covalent bond and is not enough to break a single hydrogen bond either. Under such conditions, the chemical bonds are slightly extended and behave as small molecular springs.
To meet the specific research of interest, a large variety of magnetic tweezers have been developed. In principle, there are two major types of magnetic tweezer techniques: permanent magnets and electromagnet-based magnetic tweezers.104 Permanent magnet-based magnetic 15 tweezers are the simplest and most straightforward form of magnetic tweezers.16,76,103,113,114
Typically, in such configurations, permanent magnets are placed above the sample plane with a distance in the millimeter range. A permanent magnet generates a static magnetic field, exerting an upward force on the studied molecule, which can be utilized to stretch/pull the DNA, biopolymer, and protein. Force is adjustable by physically changing the distance between the permanent magnets and the sample. In contrast, a sophisticated design may be incorporated to build the electromagnetic tweezers. In a typical configuration, a magnetic core (iron alloy) is coiled by conductive wires to form a solenoid, which generates the magnetic field.95,115-119 The strength of the magnetic field is determined by the current and the geometry of the solenoid. The working distance of electromagnetic tweezers is typically in a range of micrometers, which is positioned by a micromanipulator. To achieve a large magnetic force, a large current is needed, which may induce heating in coils and ultimately affect the sample. Therefore, a cooling system is often required when working with electromagnetic tweezers.
1.3.2 Selection of the Magnetic Bead
The principle of the magnetic tweezers is based on its magnetic field profile and the magnetic properties of the magnetic particles used in the experiment. Magnetic moment (m) is defined to describe the magnetic strength of the magnetic materials when responding to a magnetic field. Materials are set to be magnetic if their magnetic moment is not zero in the presence of a magnetic field. In general, there are three types of magnetic materials: ferromagnetic, paramagnetic/super-paramagnetic and diamagnetic. In the presence of a magnetic field, ferromagnetic materials are magnetized, are attracted to the magnetic field and stay magnetized even after removing the source of the magnetic field. Only a few substances are ferromagnetic and most of them are metal. The common ferromagnetic materials include Fe, Ni, 16
Co, and their alloys; some rare earth metals; as well as CrO2. In contrast, paramagnetic materials
are magnetized in the presence of a magnetic field and are weakly attracted to the direction of the
magnetic field but lose the magnetism with the removal of the external magnetic field. The
common paramagnetic materials include FeO, O2, Ti and Al. Particularly, the materials are
known to be super-paramagnetic if they have an exceptionally large magnetic susceptibility.
Diamagnetic materials are repelled to the external magnetic field.
There are two types of magnetic particles/beads developed for binding with biological
molecules for the magnetic tweezers experiment: ferromagnetic particles (often prepared by
CrO2) and paramagnetic particles. Due to the unique feature of the para-magnetism, paramagnetic beads are widely used in single-molecule magnetic tweezers. Paramagnetic beads
are typically submicron-sized spherical polymer beads with paramagnetic content (typically
FeO).120 The unpaired electrons present in paramagnetic materials have their magnetic dipole
moments due to spin. In the absence of the magnetic field, due to the thermal fluctuation, the
magnetic moment of the paramagnetic bead points in a random direction to give a net zero
magnetization. In the presence of the magnetic field, the magnetic dipole moment aligns parallel
with the magnetic field, which makes the paramagnetic beads act like tiny magnets and move
toward the magnetic field.
Table 1.2 Saturated magnetization of commonly used paramagnetic beads.
Diameter Volume (10-18 Ms (KA/m) m (M*V) 10-15 Ratio (m) (µm) m3) (A·m2) TM 1.05 0.6 43 25.8 1 MyOne M-280 2.83 11.86 34 403 15 M-450 4.40 44.6 36 1606 62 17
The magnitude of the magnetic force induced by a magnetic field depends on the magnetic moment of the paramagnetic beads, which consequently depends on the volume of the magnetic materials carried by the paramagnetic beads. Therefore, the size of the magnetic beads needs to be optimized based on the experimental force required.121 The most commonly used
superparamagnetic beads for the magnetic tweezers experiment are the micron-sized paramagnetic beads (Dynabeads® MyOne™, Invitrogen), which are also used in this dissertation. We have summarized the magnetization of the superparamagnetic beads used in this dissertation in Table 1.2. Under the same magnetic field when the magnetization reaches their saturated volume, M-280 paramagnetic beads (2.8 µm) are expected to have 15 times larger magnetic force than the 1 µm paramagnetic bead and 62 times larger for the M-450 paramagnetic bead (4.40 µm). To achieve a small constant magnetic force for force manipulation on a single
HRP enzyme in chapter 3 and chapter 4, we have used 1 µm paramagnetic beads. In chapter 5,
we have used 2.8 µm paramagnetic beads to achieve a higher magnetic force for live-cell motion
manipulation.
1.3.3 Force Calibration of the Magnetic Tweezers
To perform quantitative measurements, the magnetic tweezers need to be calibrated.
There are three main methods that can be employed for force calibration:
(1) Direct measurement. The most straightforward way of force calibration is to measure the
magnetization of the paramagnetic beads and the magnetic gradient.122 For a given paramagnetic
bead, the force can be calculated in the following equation:
= = ( ) = = Equation 1.3 𝝏𝝏𝝏𝝏 𝑭𝑭 −𝜵𝜵𝜵𝜵 −𝜵𝜵 −𝒎𝒎 ∙ 𝑩𝑩 𝑴𝑴𝑴𝑴 ∙ 𝜵𝜵𝜵𝜵 𝑴𝑴𝑴𝑴 𝝏𝝏𝝏𝝏 18
Where m is the magnetization of the paramagnetic beads, B is the magnetic field, M is the
saturated volume magnetization, V is the volume of the paramagnetic bead, and z represents the
distance between the magnet and the sample. The magnetization of the magnetic beads is
commonly measured using a superconducting quantum interference device (SQUID).122 If the magnetic field (B) of the region of interest is large enough, the magnetization will reach its saturated value, which is often used in the calculation of the magnetic force. Using a high precision gauss meter, the magnetic field is acquired at different positions by displacing the magnet in the z direction. A magnetic field strength curve can also be obtained as a function of the distance, from which the magnetic field gradient is determined.
(2) Calibration using Brownian fluctuation.100,101,123 In this method, a paramagnetic bead is
usually tethered to a big polymer (such as DNA, around several µm in length). By analyzing the
Brownian fluctuation, the applied force can be calculated from the following equation:
= Equation 1.4 𝒌𝒌𝑩𝑩𝑻𝑻𝑻𝑻 𝟐𝟐 𝑭𝑭 <𝜹𝜹𝒙𝒙 > Where kB is the Boltzmann constant, T is the temperature, l is the extension of the tethering
molecule, and <δx2> is the variance of the bead fluctuation in the x direction. The x and y
coordinates are directly measured from the image. The vertical extension of the tethering
molecule l is obtained by analyzing the diffraction pattern of the magnetic bead based on the
CCD image.
(3) Calibration using Stokes’ Law.96,124 This approach is particularly well-suited for the micro- particles of which the viscous drag motions dominated over all other hydrodynamic effects. To respond to the applied force F, the paramagnetic beads move with a velocity v = F/D, where D is 19
the appropriate drag coefficient. The velocity of the paramagnetic beads can be obtained by tracking the displacement of the paramagnetic beads under a magnetic force. According to
Stokes’ Law (D = 6πηr, where η is the dynamic fluid viscosity, r is the radius of the bead), the magnetic force applied on the paramagnetic beads can be calculated as:
= Equation 1.5
𝑭𝑭 𝟔𝟔𝟔𝟔𝟔𝟔𝟔𝟔𝟔𝟔 1.3.4 Magnetic Tweezers in Biological Systems
In single-molecule magnetic tweezers, micron-sized paramagnetic beads are often used
while the studied molecules can be ranged from DNA99,125 to protein102,124,126 and even in living cells.117,127 The first application of magnetic tweezers dates back to 1949 by Crick and Hughes,
who applied a magnetic force on magnetic particles inside living cells using a permanent
magnet.128 Later, Smith et al gave the first demonstration of single-molecule manipulation using
magnetic tweezers, in which a single DNA molecule was tethered to a cover glass at one end and
bound to a paramagnetic bead at the other end.100 Since then, single-molecule magnetic tweezers have been widely used as a single-molecule manipulation technique.129 20
Figure 1.4 (A) Experimental setup and (B) A schematic representation of a magnetic bead
undergoing Brownian fluctuation.101
In 1996, Croquette group101 first demonstrated the supercoiling of a single λ-DNA using
magnetic tweezers and calibrated the magnetic force using Brownian fluctuation (Figure 1.4).
The single linear DNA molecule was tethered to a cover glass at one end and bound to a para-
magnetic bead at the other end. Recently, protein-protein interactions have been widely
investigated by force manipulation using magnetic tweezers, for example, studying how the force
stimulus is transformed into a chemical response. In 2007, Lee et al126 studied the bond lifetime
of the immunoglobulin (IgG)-protein A under force manipulation using magnetic tweezers. IgG
was immobilized to the cover glass while protein A was bound to a paramagnetic bead. They characterized the dissociation behavior of the bond between IgG and protein A by measuring the number of magnetic beads bound to the surface. They identified a strong and a weak slip bond present in the IgG-protein A interaction. The magnetic tweezers approach has been demonstrated to be an informative technique to identify both weak biomolecular interactions and strong biomolecular interactions. 21
Figure 1.5 Representation of the device used to measure the binding events. The Talin Rod
(PDB ID 1u6u) and Vinculin head (PDB ID 1sj8) structures are represented in green and grey,
respectively. The arrow shows the direction of the movement of the beads when they are pulled
using the magnetic tweezers.124
In 2009, Sheetz group124 offered the experimental evidence that stretching of the protein
molecule might expose more binding sites for other proteins (Figure 1.5). They investigated the effect of the force on the interaction between two proteins, Talin Rod (TR) and Vinculin. As has
been well known in previous studies, one active binding site and four buried inactive binding
sites are present in TR. In the absence of the force manipulation, only one binding event was
observed, which indicated that TR contains one active binding site. However, in the presence of
a force manipulation, a 12pN stretching force made the TR protein partially unfolded and
exposed two more buried binding sites to Vinculin. Recently, using ultra magnetic tweezers, the
first folding/unfolding equilibrium of the I27 protein was reported in 2015.102 Both folding and
unfolding processes were observed in the same extension trajectory under a low constant force. 22
Furthermore, the critical force is determined around 5.4 pN, at which the unfolding and folding processes have an equal probability. A force-dependent free energy landscape of unfolding/folding transitions was built based on the measurement of the free energy cost of the unfolding. Lu group also reported their findings that a single HPPK enzyme under force pulling manipulation is shown to have less flexibility when binding to the substrates.103 Later, they
investigated the enzyme activity and the product release mechanism under a small constant force
manipulation by single molecule magnetic tweezers.16,51
We have summarized a few important applications of single-molecule magnetic tweezers
in studies of DNA, protein-protein interactions and protein folding. For sure, there are many
more significant implications in the fields of enzymology and biological research. Technically,
force manipulation by magnetic tweezers is a novel approach capable of unfolding or deforming
an enzyme under the enzymatic reaction condition in real time when the enzyme thermal fluctuation is fully maintained. In addition to providing deep insight on the mechanical properties of the studied molecules, single-molecule magnetic tweezers are also capable of shedding light on the interplay between force and the underlying behaviors. Besides, new designs of the magnetic tweezers that are equipped with more complex spatial and temporal field distributions and manipulations is required to adapt into the specific research fields.
1.4 Research Objectives
This dissertation focuses on the development of advanced magnetic tweezers techniques
and utilizes the home-developed magnetic tweezers to address significant and fundamental
biological questions, i.e. how the protein dynamics profile plays a role in the protein functioning,
and how the mechanical properties of the proteins impact the protein conformational dynamics. 23
We have developed a few generations of magnetic tweezers and further utilized them for the studies of the specific area of interest in our research project. In summary, there are four research projects presented in this dissertation, where the first two projects focus on unraveling the force manipulation impact of the protein dynamics during the enzymatic reactions from a single molecule perspective. In chapter 3, we studied conformational dynamics of the enzymatic active site by an in-situ single fluorogenic probe under the piconewton constant force manipulation. In chapter 4, we generated oscillating magnetic tweezers to investigate the oscillating force manipulation on single-molecule enzymatic conformational and reaction dynamics. In chapter 5, we developed an integrated double-ring magnetic tweezers imaging microscope for bidirectional manipulation of living cell motions. In chapter 6, we demonstrated lock-in amplifier-coupled rotating magnetic tweezers to synchronize the magnetic force response of the oscillation force.
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CHAPTER 2. EXPERIMENTAL SECTION
2.1 Single-Molecule Techniques
2.1.1 Principles of Total Internal Reflection Microscopy
Total internal reflection microscopy (TIRM) was developed by Daniel Axelrod at the
University of Michigan, Ann Arbor in the early 1980s for the studies of the cell-substrate
contact.1 In fact, the concept of total internal reflection for illumination had already been used by
E.J. Ambrose in 1956 to study cell movement by illuminating the cell contact area.2 Nowadays,
TIRM has been widely used as a technique of fluorescence microscopy (TIRFM) with a high
axial resolution (~100 nm) and wide focal plane.3,4 The working principle of TIRFM is the evanescent wave induced by the total internal reflection. Total internal reflection occurs only
when the light travels from a transparent medium with a higher reflective index to another
transparent medium with a lower reflective index; for example, from cover glass (n=1.515) to aqueous solution (n=1.33). As shown in Figure 2.1, when the light travels through the interface of two mediums with different reflective indices, the light will be transmitted and internally
reflected depending on the incident angle. There are three circumstances: if the incident angle
(Ɵ) is small enough, the ray is mostly transmitted and partially reflected. Due to the bent or the
refraction of the light, the transmitted light exits in a larger angle than the original incident angle.
As the incident angle (Ɵ) increases to a certain value, called the critical angle (Ɵc), the
transmitted light exits at 90° to achieve a total internal reflection. The critical angle is also
known as the smallest angle to achieve total internal reflection. If the incident angle continues to
increase to be greater than the critical angle, the ray is total internally reflected and none is
transmitted. 33
Figure 2.1 Conceptual representation of Total Internal Reflection.
As the smallest incident angle that yields total internal reflection, the critical angle (Ɵc) is determined by the reflective indices of two mediums, which can be calculated by Snell’s Law in
Equation 2.1.
= ° Equation 2.1
𝒏𝒏𝒊𝒊𝒔𝒔𝒔𝒔𝒔𝒔𝜽𝜽𝒄𝒄 𝒏𝒏𝒕𝒕𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔𝒔 To achieve total internal reflection in the experiment, the incident angle is deviated slightly away from the critical angle. The total internal reflected light generates an evanescent electromagnetic field near the interface, which is converted from the energy of the incident light.
The evanescent electromagnetic field has the same frequency as the incident light, while the intensity decays exponentially with distance from the interface, which only illuminates the sample up to ~200 nm. The relationship between the energy of the evanescent field and the distance from the interface is described by Equation 2.2:
( / ) ( ) = ( ) Equation 2.2 −𝒛𝒛 𝒅𝒅 𝑬𝑬 𝒛𝒛 𝑬𝑬 𝟎𝟎 𝒆𝒆𝒆𝒆𝒆𝒆 34
Where E(z) is the energy of evanescent field at distance z, E(0) is the energy of evanescent field
at interface, z is the distance from the interface and d is the penetration depth of the illumination.
The penetration depth is defined by the wavelength of the incident light (λ(i)), incident angle (Ɵ),
as well as the reflective indices of two mediums (n1 and n2), which is described in Equation 2.3:
( ) = × Equation 2.3 𝝀𝝀 𝒊𝒊 𝟏𝟏 𝟐𝟐 𝟐𝟐 𝟐𝟐 𝒅𝒅 𝟒𝟒𝟒𝟒 �𝒏𝒏𝟏𝟏 𝒔𝒔𝒔𝒔𝒔𝒔 𝜽𝜽−𝒏𝒏𝟏𝟏 Therefore, TIRFM has its unique advantage on eliminating the background from the area above
200 nm, which ultimately increases the signal to noise ratio and improves the spatial resolution
of imaging.5,6 Moreover, the induced evanescent field has a larger focal plane, 104 times larger than the focal volume in confocal microscopy, which is ideal for wide-field imaging∼ using a charged coupled device (CCD) camera.
Figure 2.2 Conceptual scheme of objective-based total internal reflection microscopy. The
evanescent field generated at the interface decays exponentially with the distance z from the
interface. 35
There are two common approaches to achieve TIRFM: prism based and objective based.
Modern TIRFM systems are often objective based (Figure 2.2). In comparison to the prism based
TIRFM, objective based TIRFM is more convenient and the incident angle can be adjusted easily.7 Objective based TIRFM requires a high numerical aperture objective with NA>1.45 to allow a large deviation from the critical angle to achieve a flatter reflection, which induces a thinner penetration depth for a higher spatial resolution. Since TIRFM is an ideal approach for studies of interfaces, it has been widely used for fluorescence imaging in biological systems, such as cell surface and membrane studies.8,9 Besides, it has also been incorporated with single- molecule fluorescence spectroscopy, which serves as a powerful approach for single-molecule fluorescent studies in protein and DNA.10-12
2.1.2 Principles of Confocal Microscopy
Confocal microscopy is an optical imaging technique that has been widely used for fluorescence imaging in biological research through fluorescent labeling.13 The first development of confocal scanning microscopy dates back to 1950s by Marvin Minsky who utilized stage scanning for confocal microscopy.14 The principle of confocal microscopy is to use two pinhole apertures to reduce the background of its out-of-focus properties. As shown in Figure 2.3, the coherent light from the laser source needs to pass through the first pinhole aperture, which is located just before the objective lens with a scanning point on the specimen. Then the laser is reflected by a dichroic mirror to the objective lens with point scanning on the sample plane. The emission is collected by the same objective, travels back, passes through the dichroic mirror and continues to propagate to the second pinhole filter before being sent to the detector. In such a configuration, the two small pinhole apertures serve as the spatial filters, which can significantly reduce unwanted background noise and provide high contrast imaging. 36
Figure 2.3 Conceptual representation of confocal microscopy.
Compared to conventional wide-field imaging that usually suffers serious out-of-focus background issues, confocal imaging features several unique advantages, which include reducing the background from outside the focal volume and narrowing the focal volume to achieve high spatial and lateral resolution. Figure 2.4 provides the conceptual difference between wide-field scanning from conventional wide-field microscopy and point scanning from confocal microscopy. In wide-field microscopy, the entire specimen is entirely bathed in the illumination light, which excites the entire area; therefore, all the background information is collected.
However, point scanning is achieved by scanning the sample plane, resulting in a point being illuminated in confocal microscopy, which improves the signal to noise ratio.15 The tightly focused laser beam can reach an ultra-small size about half of the excitation wavelength, providing diffraction limited imaging resolution. 37
Figure 2.4 Comparison between wide-field scanning of epifluorescence microscopy and point scanning of confocal microscopy.
We have employed fluorescence confocal microscope in this dissertation, and the details of the setup will be discussed further later. Fluorescence confocal microscopy has become a
popular and powerful approach in single-molecule imaging because of its high spatial
resolution.16-20 The width of a confocal beam size is about the half of the excitation wavelength,
typically ~250-300 nm. Thus, the laser light selectively excites molecules that are located within
the small detection volume, excluding the background photons that are outside the detection
volume, which is extremely useful in studies of weak signals of single fluorophores in biological
studies.21,22 With the advancement of photon detection systems, such as single photon avalanche
diode (SPAD) and time-correlated single-photon counting module (TCSPC), it provides new
opportunities to study complex biological systems from the single-molecule perspective, such as
conformational dynamics of protein.23-26
2.1.3 Time Correlated Single Photon Counting
Time-correlated single photon counting (TCSPC) is a technique of fluorescence
spectroscopy which detects single photons with precisely time dependent information by a 38
repetitive excitation-emission cycle.27,28 Figure 2.5A illustrates the conceptual diagram of
TCSPC. An ultrafast laser, typically a picosecond or femtosecond laser, is used to provide
periodic laser pulses and excites the fluorophore, then the single emission photon is registered
with two characteristics of time, the chronic arrival time and the delay time. Chronic arrival time represents the actual arrival time of the photon being detected and registered. Delay time represents the time difference between the photon emission and the corresponding pulse
excitation. The electrical synchronization signal of the excitation pulse provides the reference of
the timing, and the photon emission is measured by the photon detectors, such as single photon
avalanche diode (SPAD). To achieve single photon counting, no photons in an empty cycle are
highly possible in some repetitive excitation-emission cycles. For example, in Figure 2.5A, the second pulse cycle shows no photon being detected. The situation of single photon or no photon being detected is completely a random process, which can only be described in terms of probability. The reason why to maintain a low probability of photon detection is to avoid the
“pile-up” effect. There is dead time, at least a few nanoseconds for the optical detectors and the electronics after a photon detection.29 During the dead time, no photon can be registered. If more photons are generated in a cycle, only the first photon will be registered and the rest of them will be missed, resulting in the “pile-up” effect. Consequently, the average measured fluorescence lifetime would be shorter, and the feature of the exponential decay may be lost, such as mono- exponential becoming bi-exponential. 39
Figure 2.5 (A) Time-correlated single-photon counting module detects the photon with two parameters in the time domain: chronic arrival time and delay time between the laser excitation
and photon detection. (B) The typical raw data of single-molecule photon time-stamping
spectroscopy. Each data point represents a detected photon plotted by its arrival time (t) and
delay time (∆t). (C) Fluorescence intensity trajectory derived from (B). 40
During the signal processes in TCSPC, the optical events are converted into the electric
signals transmitting through the components of TCSPC. There are three main components in
TCSPC: Constant Fraction Discriminator (CFD), Time to Amplitude Converter (TAC) and
Analog to Digital Converter (ADC). CFD is used to extract the timing information from the
photon detector, such as APD. Similarly, the synchronization signal from the excitation pulse is
also fed into the CFD for the time measurement of the reference. Then the electrical signals are
further fed into TAC, a linear ramp generator, which will be started by one signal and stopped by
another. After processing by TAC, a resultant voltage signal is produced, which is proportional
to the time difference between excitation signal and emission signal. In this manner, the delay
time is being measured. ADC is further used to convert the voltage signal from TAC to digital
time values, which can be easily used for fluorescence analysis.
Figure 2.6 Histogram of delay time to give fluorescence lifetime in time-resolved fluorescence measurement with TCSPC. 41
As has been mentioned before, for each registered/detected photon, both real arrival time and delay time are recorded, known as the photon time stamping spectroscopy (Figure 2.5B), which provides rich information of the fluorophores and their surrounding environment.30-33
Photon time stamping is analogous to the mail having two stamps: one stamp for the sending date and one stamp for the actual receiving date.33-37 Figure 2.5C illustrated the fluorescence intensity trajectory, which is obtained by building the histogram of the chronic arrival time. The histogram of the delay time of the photons measured in TCSPC provides the fluorescence lifetime of fluorophores (Figure 2.6).38 In this dissertation, we have combined TCSPC with fluorescence confocal microscopy to investigate the conformational dynamics in HRP catalyzed enzymatic reactions.
2.2 Fluorescence Anisotropy
Fluorescence anisotropy analysis on the fluorescent probe is essential to provide information on the size and shape of its attached proteins and their steric constraints, such as the conformational change and the enzyme flexibility.39-41 The principle of anisotropy is polarized excitation and emission. Under thermal fluctuation, the fluorophores are randomly oriented.
Upon excitation by a polarized light, the fluorophores that have the dipole moment parallel to the polarized light are preferentially excited and emit photons, resulting in a partially polarized emission. Anisotropy is defined as the relative angle between these moments to describe the extent of the polarization of the emission.27 There are several processes that can lead to the depolarization of the emission, which further decreases the anisotropy value. Hence, rich information can be extracted from the fluorescence anisotropy, such as the size and the shape of the fluorophores, as well as their surrounding environment. 42
In particular, the most common cause of depolarization is the rotational diffusion within the time scale of the lifetime of fluorophores, which can have a significant impact on the anisotropy. Due to the thermal fluctuation, the rotational diffusion occurs typically in around 100 ps in a fluid solution. Therefore, during the fluorescence lifetime, typically a few nanoseconds, the fluorophores have rotated many times. As shown in Figure 2.7A, because of fast rotational diffusion, the feature of the polarized emission will be lost, resulting in a zero anisotropy.
However, in the case of fluorophores that are bound to biological macromolecules, such as proteins, the rotational diffusion can be significantly decreased, which results in a higher anisotropy. Therefore, anisotropy and rotational correlation time are key parameters to probe the conformational dynamics of protein. 43
Figure 2.7 Conceptual diagram of the polarized emission and rotational diffusion. (A) The fluorophore in a non-viscous fluid solution. (B) Fluorophore bound to a macromolecule
(protein).
In our experiments, the fluorescence emission is separated into two polarization components by a polarization beam splitter: parallel and perpendicular components. Two channels (T-format) are used to measure the intensities of the parallel ( ( )) and perpendicular
∥ ( ( ))) components simultaneously. As the sensitivities of the two detectors𝐼𝐼 𝑡𝑡 are typically
⊥ distinct,𝐼𝐼 𝑡𝑡 G factor is used to compensate the difference between the two channels. G factor is
determined by the fluorescent intensity response of two channels with different excitation
polarizations. The total intensity (IT(t)) and the anisotropy (r) can be given by equation 2.4 and
equation 2.5, respectively
( ) = ( ) + ( ) Equation 2.4
𝑰𝑰𝑻𝑻 𝒕𝒕 𝑰𝑰∥ 𝒕𝒕 𝟐𝟐𝟐𝟐𝑰𝑰⊥ 𝒕𝒕 44
( ) ( ) ( ) = Equation 2.5 ( ) ( ) 𝑰𝑰∥ 𝒕𝒕 −𝑮𝑮𝑰𝑰⊥ 𝒕𝒕 𝒓𝒓 𝐭𝐭 𝑰𝑰∥ 𝒕𝒕 +𝟐𝟐𝟐𝟐𝑰𝑰⊥ 𝒕𝒕 Rotational correlation time is used to describe the restricted rotational diffusion of the protein-
attached fluorescent product, which largely depends on the size and shape of the molecules, as
well as the molecular confinement status and their local environment. Assuming there are no
other processes causing the loss of anisotropy, the Perrin equation is used to define the rotational
correlation time in equation 2.6:
= + Equation 2.6 𝟏𝟏 𝟏𝟏 𝝉𝝉𝒇𝒇 𝒓𝒓 𝒓𝒓𝟎𝟎 𝒓𝒓𝟎𝟎𝝉𝝉𝒓𝒓
Here, r is the steady-state anisotropy; r0 is the fundamental anisotropy in the absence of
molecular rotation; τf is the fluorescence lifetime; τr is the rotational correlation time.
2.3 Force Calibration of the Magnetic Tweezers
A cylinder permanent magnet is the key component of our magnetic tweezers, upon which we built an independent 3D rotational movement stage to control the movement of the magnet. Force is generated upon the paramagnetic beads using an external magnetic field gradient. In the absence of the magnetic field, the magnetic moment of each atom in paramagnetic beads points in a random direction. However, in the presence of an external magnetic field, the magnetic bead is magnetized and acts as a tiny magnet and moves towards the higher magnetic flux. We controlled the magnitude of the force by displacing the magnets vertically. To determine the force, we measured the magnetic field by placing the probe of a gauss meter at different distances away from the permanent magnet. A magnetic field strength curve (Figure 2.8) was obtained as a function of the distance. 45
Figure 2.8 Magnetic Field-distance curve of the magnetic tweezers. A home built-in 3D
rotational stage is used to control the distance between the sample and permanent magnet.
Force is calibrated by the magnetic field gradient. For a given magnetic bead, the force
can be calculated in the following equation:
= = ( ) = = Equation 2.7 𝝏𝝏𝝏𝝏 𝑭𝑭 −𝜵𝜵𝜵𝜵 −𝜵𝜵 −𝒎𝒎 ∙𝑩𝑩 𝑴𝑴𝑴𝑴 ∙ 𝜵𝜵𝜵𝜵 𝑴𝑴𝑴𝑴 𝝏𝝏𝝏𝝏 Where, m is the magnetic moment of the magnetic bead and B is the magnetic field. Both volume Magnetization (M) and volume (V) are the properties of the magnetic bead, which can be determined by the specific magnetic bead. For the super-paramagnetic bead (Dynabeads®
MyOne™ Streptavidin T1, 1.05 mm diameter, Invitrogen Company), the volume magnetization
(M) is 43*103 A m-1 and the volume (V): 0.6*10-18 m3. Thus, according to the calculation, an
external force of 1.5 pico-Newton generated by the magnet can be applied on the para-magnetic
bead. 46
2.4 Finite Element Method Magnetics (FEMM) Simulation
Finite element method magnetics (FEMM) simulation has been employed to simulate the
magnetic field strength of the permanent magnets that have been used for magnetic tweezers,
including cylindrical permanent magnets (chapter 3,4 and 6) and ring magnets (chapter 5). The
underlying principle of FEMM is the finite element method (FEM), a general mathematic
method for solving partial differential equations. In detail, the studied problem is subdivided into
small simple finite elements, which are further described by a set of mathematic equations. FEM
recombines all these equations into a global system of equations that models the entire domain of
the studied system. For final solution approximation, variational methods are employed, which
were introduced by the father of FEM, R Courant, in 1943.42 Applications of FEM have
demonstrated several advantages in the analysis of complex systems, including providing an
accurate representation for complex geometry by creating the finite elements and offering a great
approximation for the complex systems using variational methods.43 FEM has been widely used
in a range of applications in studies of electromagnetic field, heat flow, optics and
nanotechnology.44-49
To theoretically characterize the magnetic field exerted by our home-developed magnetic tweezers, we have used FEMM to simulate the magnetic field distribution, which is further used
to calculate the magnetic force applied on the sample plane. FEMM is a widely available
software package used for 3D axisymmetric magnetic field simulation in low frequency. The
FEMM simulation is performed in three steps: 1) Generating the geometry of a particular
problem in a CAD-like interface and defining the material property and boundary conditions in
an interactive shell. 2) Subdividing the problem into a large set of triangles (finite elements). 3) 47
Using the high precise solver (Newton AC solver) with 10-8 precision for solving the partial differential equations and approximating the solution of the problem.50
Figure 2.9 (A) A typical example of model construction and finite mesh generation of a permanent magnet. (B) Simulated magnetic field distribution by FEMM.
Figure 2.9 illustrates the typical processes for the magnetic field simulation of a permanent magnet using FEMM. Based on the geometry of the permanent magnet, the model is constructed in Figure 2.9A, the material property of the magnet is defined as N42 and the surrounding area is defined as air, following by the finite mesh generation. Using Newton AC solver with 10-8 precision, the simulated magnetic field distribution is shown in Figure 2.9B. We have followed the similar procedure for magnetic field simulation of our home-developed magnetic tweezers, which will be described in detail in chapters 4 and 5. 48
2.5 Principle of Lock-In Amplifier
The lock-in amplifier is commonly used as a phase-sensitive instrument which extracts
weak signals from a large background/noise environment. The working principle of the lock-in
amplifier relies on the demodulation of two sinusoidal functions, singling out the signal at a
specific reference frequency but rejecting the noise signals at the other frequencies, which allows
a weak signal to be recovered from a large noise power. The demodulation of the signals is
achieved by multiplying the received signal by the reference signal and then integrating over
time. When the frequency of signal and reference signal is not equal (ωs≠ωr), the demodulation
gives a result of zero. In contrast, when the frequency of the signal is equal to the frequency of
the reference signal (ωs=ωr), the integration is half of the amplitude of the product. Therefore, a majority of the noise signal is eliminated, and the signal is also amplified, which significantly
increases the signal to noise ratio. The development and commercialization of the lock-in
amplifier devices opens a new window for implications in optical detection with high
sensitivity.51-57
Figure 2.10 Typical experimental set-up of lock-in amplifier. 49
Figure 2.10 illustrates a typical experimental setup of a lock-in amplifier. To uncover the
weak signal from a large noise power using a lock-in amplifier, a strong and clean reference
signal that has the same frequency as the signal is required. The reference signal can be obtained
through an oscillator or function generator. In a typical scenario in Figure 2.10, a chopper wheel
is used to chop the laser source at a certain frequency, adjusting by the chopper controller to
generate a reference signal at a frequency (ωr). The reference signal can be a square wave or a sine wave, here the reference is described by a sine function in equation 2.8:
( ) = ( ) Equation 2.8
𝑽𝑽𝒓𝒓 𝒕𝒕 𝑽𝑽𝒓𝒓 𝑺𝑺𝑺𝑺𝑺𝑺 𝝎𝝎𝒓𝒓𝒕𝒕 A photon detector is used to collect fluorescence response of the laser excitation at the reference
frequency to give a signal input of the lock-in amplifier. The signal can also be described by
another sine function as equation 2.9:
( ) = ( + ) Equation 2.9
𝑽𝑽𝒔𝒔 𝒕𝒕 𝑽𝑽𝒔𝒔 𝑺𝑺𝑺𝑺𝑺𝑺 𝝎𝝎𝒔𝒔𝒕𝒕 𝝋𝝋 Since the noise is often spread in a wide range of frequencies, the weak signal is buried in a large
noise power. The lock-in amplifier only singles out the weak signal at the reference frequency
but rejects the noise at any other frequencies by multiplying the signal with the reference signal.
When the frequency of the signal equals the frequency of the reference signal, the lock-in
amplifier gives the result of half of the amplitude of the product, described in equation 2.10:
( ) ( ) = [ [( ) + ] [( + ) + ]] 𝟏𝟏 𝑽𝑽𝑺𝑺 𝒕𝒕 𝑽𝑽𝑹𝑹 𝒕𝒕 𝑽𝑽𝑿𝑿 𝑽𝑽𝑹𝑹 𝑪𝑪𝑪𝑪𝑪𝑪 𝝎𝝎𝑺𝑺𝒕𝒕 −𝝎𝝎𝑹𝑹𝒕𝒕 𝝋𝝋 − 𝑪𝑪𝑪𝑪𝑪𝑪 𝝎𝝎𝑺𝑺𝒕𝒕 𝝎𝝎𝑹𝑹𝒕𝒕 𝝋𝝋 𝟐𝟐 Equation 2.10 50
A calibration experiment has been performed to demonstrate the idea of the lock-in amplifier. A
laser source is chopped by the chopper wheel to generate an excitation laser at a certain
frequency, which illuminates the fluorescent microspheres. The fluorescence is collected by a
single photon avalanche diode and further fed into a lock-in amplifier. In the absence of a lock-in amplifier, the fluorescence is buried in the background, especially at a high frequency regime.
However, the lock-in amplifier has shown its ability to extract weak fluorescence signal from the background (Figure 2.11). We have developed lock-in amplifier coupled rotating magnetic tweezers in chapter 6, aiming to monitor the magnetic force response under oscillation force manipulation by measuring the fluorescence change.
Figure 2.11 Typical raw data from the lock-in detection. Signal input(upper panel), reference
signal (medium panel) and lock-in output (lower panel) of fluorescent microspheres @ 10 Hz (B)
30 Hz and (C)50 Hz. 51
2.6 Experimental Setups of Total Internal Reflection Imaging Guided Confocal Microscopy
A home-built Total Internal Reflection Fluorescence Microscopy imaging-guided
Confocal Fluorescence Spectroscopy (TIRFM-CFS)58-61 was used to capture the fluorescence
signals from Resorufin (nascent-formed fluorescent product) in the active site of HRP. We have
combined the advantages of the wide-field imaging of objective type TIRFM with the high
spatial and lateral resolution of confocal microscopy for the studies of conformational dynamics
of a single HRP protein. In fact, the combination of TIRFM and confocal microscopy has been demonstrated in previous studies.62-70 TIRFM images the surface-immobilized HRP enzymes in a
wide field, which facilitates the probing of stochastic events of the enzymatic reaction and the
localization of the single HRP molecules.58,59,71 Confocal microscopy provides time-resolved single photon detection, which is extremely useful to unravel the dynamics of single enzymes.
The experimental setup is based on an objective-type TIRFM with an inverted microscope
(Axiovert 200M, Carl Zeiss) in an epi-illumination configuration which combines the TIRFM
mode and the confocal mode. TIRFM imaging-guided CFS is achieved by recording images in
TIRFM mode and shifting the pinpoints of interest to the confocal mode for single-molecule
fluorescence and anisotropy measurement through time-correlated single photon modules. 52
Figure 2.12 Schematic representation of the Total Reflection Fluorescence Microscopy imaging- guided Confocal Fluorescence Spectroscopy. DM1 and DM2: Dichroic mirror beam splitters,
TL: Tube Lens, SPP: Side port prism, M1: Reflection mirror, EF1 and EF2: Emission filters, L1 and L2: Lens, PBS: Polarizer beam splitter, SPAD1, and SPAD2: Single-photon avalanche photodiode. 53
In detail, in TIRFM mode, wide-field images are recorded by an electron multiplying
charge-coupled device (EMCCD Photomax 512B, Princeton Instruments), from which the
positions of the fluorophores are localized by the on-off burst behavior of the enzymatic reactions. Facilitated with a home-written program with the transformation matrix, the
corresponding coordinates in the confocal mode are calculated. Finally, the images in confocal
mode are acquired and by focusing on one particular spot, a single photon trajectory is collected.
Figure 2.12 shows the schematic set-up in this experiment. Details of the experimental set up are
similar as that described in a previous publication.58 In TIRFM mode, a cw laser (GCL-050-L,
Crystal laser) with 532 nm is expanded by the telescope lens 1 and lens 2, aligned to the side port
by a dichroic mirror beam splitter (DM1) with the axis of rotation located in the intermediate
image plane, reflected by a side port prism (SPP), passed through an empty filter cube, and
focused by a tube length (TL) on the back focal plane of the objective (Plan-Fluar, 1.45 NA,
100×, Carl Zeiss). By adjusting the incident angle at the cover glass/solution interface to slightly
exceed the critical angle to generate an evanescent field for total internal excitation, we slightly
deviate DM2 from 45° relative to the incident light. In our experiment, the refractive index of the
aqueous solution and cover glass is 1.33 and 1.515, respectively. Here, a 0.68° deviation from
the 45° incident angle of the DM2 provides total internal reflection with evanescent field depth
about 100 nm. An evanescent electromagnetic field with exponential intensity decay in the
normal direction is generated at the cover glass-solution interface, which only excites the
fluorescence molecules within the evanescent field. The fluorescence emission is collected by
the same objective and passes a mirror M1 and a filter before detection by EMCCD. 54
Figure 2.13 Calibration of TIRFM imaging-guided confocal single-molecule fluorescence spectroscopy. (a) Wide-field fluorescence image of the microspheres in EMCCD coordinates. (b)
Confocal fluorescence image of the microspheres in two polarization channels.
In the confocal mode, a pulsed laser (Chameleon Discovery, Coherent, ~100 fs fwhm) is used. After an optical parametric oscillator (OPO BASIC, Coherent Inc.) and frequency doubling by a β-barium borate crystal (BBO), the linear-polarized pulse laser is aligned through a lens- pinhole lens system to the backport and then reflected by a dichroic mirror beam splitter (DM1) in the filter cube and focused on the cover glass/solution interface by the objective. The confocal images are acquired by an x-y 2D piezoelectric scanning stage (Nano-H100, MCL) with a positioning resolution of 0.2 nm for fine-tuning. By focusing on the pinpoint of interest, the fluorescence emission is collected by the same objective and then separated by a polarizer beam splitter (PBS) into parallel and perpendicular polarization components, which are detected by a pair of single photon avalanche diodes (SPAD1 and SPAD2) simultaneously. Finally, HRT-82 55
(Becker＆Hickl’s Time Resolved Single Photon Counting Router Module) is used to transmit the
signals from two SPAD detectors to one single photon counting module. Both the arrival time (t)
and delay time (∆t) between the pulse excitation and emission are recorded for each single enzymatic turnover event.
To combine TIRFM and CFS into one experimental setup, there are some common optical paths lying between DM1 and the cover glass for two detection modes. Switching between TIRFM mode and CFS mode is achieved by toggling the filter cube turret and the side
port prism. Moreover, a transformation matrix is developed for determining the coordinates of
the spots of interest from the confocal image from the guidance of the TIRFM image. The switch
between two modes is completely independent to the sample stage, which ensures the system is
stable during the experiment and the switch. The transformation relationship between the
coordinates from two modes is as follows:
= + Equation 2.11 𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄𝒄𝒄 𝒙𝒙 𝒙𝒙𝟎𝟎 𝒙𝒙 −𝒙𝒙𝟎𝟎 𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄𝒄𝒄 �𝒚𝒚 � �𝒚𝒚𝟎𝟎 � 𝑻𝑻 ∙�𝒚𝒚 −𝒚𝒚𝟎𝟎 �
𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄 𝒙𝒙𝟏𝟏 −𝒙𝒙𝟎𝟎 𝒙𝒙𝟐𝟐 −𝒙𝒙𝟎𝟎 = � 𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄� Equation 2.12 𝒚𝒚𝟏𝟏 −𝒚𝒚𝟎𝟎 𝒚𝒚𝟐𝟐 −𝒚𝒚𝟎𝟎 𝒄𝒄𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄𝒄𝒄 𝑻𝑻 𝒙𝒙𝟏𝟏 −𝒙𝒙𝟎𝟎 𝒙𝒙𝟐𝟐 −𝒙𝒙𝟎𝟎 � 𝒄𝒄𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄𝒅𝒅 𝒄𝒄𝒄𝒄𝒄𝒄 𝒄𝒄𝒄𝒄𝒄𝒄� 𝒚𝒚𝟏𝟏 −𝒚𝒚𝟎𝟎 𝒚𝒚𝟐𝟐 −𝒚𝒚𝟎𝟎 where (xccd, yccd) is the x-y coordinate of the spot of interest in the TIRFM image, and (xcf,
ycf) is the x-y coordinate of this spot in the confocal image. T is the transformation matrix that
can be determined from any three non-collinear patterned spots’ coordinates. Figure 2.13 shows 56
the calibration image by 0.1mm fluorescent microspheres from two modes, from where we can
calculate the transformation matrix.
2.7 Materials and Sample Preparation
2.7.1 Immobilization of Single HRP Enzyme
Figure 2.14 Conceptual scheme for single HRP sample preparation.
Horseradish peroxidase (HRP) is a glycoprotein with 6 lysine residues, which can be used
for tethering (Figure 2.14). There are 2 lysine residues partially/totally buried inside of the protein matrix structure and 3 lysine residues on the same surface of the protein. In a typical surface immobilization, only the exposed residues are likely subject to the tethering. From the
Steered Molecular Dynamic (SMD) simulation, we studied all the possible configurations to help
us understand the mechanical force effect on the active site conformations of a single enzyme
molecule and have found that a pN weak force has the capability to perturb the enzyme active
site conformations under different tethering geometries.72 The procedure of sample preparation is 57
shown in Figure 2.15, which is similar to the previous publication.72 The HRP molecules were
tethered to the cover glass at one end by 3-aminoproplytriethoxy Silane and Dimethyl
Suberimidate·2HCl (DMS) linker and attached to a streptavidin-coated super-paramagnetic bead
(Dynabeads® MyOne™ Streptavidin T1, 1.05 mm diameter, Invitrogen Company) via a biotin- streptavidin noncovalent interaction at the other end. All the attachment to HRP molecules, either Dimethyl Suberimidate·2HCl (DMS) or Biotin are bound to the amine group of lysine residues in the amino acid sequence.
Figure 2.15 Protocol for single HRP sample preparation. Single HRP is immobilized on the
cover glass surface and bound to the streptavidin-coated paramagnetic bead at the other end.
First, the cover glass (Gold seal, 3406) was washed in sulfuric acid dichromate cleaning
solution for 5 mins to eliminate grease and possible fluorescent spots. The cover glass was next
sonicated in distilled water, acetone and methanol for 15 mins separately. After being washed
with distilled water and dried by nitrogen gas, the cover glass was incubated overnight in a 58
DMSO solution containing a 10% concentration mixture, which consists of 3- aminoproplytriethoxy Silane and isobutyltrimethoxy Silane in ratio of 1:10000. The cover glass was then washed with distilled water and consecutively transferred and incubated for 4 hours in the following systems: 10nM Dimethyl Suberimidate·2HCl (DMS) in PBS buffer solution (PH =
8.0); 1nM HRP in PBS buffer solution (PH = 7.4); 10nM NHS-PEO12-Biotin in PBS buffer solution (PH = 7.4); 1ul magnetic bead in 15 ml PBS buffer solution, PH = 7.4.
2.7.2 Cell Cultures
Cell culture was performed in an incubator with an atmosphere of 5% (v/v) enriched CO2 air at 37 °C. Firstly, cells were cultured in a T-75 flask in DMEM (Dulbecco's Modified Eagle
Medium, 11995, Gibco/Life Technologies) supplemented with 10% fetal bovine serum (Sigma-
Aldrich) and 1% penicillin-streptomycin (Gibco/Life Technologies). The cell population reached
~75% coverage on the surface of the T-75 flask in 2-3 days, then the cells were sub-cultured in a new T-75 flask for future use and in a 35 mm petri-dish containing a 25 mm round glass slide, which is further used for our experiment. Meanwhile, the paramagnetic beads (Dynabeads® M-
280 Streptavidin) were dispersed in the same culture medium before use. When the cell population in the petri-dish reached ~50%, the previous prepared paramagnetic beads suspension was added into the cell culture 2 days before the experiment to allow for phagocytosis. Finally, the adherent cell sample was trypsinized by Trypsin EDTA and then transferred to the cell chamber for live-cell imaging and further measurement.
2.7.3 Rhodamine 6G Stained Paramagnetic Beads
The paramagnetic beads were incubated in the Rhodamine 6G (R6G) solution for 12 hours and then withdrawn by permanent magnet. Then, three consecutive washes with distilled 59 water followed to remove the excess unbound R6G. An aqueous solution of R6G stained paramagnetic beads was prepared just prior to the experiment.
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CHAPTER 3. PROBING CONFORMATIONAL DYNAMICS OF AN ENZYMATIC ACTIVE
SITE BY AN IN SITU SINGLE FLUOROGENIC PROBE UNDER PICONEWTON FORCE
MANIPULATION
Unraveling the conformational fluctuation of an enzyme during the catalytical steps of an enzymatic reaction, particularly during the product release, an essential step which directly determines the productivity of a turnover, is challenging due to the transient nature of intermediate conformational states, conformational fluctuations, and the associated complex dynamics. Here, we report our study on the conformational dynamics of horseradish peroxidase using single-molecule multiparameter photon time-stamping spectroscopy with mechanical force manipulation. It is highly informative to manipulate the enzyme under the enzymatic conditions and simultaneously monitor in real-time the conformational fluctuations of the enzymatic active site. A fluorogenic enzymatic reaction is used as a model system, having a nascent-formed fluorescent product molecule at its perfect fitted position at the enzymatic active site, which serves as an in-situ probe to report the real-time active-site configuration and its fluctuations.
Interestingly, the product-releasing dynamics shows the complex conformational behavior with multiple product-releasing pathways. However, under magnetic force manipulation, the complex nature of the multiple product-releasing pathways disappears, and more simplistic conformations of the active site are populated.
3.1 Introduction
3.1.1 Significance of the Protein Conformational Dynamics
Enzymes are macromolecular biological catalysts capable of enhancing the reaction rate with millions of time faster to maintain the metabolic processes and sustain the life.1,2 In modern 65 enzymology, enzymes have been known to increase the reaction rate by changing the energy landscape and reaction pathways.3-5 Moreover, it has been extensively proven that the 3- dimensional structure, especially the conformational dynamics plays a crucial role in protein functioning, including protein folding/unfolding, misfolding, aggregation processes, as well as in enzyme catalysis and cell signaling functioning.6-8 Instead of being the static structures, the enzymes need to possess a variety of conformational states and fluctuate between the conformational states to complete the enzymatic reaction, which has been studied and proven by theoretical simulations and single-molecule microscopic techniques functioning.9-14 Enzymes are intrinsically dynamics and inhomogeneous, which may have a conformational fluctuation ranging from ns to s and 10-2 Å to Å, temporally and spatially.15,16 These spatial and temporal fluctuation largely defines the conformational states that the enzyme adopts, which ultimately controls the function of the enzyme, including the enzyme potential energy surface, reaction coordinates as well as the reaction rates.17-19 Not only do the active site relevant amino acids determine the enzyme catalysis, but the overall enzyme matrix, particularly the conformational dynamics also govern the enzyme activity.20-24
A key feature of the protein conformational dynamics is the fast interconversion between different conformational states, including high energy states and low energy states, with different population and lifetime.25 Understanding how the enzyme dynamics of the conformational fluctuation relates to the enzyme activity can shed light on the fields of the enzymology.26
Therefore, protein dynamic profile has been used to fully describe a protein with the information regarding all the conformational states and their population. However, due to the transient nature of these conformational states, it is still challenging to accurately capture and probe each 66
intermate conformational state by the conventional experimental techniques even with the
advanced the site-specific dye labeling.
3.1.2 Product Releasing in an Enzymatic Reaction
Despite the traditional understanding which is mostly rooted on deep comprehension of
the transition states and reaction rates, the mechanism of the enzymatic reaction has been well
explained by Michalis-Menten mechanism, where multiple catalytical steps are involved.27-29
Enzyme (E) interacts with the substrate (S) by a non-covalent bond to form an enzyme-substrate complex [E·S], further get converted to activated complex and followed by the conversion to enzyme-product complex [E·P]. Finally, undergoing the product release process, the product (P) was released from the active site and the enzyme would go back to the cycle of enzymatic reaction.27,30 Nevertheless, all the catalytical steps are intrinsically reversible processes, a productive enzymatic turnover occurs only when the product successfully released from the enzymatic active site.31,32 More importantly, the product releasing is often the rate limiting step.
Therefore, it is critical to understand the mechanism how the product released from the
enzymatic active site and how the conformational dynamics play the role in facilitating the product releasing. Each catalytical step involves different intermediates and conformers, which makes the system complicated and hard to characterize due to the transient nature of these intermediates.33
There are few studies so far to study the release of an enzyme product.33-36 Vendruscolo
and coworkers has reported that a low-population “unlocked” intermediate state facilitates the
product release from the human lysozyme and illustrated that conformational dynamics play a
central role in enzymatic reaction.33 Further, Wright and coworkers have demonstrated a study 67
on the enzyme dihydrofolate reductase that there are two parallel pathways involved in the
product release process, an intrinsic spontaneous dissociation pathway and an allosteric mediated
pathway.34 Recently Lu group reported that horseradish peroxidase (HRP) adopts multiple
intermediate conformational states and enzymatic product releasing shows two pathways: a
solvation-mediated diffusion releasing pathway and a sudden spilling-out releasing pathway. A
significant pathway is that the product molecules are spilled out from the enzymatic reaction site
with tightly bound enzyme active-site conformation, although, an open-up conformational active
site releasing the product molecules through a loosely bound enzyme-product state remains the
significant parallel product releasing pathway.36
3.1.3 Force Manipulation on Enzyme Dynamics
Protein conformational states can be easily perturbed or controlled by physical or chemical perturbation. Among them, mechanical force has been used to manipulate protein and further utilized to provide deep understanding of ultimate relationship between structure- dynamics-function. It is highly informative to manipulate the enzyme under the enzymatic conditions and simultaneously real-time monitor the conformational fluctuations of the enzymatic active site as well as the reaction activity change. Extensive efforts have been made by traditional approaches, such as NMR studies,37 molecular dynamics simulations,19,38 and
theoretical modeling,39-44 to provide understanding on the associated structural motions.
However, because of the complex and heterogeneous feature of the involved enzymatic dynamics and its external surroundings, we still lack systematic understanding, particularly regarding the complex mechanical force stimulation. For example, how the force stimulus is transformed into a chemical response and what are the interplay between conformation dynamics and the external mechanical force? As reported in the previous publication in our group, Cy3- 68
Cy5 dye labeled HPPK protein has been studied as a model system to investigate the
conformational dynamics using magnetic tweezers.45 The enzyme in the condition of force pulling shows to have less flexibility when binding to the substrate. Later, a recent work on manipulating single-molecule enzymatic activity using magnetic tweezers has been reported in
2015.46 HRP catalyzed enzymatic reaction has shown to be successfully manipulated by external
force using magnetic tweezers. Furthermore, the deformed enzyme molecules under force
pulling still show significant activities.
Here, we utilized a fluorogenic enzymatic reaction with the nascent formed fluorescent
product in the active site of the enzyme as a perfect in-situ molecular probe to real-time report
the conformational dynamics of the enzymatic active site. The typical fluorogenic feature of this
reaction makes the nascent-formed product molecule at its perfect fitted position at the
enzymatic active site, serving as an in-situ probe to report the real-time active-site configuration
and its fluctuations. Particularly, we aim to study the effect of external force on the product
release under mechanical force manipulation using magnetic tweezers technique. We chose
single HRP enzyme molecule as our model because many aspects of the enzymatic reaction have
been well studied in both experimentally and theoretically, including our previous works,36,46,47
which form a solid foundation for our data interpretations.11,36,48-53 More importantly, it is
conceptually technical advancement that utilizing the feature of the fluorogenic reaction that the
nascent formed product molecule at its perfect fitted position at the enzymatic active site, serving
as an in-situ probe to report the real-time active site configuration and its fluctuations.
Meanwhile, a home built in total internal reflection fluorescence microscopy imaging-guided
confocal fluorescence spectroscopy is used to characterize the conformational dynamics of the
nascent fluorescent product releasing from the enzymatic active site. 69
We have studied the mechanical force manipulation on HRP catalyzed fluorogenic
enzymatic reaction using home-developed magnetic tweezers to unravel the conformational
dynamics of the enzymatic active site. The remarkable advantage of single-molecule magnetic
tweezers is that a small pN scale applied force provides the edge of pausing the deformation and
unfolding of a biomolecule at any point under a physiological condition without adding any
chemical denaturant.46
3.2 Experimental Sections
3.2.1 Sample Preparation
Maleimide-activated HRP enzyme was purchased from Thermo Scientific (31485). EZ-
Link NHS-PEG12-biotin was purchased from Thermo Scientific (21312). Streptavidin coated
paramagnetic beads were purchased from Invitrogen (Dynabeads MyOne Streptavidin T1).
Amplex Red was purchased from Life Technologies (A22188). Cover glass was purchased from
Gold Seal (3406). 3-mercaptopropyl-trimethoxysilane (175617) and isobutyltrimethoxysilane
(444065) are purchased from Sigma-Aldrich. All other chemicals were purchased from Sigma-
Aldrich and used without further purification. PBS buffer was prepared by mixing potassium
phosphate dibasic solution (P8584; Sigma-Aldrich) and potassium phosphate monobasic solution
(P8709; Sigma-Aldrich). 70
Figure 3.1 Conceptual scheme of Horseradish peroxidase (HRP, PDB 1HCH) as an enzyme to catalyze the non-fluorescent Amplex Red to fluorescent product Resorufin in presence of H2O2.
The active site involved amino acids are highlighted in Red. 71
Figure 3.1 shows conceptual scheme of a single immobilized HRP enzyme molecule with a tethered magnetic bead, which is the sample used in our experiments. Single HRP molecules were tethered to the cover glass at one end by 3-mercaptopropyl-trimethoxysilane with
sulfhydryl crosslinker through Maleimide thiol reaction and attached to a streptavidin-coated
superparamagnetic bead via a biotin−streptavidin bond at the other end. First, the cover glass was
washed in sulfuric acid dichromate cleaning solution for 5 min to eliminate grease and possible
fluorescent spots. The cover glass was next sonicated in distilled water, acetone, and methanol in
15 min separately. After being washed with distilled water and dried by nitrogen gas, the cover
glass was incubated overnight in a dimethyl sulfoxide solution consisting of 3-mercaptopropyl-
trimethoxysilane and isobutyltrimethoxysilane with a ratio of 1:10000. The cover glass was then
washed with distilled water and consecutively transferred and incubated for 4 h in the following
systems: 1nM HRP in PBS buffer solution (pH = 7.4); 10 nM NHSPEO12-biotin in PBS buffer
solution (pH = 7.4). After washing the coverslips, we incubated them in 2 ul streptavidin-coated
paramagnetic bead in 15ml PBS buffer solution (pH = 7.4) for another 4 h.
The reaction solution was prepared just prior to the experiment, which contains 200 nM
Amplex Red (10-acetyl-3,7-dihydroxyphenoxazine) and 2 mM hydrogen peroxide (H2O2). A sample immobilized cover glass was attached at the bottom of the reaction chamber and about
200 μL of the reaction solution was filled in for the experiment, while a clean cover glass is put on the top of the reaction chamber as a lid to avoid the evaporation. 72
3.2.2 Magnetic Tweezers Coupled Total Internal Reflection Imaging Guided Confocal
Microscopy
Figure 3.2 (A) Typical snapshot of an image from an electron-multiplying charge-coupled
device (EMCCD) in Total-internal reflection mode under an applied magnetic field. (B)
Fluorescence confocal images of two polarization components from the single-photon avalanche
photodiodes (SAPD1 and SAPD2) in the confocal mode under an applied magnetic field.
Corresponding single-molecule photon time-stamping data will be collected further. PBS:
Polarization beam splitter. 73
To address the conformational dynamics impact on the enzymatic product release under
force manipulation, we carried our experiment with an HRP-catalyzed fluorogenic assay by a
home-built TIRFM-guided confocal fluorescence spectroscope combined with magnetic
tweezers based on an inverted microscope (Axiovert 200M; Carl Zeiss) equipped with a 63× oil-
immersion objective (N.A. 1.4, Plan-Apochromat; Zeiss). The detailed experimental setup has
been discussed in Chapter 2. In brief, single HRP enzyme is identified and located through the
stochastic on−off burst of the fluorescence signals detected by EMCCD in the TIRF mode
(Figure 3.2A). Then, the corresponding coordinates of the fluorescence spot of interest are
calculated based on the localization by two-dimensional Gaussian fit and coordinates transformation.54 In the confocal mode, fluorescence confocal images of two polarization
components were acquired from a pair of single photon avalanche photodiodes through a
computer-controlled close loop piezoelectric x-y scanning stage (Figure 3.2B). Finally, the
excitation focus in the confocal mode is shifted on single enzyme and single photon time
stamping data is further collected for two polarization components while simultaneously
applying a pulling magnetic force via magnetic tweezers.
3.2.3 Data Analysis
Fluorescence anisotropy analysis on the fluorescent probe is essential to provide
information on its attached protein dynamics and steric constraints, such as the conformational
change and the enzyme flexibility.55-57 Upon excited by a vertically polarized pulsed light in the
experiment, there are a number of excited state molecules that have absorption transition moment
oriented along the electric vector of the polarized exciting light, thus the emission is also
polarized. Anisotropy is used to describe the extent of the polarization of the emission 58.Two
channels (T-format) are used to measure the intensities of the parallel ( ( )) and perpendicular
𝐼𝐼∥ 𝑡𝑡 74
( ( ))) components simultaneously. As the sensitivities of the two detectors are typically
⊥ distinct,𝐼𝐼 𝑡𝑡 G factor is used to compensate the difference between the two channels. G factor is
determined by the fluorescent intensity response of two channels with different excitation
polarizations. Here, the measured G factor is 1.79. The total intensity (IT(t)) and the anisotropy
(r) can be given by equation 3.1 and equation 3. 2, respectively
( ) = ( ) + ( ) Equation 3.1
𝑰𝑰𝑻𝑻 𝒕𝒕 𝑰𝑰∥ 𝒕𝒕 𝟐𝟐𝟐𝟐𝑰𝑰⊥ 𝒕𝒕 ( ) ( ) ( ) = Equation 3.2 ( ) ( ) 𝑰𝑰∥ 𝒕𝒕 −𝑮𝑮𝑰𝑰⊥ 𝒕𝒕 𝒓𝒓 𝐭𝐭 𝑰𝑰∥ 𝒕𝒕 +𝟐𝟐𝟐𝟐𝑰𝑰⊥ 𝒕𝒕 As the fluorescent product (Resorufin) is still attached to protein before the product is released
from the enzymatic active site, the motions of the molecule are confined. Rotational correlation
time is used to describe the restricted rotational diffusion of the protein-attached fluorescent product, which largely depends on the size and shape of the molecule, as well as the molecular confinement status and the local environment. The Perrin equation is used to define the rotational correlation time in Equation 3.3:
= + Equation 3.3 𝟏𝟏 𝟏𝟏 𝝉𝝉𝒇𝒇 𝒓𝒓 𝒓𝒓𝟎𝟎 𝒓𝒓𝟎𝟎𝝉𝝉𝒓𝒓
Here, r is the steady-state anisotropy; r0 is the fundamental anisotropy in the absence of
59,60 molecular rotation (0.318 in current experiment, measured in 80% glycerol solution); τf is the
fluorescence lifetime; τr is the rotational correlation time. 75
3.2.4 Force Calibration for Magnetic Tweezers
A cylinder permanent magnet is the key component of our first generation of the magnetic tweezers. The magnet (D4C-N52, 1/4″diam ×3/4″thick, K&J Magnetics) we used in the experiment is a widely used rare-earth strong neodymium magnet with a grade of 52. We have built an independent 3D rotational stage with height adjustment to accurately position the magnet right on top of the sample. In the experiment, the magnetic bead (Dynabeads® MyOne™
Streptavidin T1, 1.05 mm diameter, Invitrogen Company) is bound to single protein sample to generate the magnetic force, which is the driving force to manipulate the conformational dynamics of single protein. In the absence of the external magnetic field, the magnetic moment of each atom in paramagnetic bead point in random direction because the magnetic beads are superparamagnetic. However, in the presence of external magnetic field, the paramagnetic bead is magnetized and acts as a tiny magnet and moves towards the higher magnetic flux. The magnitude of the magnetic force is adjusted by displacing the magnets vertically to change the distance between the magnet and the sample. The magnetic force is calibrated before the experiment by measuring the magnetic field gradient using the following equation. The magnetic field strength curve (upper panel, Figure 3.3) was obtained by placing the probe of gauss meter at different distances away from the permanent magnet. 76
0.6 0.4 0.2 0.0 strength(T) Magnetic field 4 2 0 Force (pN) 0 2 4 6 8 10 12 14 16 Distance (mm)
Figure 3.3 Magnetic force calibration curve of the magnetic tweezers.
For a given paramagnetic bead, the force can be calculated in the following equation 3.4:
= = ( ) = = Equation 3.4 𝝏𝝏𝝏𝝏 𝑭𝑭 −𝜵𝜵𝜵𝜵 −𝜵𝜵 −𝒎𝒎 ∙𝑩𝑩 𝑴𝑴𝑴𝑴 ∙ 𝜵𝜵𝜵𝜵 𝑴𝑴𝑴𝑴 𝝏𝝏𝝏𝝏 Where, m is the magnetic moment of the paramagnetic bead, which can be expanded to the
product of the volume Magnetization (M) and volume (V) of single paramagnetic bead. B is the magnetic field. In our experimental setup, the magnet is kept ~2 mm away from the sample place to impose a magnetic field of ~2000 G, which allows the magnetization of the paramagnetic bead to be close to its saturation value. For the specific paramagnetic bead that we used, the saturation magnetization (Ms) is given by 43×103 A m-1 and the volume (V) is 0.6×10-18 m3. Therefore,
according to the force calibration (lower panel, Figure 3.3), an external force of ~2 pN generated
by the magnet can be applied on the paramagnetic bead bound single protein. 77
3.3 Results and Discussions
3.3.1 Time-Correlated Single Photon Time Stamping Spectroscopy
The metalloenzyme HRP is a 44 KDa glycoprotein, catalyzes the oxidation of a broad range of substrates such as aromatic amines, indoles, phenols, and sulfonates in the presence of hydrogen peroxide as an oxidizing agent.48,52 Extensive efforts have been made previously
toward the crystal structure and the catalytic mechanism of HRP, which only limited to the
discussion of the reaction rate and dynamic disorder.52,53,61-63 Here, we used a mode system that
HRP catalyzes the nonfluorescent Amplex Red to fluorescent resorufin in the presence of H2O2 to investigate the conformational dynamics impact in the product release under force manipulation. Generally, the substrate Amplex Red in the solution diffuses and binds to the active site of the enzyme, further gets converted to fluorescent product, and finally gets released from the active site and diffuse away in milliseconds, which completes an enzymatic reaction turnover cycle.
Fluorescence from the product in the active site of protein is used as a key parameter to address the product releasing mechanism in a fluorogenic enzymatic reaction. To investigate the impact of external force on the product releasing process, we collected single photon counting data of the enzymatic reaction with and without the force manipulation by combining with single-molecule magnetic tweezers technique. TIRFM image-guided confocal spectroscopic technique allows the fluorescence signal of Resorufin to be detected only when it is still confined in the active site of HRP enzyme molecule. It is due to the fact that the fluorescent Resorufin molecule diffuses into solution quickly in millisecond after being released from the active site of
HRP.64 In a single-molecule photon time stamping measurement, both the arrival time (t) and 78 delay time (∆t) are recorded for each detected photon. Thus, each photon will have two characteristics, among which chronic arrival time (t) is the real time when photon is detected and the delay time (∆t) is the time interval between the pulse excitation and the photon emission.
Moreover, histogram of delay time provides the fluorescence decay of a turnover event, which yields the fluorescence lifetime.65,66 The intensity trajectory is calculated from the distribution of arrival time (t) within a given bin time. A typical single-molecule photon time stamping raw data in parallel and perpendicular polarization have been shown in Figure 3.4. Furthermore, fluorescence intensity trajectories and lifetime decays have been also plotted for the corresponding enzymatic turnover event.
Figure 3.4 A typical raw data of single-molecule photon time-stamping spectroscopy in the parallel channel (A) and perpendicular channel (B). Meanwhile, the fluorescence decay and fluorescence intensity trajectory are plotted in the corresponding figure as well.
We have combined the anisotropy measurement with our single-molecule time stamping spectroscopy to acquire information regarding the properties of the fluorescent product and its surrounding environment, which improves the resolution of the optical detection.67-71 Figure 3.5 79 provides a portion of fluorescence intensity trajectories of parallel and perpendicular polarization with a bin time of 20 ms, from which the anisotropy trajectory is calculated using Equation 3.2.
We have conducted the single-molecule enzymatic reaction assay in a low substrate concentration to achieve isolated single turnover events, which still serve the purpose of our study, although a relative low turnover rate ( 0.1–0.3 s−1) is recorded. Additionally, we have only considered and analyzed those isolated eve∼ nts with a high fluorescence photon count
(>1,000 photons over the entire catalytic event) to eliminate the fluorescence fluctuation from the background. As can been seen in Figure 3.5A, anisotropy fluctuates around zero between fluorescent photon bursting while there is a substantial increase of anisotropy during the photon burst in an enzymatic turnover. The increase of anisotropy indicates that the active sites is relatively confined for the fluorescent product to freely rotate. For further quantitative analysis of the enzyme motion in the enzymatic reaction, the detected photon stamping signal associated with a turnover event is divided into three regions: rising edge, on time, and falling edge. The rising edge corresponds to the formation of the enzyme−product complex. ON time represents the duration of the enzymatic reaction when the high fluorescent intensity of the product is above the background noise. The falling edge is the final step of an enzymatic turnover, which corresponds to the product releasing process. The limits of the rising edge, ON time, and falling edge are identified as the time interval between the intensity values I1 and I2, I2 and I3, and I3 and I4, respectively (Figure. 3.5B). I1 and I4 are defined as the mean value of the background intensity, whereas I2 and I3 are defined as the mean value of on-time intensity of an enzymatic turnover event. 80
Figure 3.5 (A) Single molecule fluorescence intensity and anisotropy trajectories of HRP- catalyzed oxidation of Amplex Red, binning with 20 ms. Parallel polarization component (red) and perpendicular polarization component (black) after compensation by the G factor;
Corresponding anisotropy fluctuation (green, upper panel). (B) Identification of the rising edge,
ON state, and the falling edge of a catalytic event.
3.3.2 Conformational Dynamics Without Force Manipulation
To quantitatively study the conformational dynamics during each catalytical step in an enzymatic reaction, we have employed multi-parameter analysis: Fluorescence lifetime,
Anisotropy and Rotational correlation time, which provides the photophysical properties of the nascent formed fluorescent product and the surrounding environment.72-77 Fluorescence lifetime is directly obtained from single-molecule photon time stamping measurement by averaging the delay times of all the photons in 20ms bin time for both polarizations. Anisotropy is measured by splitting the fluorescence emission using a polarization beam splitter and the emission photons in both parallel and perpendicular polarization are detected the by a pair of single photon avalanche diodes (SPAD). Anisotropy and rotational correlation time are calculated based on the equation 81
3.2 and 3.3. Particularly, rotational correlation time represents the average time of a molecule to
rotate one radian, which is used to study the rotational motions of fluorescent product.
Figure 3.6 Two-dimensional joint distribution of anisotropy and lifetime (Upper) and rotational
correlation time and lifetime (Lower) calculated from 281 catalytic turnovers. Two-dimensional
joint distribution of anisotropy and lifetime at (A) the rising edge, (B) ON time, and at (C) the falling edge. Two-dimensional joint distribution of rotational correlation time and lifetime at
(A1) the rising edge, (B1) ON state, and (C1) the falling edge.
To explore the detailed conformational dynamics, we have conducted multi-parameter
analysis: 2D correlation plots between lifetime and anisotropy, as well as 2D correlation plots
between lifetime and rotational correlation time. The advanced data collection and analyses
method is capable of identifying multiple product-releasing and clearly resolves the multiple
channel enzymatic active-site conformational dynamics during enzymatic product releasing
process, which could be difficult to resolve while using only one observable parameter, e.g., 82 either anisotropy or lifetime alone.78-80 Figure 3.6 show the 2D joint distribution of lifetime and anisotropy, as well as lifetime and rotational correlation time at the rising edge, during on time and at the falling edge from 281 catalytical events, respectively. Only one domain is populated in fluorescence lifetime-anisotropy correlation at both rising edge (domain 1’) and during on time
(domain 1’’). However, interestingly, three distinct domains are distributed at the falling edge
(domain 1, 2 and 3). The presence of multiple distinct domains at the falling edge indicates that multiple distinct conformations of active site of the enzyme are populated during the product releasing. To get a quantitative knowledge of the enzyme conformational dynamics during enzymatic reaction, we further calculated the corresponding fluorescence lifetime, anisotropy and rotational correlation time for the domains populated at the rising edge, during the on time and at the falling edge (Table 3.1). As has been reported in the table, domain 1’ exhibited at the rising edge centers on τf=3.3±0.2ns, r=0.11±0.04 and τr =1.8 ns. Error bars are calculated as two standard deviations from the data that fall within the black circles. A similar conformation is present during the on time as the enzymatic reaction goes on because of the similar domain present during on time, which centers on τf =3.4±0.2 ns, r=0.12±0.06 and τr =2.0 ns. The result strongly suggests that the active site conformation populated at the rising edge, i.e., when the nascent product is formed, continues to persist as the reaction proceeds. The large standard deviations of domain 1′ and 1′′ may indicate that large scale conformational motions are involved. However, besides the similar domain (domain 2 centers on τf=3.5±0.04ns, r=0.10±0.04 and τr =1.5 ns) that exists at the beginning of the enzymatic reaction and go through the rising edge and falling edge, two more domains appear at the falling edge. It is evident that multiple conformations are populated during the product release although they are not so pronounced as in the previous publication. Moreover, it is interesting that the two more new domains show 83
lower anisotropy. In details, domain 1 centers on τf=3.3±0.1ns, r=0.07±0.04, and τr = 0.9 ns and
domain 3 centers on and domain 3 is centered on τf=3.5±0.04ns, r=0.05±0.02 and τr =0.6 ns.
Table 3.1 Summarized Rotational Correlation time of all the domains present in the catalytic event with and without force manipulation (τf: Fluorescence Lifetime; r: Anisotropy; τr: Correlation Rotational time.)
Falling Edge Rising Edge On time Domain 1 Domain 2 Domain 3
τf =3.3±0.2 ns τf =3.4±0.2 ns τf =3.3±0.1 ns τf =3.5±0.04 ns τf =3.15±0.04 ns No r=0.11±0.04 r=0.12±0.06 r =0.07±0.04 r=0.10±0.04 r=0.05±0.02
Force τr=1.8 ns τr =2.0 ns τr =0.9 ns τr =1.5 ns τr =0.6 ns (0 pN) (54%) (27%) (18%)
τf = 3.45ns τf = 3.5ns τf = 3.5ns Constant r =0.12 r = 0.12 r = 0.07
Force τr = 2.1 ns τr = 2.1 ns τr = 1.0 ns (1.5 pN)
According to the previous work, Resorufin has a rotational correlation time of 0.1 ns in
water.81 Domain 2 with higher anisotropy and rotational correlation time (1.5ns) suggests a
tightly bound state or an confined active site environment for Resorufin. Domain 1 and 3 has
similar lifetime values as other domains but has much lower anisotropy and rotational correlation
time, which suggests a loosely bound state or an open-up conformation of the HRP active site.
We further calculated the population of each domains populated during the product release.
Loosely bound domains 1 and 3 have 54% and 18% relative population, respectively, and tightly
bound domain 2 has 27% relative population (Population percentages are calculated for the data
that fall within the black circles in Figure 3.6). The significant population of domain 2 indicates that tightly bound conformations may have favorable electrostatic interactions and other possible 84 interactions. Taking together, the conformational dynamics during the product release can be explained in two situations: (1) domain 2 represents intermediate conformational states of the enzymatic active site but still waits for the right orientation of the amino acid residues necessary to open the active site for product release (domain 1 and 3). (2) three domains are populated as parallel pathways. The presence of tightly bound states (domain 3) suggests that the product is expelled out from the active site into the solution. Although multi-parameter data analysis is powerful enough to unravel the existence of structurally distinct conformational states of active site in the product releasing step, information regarding the sequence of appearance of the intermediate conformations during the product release is missing due to the low time resolution of data. A higher spatial resolution may be required in future for definite conclusion.
Nevertheless, both situations point to complex conformational dynamics of the active site during the product release.
Our results have shown a great agreement with the previous publications that a low- population “unlocked” intermediate state facilitates the product release from the human lysozyme and large-scale conformational fluctuations involved when the product is necessary in releasing it.33 Further, Wright and coworkers have demonstrated a study on the enzyme dihydrofolate reductase that there are two parallel pathways involved in the product release process, an intrinsic spontaneous dissociation pathway and an allosteric mediated pathway.34 85
3.3.3 Conformational Dynamics Under Force Manipulation
Figure 3.7 Two-dimensional joint distribution of anisotropy and lifetime (Upper) and rotational correlation time and lifetime (Lower) calculated from 356 catalytic turnovers under 2 pN of magnetic pulling force. Two-dimensional joint distribution of anisotropy and lifetime∼ at (A) the rising edge, (B) ON state, and (C) the falling edge. Two-dimensional joint distribution of rotational correlation time and lifetime at (A1) the rising edge, (B1) ON state, and (C1) the falling edge. 86
To acquire deeper insight on the conformational dynamics in the enzymatic reaction, we
characterized the effect of mechanical perturbation on the conformational dynamics by applying
a constant pulling force of ~2pN using magnetic tweezers. Although the applied force is much
weaker than the force needed to break a hydrogen bond (6-9pN), previous reports suggest that
the 1-2pN external pulling force can deform or unfold an enzyme by 30–100% thereby imposing
an significant perturbation on enzymatic reactions.45,46 Figure 3.7 show the 2D joint distribution
of lifetime and anisotropy, as well as lifetime and rotational correlation time at the rising edge,
during on time and at the falling edge from 356 catalytical events, respectively, under a constant
force manipulation. Different from the situation in Figure 3.6 without force manipulation, only
one domain is present in both at the rising edge, during on time and at the falling edge. The
domain existing at the rising edge centers on τf=3.4±0.10 ns, r=0.12±0.06 and τr=2.1 ns and the
same conformation continues to persist until on time because of the similar domain populated
during the on time, which centers on τf=3.5±0.14 ns, r=0.12±0.04 and τr=2.1 ns. However, falling edge consists of only a single domain centering on τf=3.5±0.2 ns, r=0.07±0.04 and τr=1.0
ns, which suggests only one conformational state is dominate during the product release under
the constant force manipulation. Furthermore, the domain populated at the falling edge has lower
anisotropy and shorter rotational correlation time than the domain populated during on time and
at the rising edge, which suggests that the enzyme is partially deformed under force manipulation and loses its native active site conformational dynamics. After being perturbed by a small constant force, the enzyme still retains the ability to explore possible subsets of active site conformation due to the thermal fluctuation. Upon losing the folded/rigid active site structure under force manipulation, the tightly bound state shifted to a loosely bound active site conformation when the product exists the active site. Therefore, we have shown that 87
conformational dynamics of the enzyme due to thermal fluctuation plays a critical role in
regulating the enzymatic reaction and a small constant force (~2 pN) has profound impact on
enzymatic conformational fluctuation.
We have compared the domains populated during each catalytical step in an enzymatic
reaction with and without force manipulation in Table 3.1. Interestingly, under the mechanical
force manipulation, the feature of exhibiting multiple distinct conformational states is lost while
only one conformation is populated at the falling edge. Particularly, anisotropy and rotational
correlation time of the domain populated during product release under the force manipulation is
smaller than that of native enzyme. This signifies that under the external force pulling, active site
is deformed or open-up and the product has larger free space to rotate, giving rise to a lower
anisotropy and rotational correlation time. In contrast, no significant change has been observed at
the rising edge and on time regardless of the force manipulation. It has been known well that the
interaction between the enzyme and transition state is much stronger than the interactions
between the enzyme and substrate or product.82 This is possibly due to that the weak constant
force (~2 pN) we applied in this work is even weaker than a force that needed to break a
hydrogen bond, which is not comparable to the interactions between involved at the falling edge
([E·P]→[E·P]) and during on time ([E·P]) but enough to perturb the conformational dynamics when the product exists the active site ([E·P]→E+P). This is also supported by the high
rotational correlation time and anisotropy at the rising edge and during ON time under force
pulling ( 2.0 ns). Moreover, the conformational dynamics during the product release is intrinsically∼ weak and a large scale of conformation fluctuation is involved. Therefore, under the pulling force, the interaction weakens further, and the loosely bound state becomes the dominant product-releasing state, which is reflected by smaller anisotropy and faster rotational correlation 88
time. We believe that the change of the conformational dynamics would impose a significant
impact on the mechanism of the product releasing. Previous work reported that HRP adopts
multiple intermediate conformational states and enzymatic product releasing shows two
pathways: a solvation-mediated diffusion releasing pathway and a sudden spilling-out releasing
pathway.36 Our results from the conformational dynamics without force manipulation has a great
agreement with the previous report. Moreover, we have also found that under magnetic force
manipulation, the complex nature of the multiple product-releasing pathways disappears, and more simplistic conformations of the active site are populated.
3.3.4 Product Release Mechanism
As shown in Figure 3.8, we have proposed a possible mechanism of the product releasing, showing the possible pathways that may be involved when the product is about to exist the active site of HRP enzyme. The unperturbed HRP adopts multiple conformations to release the product from the active site, which may be achieved by solvation-mediated diffusion pathway through the opening-up active site conformation after exploring tightly bound conformation or by two parallel pathways: solvation-mediated diffusion pathway and spilling out pathways where the product is expelled from a tightly bound active site. Under a constant weak external pulling force, HRP retains its activity but loses the complex conformational dynamics during the product release, showing a simplistic product releasing pathway, solvation-mediated pathway through an opening-up active site conformation.
The fundamental understanding of efficiency of catalysis in enzymology is mostly rooted on the structure of the enzyme and substrate, as well as their structural interaction. In recent years, the advancement of the single-molecule studies provides new opportunity to dynamic 89 study of enzyme fluctuation and people have found that the conformational dynamics of enzyme, even the overall protein matrix, providing the required entropy, enthalpy, and corresponding energy landscape, has a profound impact on an enzyme to ultimately possess the power of enhancing reaction rates. According to the experimental results that multiple conformational states are populated during the step of product release and the crystal structure of the HRP,61 we anticipate that the charge redistribution may trigger dramatic conformational changes in the enzyme active site. 90
Figure 3.8 Schematic representation of the product-releasing pathways. 91
3.4 Conclusions
We have reported a study on the conformational dynamics of HRP using single molecule
multi-parameter photon time-stamping spectroscopy with mechanical force manipulation, a
newly developed single-molecule fluorescence imaging magnetic tweezers approach. HRP-
catalyzed fluorogenic enzymatic reaction serves as a powerful tool to probe the conformational
dynamics of the enzymatic active site. To characterize the details of the conformational
fluctuations, we employed multiparameter correlation analysis: fluorescence lifetime, anisotropy
and rotational correlation time by time-correlated single-photon counting.83-88
Interestingly, the product release dynamics shows the complex conformational behavior with multiple conformational states populated when the Resorufin is released from the active site. Specifically, we have identified that there are two product releasing pathways: the solvation- mediated diffusion releasing pathway and the spilling-out releasing pathway. The solvation- mediated releasing pathway is associated with loosely bound enzyme active-site opening-up conformations which have low anisotropy and small rotational correlation time. While the spilling-out releasing pathway is associated with tightly bound enzyme active-site conformations which have high anisotropy and large rotational correlation time. Under small external constant pN force manipulation, the complex nature of the multiple product-releasing pathways disappears, and the solvation-mediated releasing pathway with the simplistic conformations is dominated. We have experimentally observed the transient conformational states under the condition of enzymatic reaction as well as altered the fluctuation dynamics and product releasing pathways by force manipulation. This finding has significant implications in the fields of enzymology, i.e. unraveling the ultimate relationship between conformational dynamics and enzyme kinetics. 92
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CHAPTER 4. OSCILLATING PICONEWTON FORCE MANIPULATION ON SINGLE-
MOLECULE CONFORMATIONAL AND REACTION DYNAMICS
The intrinsic protein dynamics involved in enzymatic reactions occur in a range from ns
to s and 10-2 Å to Å in time and space, respectively. Oscillation force has been demonstrated in
theoretical studies as a critical role in unraveling the comprehensive enzymatic dynamics and
addressing its regulation on enzyme activity. Utilizing the oscillating magnetic force by our newly developed magnetic tweezers-coupled single-molecule photon-stamping spectroscopy, we experimentally studied a millisecond scale oscillation force manipulation on single Horseradish
Peroxidase (HRP) enzymatic conformational and reaction dynamics. The enzyme activity decreases with the oscillatory frequency of the oscillatory force manipulation. Moreover, the oscillation force shows the capability of manipulating the enzyme active-site conformational state as well as the nascent-formed product’s interaction with the enzymatic active site, which impacts the product release pathways. Specifically, we have identified two product-releasing pathways: the solvation-mediated diffusion-releasing pathway and the spilling-out releasing pathway. We have observed that the spilling-out pathway can be significantly perturbed by oscillatory force manipulation. Our correlated interpretation of enzymatic conformational and reaction dynamics provides a new insight into the comprehensive understanding of the complex conformational dynamics involved in an enzymatic reaction. Technically, we have also demonstrated a novel approach capable of unfolding an enzyme under an enzymatic reaction condition in real-time. Furthermore, the enzyme’s thermal fluctuation is fully maintained by using an oscillatory mechanical weak pN force to manipulate enzyme conformations. The real- time in-situ fluorescence probe at the enzymatic active site reports the active-site conformational dynamics through each enzymatic reaction turnover. 99
4.1 Introduction
4.1.1 Conformational Dynamics in Enzyme Catalysis
As has been illustrated in Chapter 3, enzyme catalyzes biochemical reaction with
enhanced rates by lowering the activation energy through changing reaction pathways and
energy pathways.1-6 The catalytical power of enzyme is largely depends on its ability to lower the
energy barrier of the transition state, activate the active site, orient the substrate into the optimal
geometry and facilitate the product release. These complicated functionalities of the enzymes are
accomplished by conformational dynamics by exploring different conformational states.7-11 A key feature of the enzyme under the physiological condition is the high degree of interconversion between different conformational states, including high-energy and low-energy conformational states.12 Furthermore, not only the low energy conformational states but also the transient high
energy conformational states, even with low population or a short lifetime, may play a crucial
role in controlling the enzyme activity and reaction pathways.13 However, because of the
transient nature of the conformational dynamics, it is hard to capture and directly probe the
comprehensive dynamics, especially these significant high-energy states due to their low
population or short lifetime.14-18
In fact, a lot of efforts have been made by traditional approaches, such as NMR studies,19
molecular dynamics simulations,20,21 and theoretical modeling22-27 to study the associated
structural motions of the conformational dynamics. However, because of the complicated and
heterogeneous feature of the involved enzymatic dynamics and its external surroundings, we still
lack systematic understanding. The development and advancement of force spectroscopy
provides the opportunity to manipulate the conformational dynamics of enzyme and further study 100 the ultimate relationship between the conformational dynamics and enzyme catalysis. Force manipulation can also provide understanding on mechanical force relevant biochemical processes, i.e. how the force stimulus is transformed into a chemical response and what are the interplay between conformational dynamics and the external mechanical force? Particularly, magnetic tweezers have been developed as a powerful technique to impose a perturbation on the enzyme due to its unique advantages, which is described in chapter 1. For example, in previous works, Sheetz group reported that force pulling of single Vinculin protein molecule induced an significant change on protein conformational dynamics, which result of exposing 2 more previously buried vinculin binding sites.28 Yanagida group have reported that the molecular fluctuations are essential to generate force in motor proteins and measured the force.29-33 Gaub group reported a AFM work that release of a stretching force may induce a higher probability of enzymatic activity at a specific time.34 Recently, our group also reported our findings that single
HPPK enzyme under force pulling manipulation shows to have less flexibility when binding to the substrates.35 Later, we have investigated the enzyme activity and the product release mechanism under a small constant force manipulation by single molecule magnetic tweezers.36,37
4.1.2 Oscillating Force Manipulation
The type of force used to manipulate the single molecule is often the single pulling force, which provides scenario under the chemical equilibrium.28,38-42 Interestingly, theoreticians have predicted that an oscillation force may have profound impact on enzyme dynamics.22,24-27 As shown in Equation 4.1, a oscillation force is often designed as follow:
( ) = + ( ) Equation 4.1
𝒙𝒙 𝒕𝒕 𝒙𝒙𝟎𝟎 𝐚𝐚𝐚𝐚𝐚𝐚𝐚𝐚 𝝎𝝎 101
Imposed external oscillation force manipulation on the enzyme has shown the ability to
drive the chemical equilibrium,25 which further developed as a new method to acquire the full
energy profile.26 Compared to a linear force ramp, oscillation force has provided a more
comprehensive data for reconstructing energy profile under same experimental effort. If the
energy barrier between states are small enough (several KBT), it is possible to select a specific
transition states by preselecting the initial values.26
Later, another single enzyme theoretical study has revealed the capability of the
oscillation force to control the enzyme activity by changing the occurrence time of a reaction-
wise favored conformation state in the scheme.22 We anticipate that a more thorough and
comprehensive understanding can be established with the experimental support. However, to the
best of our knowledge, there is no reported experimental studies in such perspective. Here, for
the first time, we experimentally investigated the enzymatic reaction dynamics under an external
periodic picoNewton force manipulation. By changing the frequency of the oscillatory force, we
have demonstrated the capability to drive the enzyme away from the equilibrium and obtain
further understanding of role of the enzyme conformational dynamics during a typical enzymatic
reaction.
As describe in chapter 1 and chapter 3, Horseradish Peroxidase (HRP) catalyzed fluorogenic reaction is used as our model because many aspects of the enzymatic reaction have been well studied in both experimentally and theoretically, which form a solid foundation for our data interpretation.9,43-47 We followed the single enzyme (HRP) molecule behaviors from the
perspective of enzymatic conformational and reactional dynamics under millisecond scale
oscillation force manipulation. By performing multi-parameter analysis (turnover counts, the
waiting time, dissociation constant and product burst counts), we investigated the enzymatic 102
reaction dynamics and found the enzyme become less active with increasing oscillatory
frequency from zero to 50 Hz. More interestingly, under the oscillation force manipulation, we
have seen a profound impact on the enzyme active-site conformational dynamics during the
product release. The weight of a tightly bound product-enzyme conformational state is
significantly perturbed and altered under different oscillatory frequencies of force, which even
changes the product release pathways and ultimately impact the completion of an enzymatic
turnover. Our unique correlation analysis of conformational dynamics and reaction dynamics
provides a comprehensive understanding of the interplay between force and involved complex
enzymatic dynamics.
4.2 Experimental Sections
4.2.1 Sample Preparation
The procedure of sample preparation is similar as described in chapter 1 and chapter 3.35-37
Single HRP molecules were tethered to the cover glass at one end by 3-aminoproplytriethoxy
Silane (TESPA)-Dimethyl Suberimidate·2HCl (DMS) linker and attached to a streptavidin- coated paramagnetic bead (Dynabeads® MyOne™ Streptavidin T1, 1.05 mm diameter,
Invitrogen Company) via a biotin-streptavidin bond at the other end. All the attachment to HRP molecules, either Dimethyl Suberimidate·2HCl (DMS) or Biotin are bound to the amine group of lysine residues in the amino acid sequence. First, the cover glass (Gold seal, 3406) was washed in sulfuric acid dichromate cleaning solution for 5 min to eliminate grease and possible fluorescent spots. The cover glass was next sonicated in distilled water, acetone and methanol in
15 min separately. After washed with distilled water and dried by nitrogen gas, the cover glass was incubated in a DMSO solution containing 10% concentration mixture consisting of TESPA 103
and isobutyltrimethoxy silane with a ratio of 1:10000. The cover glass was then washed with
distilled water and consecutively transferred and incubated for 4 hours in the following systems:
10nM Dimethyl Suberimidate·2HCl (DMS) in PBS buffer solution (pH = 8.0); 1nM HRP in PBS
buffer solution (pH = 7.4); 10nM NHS-PEO12-Biotin in PBS buffer solution (pH = 7.4); 1 ul
magnetic bead in 15 ml of PBS buffer solution (pH = 7.4).
The reaction solution was prepared just prior to the experiment, which contains 200 nM
of Amplex Red and 2 mM of hydrogen peroxide (H2O2). About 200 µl of the reaction solution was filled in a homemade reaction chamber. Sample immobilized cover glass was attached at the bottom of the reaction chamber. To avoid any thermal, vibrational and electrical/optical disturbance on the system, we carefully sealed our reaction chamber with a special black lid on top of the chamber using high vacuum grease. The special black lid is prepared by taping a black foil on a precleaned cover glass.
4.2.2 Data Analysis
We have performed a multi-parameter analysis: turnover counts, the waiting time (∆toff),
dissociation constant (Kd) and product burst counts to study single enzyme catalytical activity
under oscillation force manipulation. Turnover rate is measured by counting turnover event
counts in a 60s single-molecule fluorescence intensity trajectory. As shown in Figure 4.1, On
time (∆ton) represents the duration time of a single enzymatic turnover event and the waiting time
(∆toff) represents the time interval between two consecutive turnover events. The waiting time is
the time needed for a new enzymatic turnover cycle to start. The dissociation constant of the
enzymatic reaction is defined as the waiting time (∆toff) over on time (∆ton). Total product photon 104 bursts are defined as the sum over of emitted photons of all the turnover events within a 60s fluorescence trajectory, as highlighted in green in Figure 4.1D.
Taking the variation of the background (noise signal) into consideration, we set a threshold as two standard deviations larger than the distribution means of photon intensity.
Therefore, we have enough confidence to treat the signal above the threshold as the product photon bursts. To eliminate any possible photobleaching of Resorufin product from the solution, we have analyzed only those turnover events which have a duration beyond 100ms. 105
Figure 4.1 Fluorescence detection of HRP enzyme catalysis. (A) Typical snapshot of an image from total internal reflection microscopy by an electron-multiplying CCD (EMCCD). (B)
Confocal images (20 µm×20 µm) of single HRP enzyme in parallel and perpendicular polarization. (C) A typical example of single-molecule fluorescent trajectories. We set a threshold as two times of standard deviation more than the average value of the intensity trajectory. Bin time is 20 ms. (D) Zoomed in two individual turnover events. On-time and waiting time is defined as indicated. The product photon burst of an individual turnover event is highlighted in green. 106
Figure 4.2 A typical fluorescent trajectory of a single molecule in parallel and perpendicular
polarization, as well as the corresponding anisotropy trajectory. Bin time is 20 ms.
To quantitatively characterize the conformational states populated in each catalytical step
of an enzymatic reaction, we have also probed fluorescence lifetime, anisotropy and rotational
correlation time of the nascent formed fluorescent product, which have been described in detail in chapter 3. As shown in Figure 4.2, we recorded the fluorescence both in parallel and perpendicular polarization with respect to the excitation laser light polarization for anisotropy measurement. The detected photon stamping signal associated with a turnover event is divided into three regions: rising edge, on time and falling edge for detailed analysis. 107
4.3 Results and Discussions
4.3.1 Home-Developed Rotating Magnetic Tweezers
We have developed a generation of rotating magnetic tweezers which can generate oscillation force by incorporating several permanent magnets (n) on a rotating disk. In our experiment, we evenly distributed 8 cylinder-permanent magnets (D4C-N52, 1/4” Dia.× 3/4” thick, K&J Magnetics) on a Disk with a diameter of 5” disk (Figure 4.3), which is controlled by an electric motor. The individual magnets are separated by an angle of 45° on the rotating disk.
D4C-N52 is a strong Neodymium magnet with the grade of N52, which has a surface field of
7299 Gauss. By changing the input of the applied voltage, we can adjust the rotational speed (ω) of the disk, thus ultimately tuning the oscillatory frequency of the external force. A periodic force is obtained as the disk rotating at a certain speed, and various oscillatory frequency can be achieved by controlling the rotation speed. We would like to point out that the frequency of the periodic force is totally controlled by the current and the amplitude of the force is adjusted by displacing the whole magnetic tweezers device. We anticipate that by changing the pattern of current input, more sophisticated periodic force pattern can be achieved, which would expand the application of our home-developed rotating magnetic tweezers. 108
Figure 4.3 Conceptual scheme of our experimental system. A total of 8 permanent magnets (n=8)
are fixed on a homemade disk, which is mounted on a motor. By changing the applied voltage,
we can control the rotation speed of the disk to achieve periodic force manipulation with
different frequencies. Single HRP is immobilized on the glass surface at one end and attached to
a paramagnetic bead at the other end. HRP catalyzed the non-fluorescent substrate Amplex Red to the fluorescent product Resorufin. We detected the fluorescence signal when the product is still confined in the active site by the single-photon time stamping, which perfectly reports the real-time active site configuration. 109
The magnetic tweezers device is placed 4 mm above the sample plane and the whole
instrument is isolated from the internal and external mechanical noise and acoustic noise. Force
is generated upon paramagnetic bead by the external magnetic field gradient. In the presence of
external magnetic field, the paramagnetic bead is magnetized and acts as a tiny magnet and
moves towards the higher magnetic flux. A periodic force is obtained as the disk rotating in a
certain speed, and various oscillatory frequency can be achieved by controlling the rotation
speed. We measured the magnetic field by placing the probe of gauss meter at the sample plane
when rotating the disk (Figure 4.4). Hence, we calculated the oscillation force in Figure 4.4 based on the magnetic field strength curve, as discussed in our earlier work. 37
Figure 4.4 Magnetic force calibration curve for the cylinder-permanent magnet (D4C-N52) used
in the magnetic tweezers. 110
To further obtain a quantitative characterization of our magnetic tweezers, we have computationally simulated the magnetic field using Finite Element Magnetic Method
(FEMM).48-50 Figure 4.5A illustrates the magnetic field exerted by our magnetic tweezers. Since the magnets fixed on the disk are identical, the magnetic field exhibits by individual magnet is the same. When the disk is rotating at a certain frequency, a periodic external magnetic force is applied on the paramagnetic bead. To determine the oscillatory frequency, we have drawn 8 tiny holes at the corresponding position of the magnets on the disk. A laser source has been aligned to the hole and detected by a photodiode then the frequency was calibrated by the Oscilloscope
(Tektronix TDS 2001c). After determining the frequency at different voltage, we further compare the force momentum, the force effective time and ∆F/∆t at different frequency. Figure
4.5C shows the calculated force momentum by integrating the force within 20 ms, which is equivalence to the binning time used in our data analysis. The force momentum differs with the oscillatory frequency, which implies that the force manipulation instantaneously applied on the sample is quite different. We further compare the effective force duration time and ∆F/∆t in
Figure 4.5D, the oscillation force with low frequency has long duration time and force decrease with time slowly while the oscillation force with high frequency has short duration time and force decrease with time quickly. Oscillation force with low frequency can be understood as a mix of a constant force manipulation and zero force manipulation. However, Oscillatory force with high frequency allows a short pulse force on the single enzyme periodically, which may drive the enzyme away from the equilibrium and further facilitate full exploration of conformation states. In our experiments, we manipulated the single HRP molecule with the oscillatory forces with relatively low @15 Hz and relatively high frequency @40 Hz. 111
Figure 4.5 Conceptual representation of our developed Magnetic Tweezers system and its
characterization using FEMM simulation. (A) A total number of 8 permanent magnets are evenly
fixed on a disk, driven by a motor. The disk is rotated with a certain frequency ω, controlled by the applied voltage. (B) Schematic of the magnetic field by FEMM simulation; (C) Enzyme momentum at different frequencies; (D) Force effective time and force gradient when the disk rotated at different frequencies; (E) Force at various frequency 5 Hz, 10Hz, 15 Hz, 20Hz, 40 Hz and 100Hz. Oscillatory force @15Hz and 40 Hz were imposed on single HRP molecule for further studies on enzyme activity and conformational dynamics. 112
4.3.2 Single Enzyme Activity Analysis
Enzyme’s catalytical activity is also governed by the dynamic interconversion between conformational states. Fluorescence from the fluorogenic product in the active site of the protein is used as a key parameter to probe the enzyme behavior in a fluorogenic enzymatic reaction.
Single HRP enzyme is identified and located through the stochastic on-off burst of the fluorescence signals in TIRF mode. In the confocal mode, we detected the fluorescence of
Resorufin only when it is still confined in the active site of HRP enzyme molecule during an enzymatic reaction turnover. Typically, the fluorescent Resorufin molecule diffuses away from the tightly focused detection volume quickly in milliseconds after its being released from the active site of HRP.51 Real-time fluorescence detection right in the active site serves as a perfect in situ probes to report the enzyme active site conformation during the enzymatic reaction. 113
Figure 4.6 Enzyme activity analysis based on turnover rate mean waiting time, product burst photon counts and dissociation constant. Data collected from the same single HRP enzyme under the real-time pN oscillatory force manipulations. Around 50 trajectories, including ~1300 turnover events, are analyzed. (A) Turnover event counts of a 60 seconds fluorescent intensity trajectory as a function of the oscillation frequency. Note, the case of 0 Hz refers to no magnetic force applied, which shows the native HRP behavior. (B) Correlation plots between the mean waiting time and turnover event counts. (C) Correlation plots between product burst photon counts and turnover event counts. (D) Correlation plots between dissociation constant and turnover event counts. 114
Figure 4.7 Distribution of product burst counts of HRP under no force manipulation. (A),
oscillation force manipulation @15 Hz (B) and oscillation force manipulation @40 Hz (C).
Distribution of dissociation constants of HRP under no force manipulation (A1), oscillation force manipulation @15 Hz (B1) and oscillation force manipulation @40 Hz (C1). Data collected from
the same single HRP enzymes. Around 50 trajectories, including ~1300 turnover events, are analyzed. 115
Figure 4.6 shows the turnover event counts when single HRP enzyme is manipulated at
different oscillation force frequencies. Here frequency at 0 Hz represents the case of no magnetic
force applied. Compared to the case of no magnetic force manipulation (0 Hz, native enzyme
behavior), the turnover rate of single enzyme exhibits a descending trend with increasing
oscillation frequency, suggesting that a negative impact is induced on the enzyme activity by the
oscillation force manipulation and the activity diminished with higher oscillation frequency. To
further acquire a comprehensive understanding on the enzymatic activity, we carry out the multi-
parameter analysis under three conditions (0Hz, 15Hz, and 40 Hz): correlation between turnover
event counts vs mean waiting time, vs product burst photon counts and vs dissociation constant
(Figure 4.6). Both waiting time (∆toff) and dissociation constant (Kd) increases with a higher
oscillation frequency, consistent with the decreased product burst photon counts, which implies that enzymatic activity decreases with higher oscillation frequency. Furthermore, we build the histograms of product photon bursts and dissociation constant under oscillatory frequency of 0
Hz, 15 Hz, and 40 Hz (Figure 4.7). Distribution of product photon bursts (*104) centers at
0.27±0.06 under no force manipulation (Figure 4.7A), while a smaller value at 0.24±0.05
observed under relative low oscillation frequency @15 Hz (Figure 4.7B) and an even smaller
value 0.16±0.05 observed under high oscillation frequency @40 Hz (Figure 4.7C). The result of
product photon bursts analysis indicates product photon bursts decreased with higher oscillation
frequency manipulation, which also suggests that enzyme activity decreased. Distribution of
dissociation constant centers at 5.0±2.9 under no force manipulation (Figure 4.7A1), while a
larger value at 7.5±2.04 observed under low oscillation frequency @15 Hz (Figure 4.7B1) and an
even larger value at 11±3.36 observed under high oscillation frequency @40 Hz (Figure 4.7C1).
Consistently, the results of the increased dissociation constant with the higher oscillatory 116 frequency induce a less active enzyme, which reaches the same conclusion from the distribution of turnover event counts and waiting time (Figure 4.8). 117
Figure 4.8 Distribution of turnover event counts of HRP from 60s fluorescence trajectory under no force manipulation. (A), oscillation force manipulation @15 Hz (B) and oscillation force manipulation @40 Hz (C). Distribution of mean waiting time of HRP under no force manipulation (A1), oscillation force manipulation @15 Hz (B1) and oscillation force manipulation @40 Hz (C1). 118
To evidence that the enzymatic activity change is due to the magnetic force, we have
carried out a control experiment on the no paramagnetic bead tethered single HRP molecule,
which shows no mechanical oscillatory frequency dependent (Figure 4.9). Overall, by
performing multi-parameter analysis, we investigated the enzymatic reaction dynamics and
found the enzyme activity decreased with increasing oscillatory frequency. The control
experiment is performed under exact same experimental conditions except that the immobilized
HRP molecule is not attached to the paramagnetic bead. Therefore, no magnetic force is applied to the enzyme molecules when the magnetic tweezers device is rotating at different frequencies.
We have seen no enzyme activity change when rotating the disk at moderate @15 Hz and high frequency @40 Hz so that we can ruled out any other factors (thermal, vibrational, electrical…) from the rotating disk that could possibly induce the enzymatic activity change. Overall, by performing multi-parameter analysis, we investigated the enzymatic reaction dynamics and found the enzyme activity decreased with increasing oscillatory frequency. 119
Figure 4.9 Distribution of turnover event counts of the no paramagnetic bead tethered HRP
(control experiment) from 60s fluorescence trajectory under 0Hz, @15 Hz and @40 Hz.
4.3.3 Enzymatic Conformational Dynamics in Product Release
To explore the impact of external oscillatory force at different frequency on the
enzymatic reaction, we probed the fluorescence of nascent fluorogenic product when confined in
the active site by single-molecule photon stamping detection,37,44,52-54 capable of resolve
fluorescence lifetime, anisotropy, and hence the fluorescent product rotational time and
dynamics at the enzymatic reaction active site and beyond,37,44 under the conditions of with and
without the oscillatory force manipulation by using our home-developed magnetic tweezers. By
the single-molecule photon stamping technique, both the arrival time (t) and delay time (∆t) are recorded for each detected photon. Thus, each photon has two characteristics, among which the 120 arrival time (t) is the real chronic time when photon is detected and the delay time (∆t) is the time interval between the pulse excitation and the photon emission. Moreover, histogram of delay time provides the fluorescence decay of a turnover event, which yields the fluorescence lifetime. The arrival time provides the information about the photon flux, from which fluorescence intensity trajectory is built.
Figure 4.10 2D joint distribution of anisotropy and lifetime at the rising edge under different circumstances. Periodical force manipulation at a frequency of 0Hz (A), 10Hz (B), 15Hz (C),
25Hz (D), 40Hz (E) and 50Hz (F). Note 0Hz represents the native unperturbed enzyme behavior.
To unravel the imposed oscillation force on the enzyme conformational dynamics in each step of the enzymatic reaction, correlation analysis of fluorescence lifetime and anisotropy is conducted to characterize the conformational dynamics for each step during an enzymatic turnover event. We note that fluorescence lifetime and anisotropy might vary to some content as 121 the measurements are performed on many single HRP molecules. The focus of this work is mainly unraveling the complex feature of the enzymatic dynamics during each step of the enzymatic turnovers rather than measuring the specific lifetime and anisotropy of the confirmation states. In both cases of the rising edge (Figure 4.10) and on time (Figure. 4.11), only one main domain populated and no significant change is observed when changing the frequency of oscillation force from 0 Hz to 50 Hz. However, a substantial impact on the conformational dynamics in the product release process is evident.
Figure 4.11 2D joint distribution of anisotropy and lifetime during on-time period under periodical force manipulation at a frequency of 0Hz (A), 10Hz (B), 15Hz (C), 25Hz (D), 40Hz
(E) and 50Hz (F). Note 0Hz represents the native unperturbed enzyme behavior.
We plotted the 2D joint distribution of lifetime and anisotropy obtained from the product releasing step in Figure 4.12. Without the force manipulation (Figure 4.12A), the native unperturbed enzyme exhibits three domains, indicating that multiple conformations of the 122
enzyme active site are populated in the product release process, which has a great agreement
with our previous work.37 Interestingly, we have seen a significant change in the conformational
dynamics under oscillation force manipulation with different frequencies. When manipulating
the single HRP enzyme at a relatively low frequency @10 Hz (Figure 4.12B) and 15 Hz (Figure
4.12C), the multiple complex conformational dynamics of product release converged to form a
simple conformational state. As discussed in earlier, the oscillation force with low frequency has
a strong perturbation (relative long force duration time and small average force (∆F/∆t)), it can be treated with a combination of small constant force manipulation and no force manipulation.
Hence, the conformational dynamics during the product releasing step has the overall feature of native enzyme behavior of multiple conformational states and simple conformational dynamics due to the small constant force pulling manipulation.37 We manipulated the single HRP molecule
with a moderate oscillatory force @25 Hz, the complex conformational dynamics start to show
up again (Figure 4.12C). However, under a higher oscillation frequency manipulation @40 Hz
and 50 Hz (Figure 4.12E-F), three distinct domains are present again in the product releasing
step, which implies that high enough oscillatory frequency brings back the complex multiple conformational dynamics. We only observe the conformational changes in the product release process while no difference on the rising edge and the on time. This is primarily due to that the interaction between the enzyme and fluorescent product in the form of [E·P] is strong at the rising edge ([E·S] → [E·P]) and on time ([E·P]) and a weak oscillation pN force is not strong enough to make any difference on the conformational dynamics. During the product release process ([E·P] → E+P), the interaction between the enzyme and product is intrinsically weak and a large scale of conformational dynamics is involved so that an oscillation pN force has the capability to impose a considerable perturbation on the enzymatic reaction. 123
Figure 4.12 2D joint distribution of anisotropy and lifetime at the falling edge under the
periodical force manipulation at a frequency of 0Hz (A), 10Hz (B), 15Hz (C), 25Hz (D), 40Hz
(E) and 50Hz (F). Note 0Hz represents the unperturbed enzyme behavior. Data collected from
the same single HRP enzymes. Around 500 turnover events, including more than 2000 data
points are analyzed.
To obtain a quantitative characterization of the enzyme conformational dynamics during enzymatic reaction, we further calculated the corresponding lifetime, anisotropy and rotational
correlation time,37,44,52 for the multiple domains populated in product release step in Figure 7A,
7E, and 7F (Table S1). Three distinct domains are populated evenly under no force manipulation
(Figure 7A). Domain 1 centers on τf=3.84±0.03 ns, r=0.099±0.007 and τr= 1.73±0.21 ns, domain
2 centers on τf=3.93±0.03ns, r=0.122±0.007 and τr =2.45±0.28 ns and domain 3 centers on τf
=3.84±0.03ns, r=0.146±0.008 and τr = 3.26±0.40 ns. Lower anisotropy signifies the active site to be the loose bound conformational states while higher anisotropy signifies the active site to be 124
confined environment or tightly bound conformational states. Under high oscillatory frequencies
@ 40 Hz and @50 Hz, the multiple conformational dynamics populated again. In details, in case
of the manipulation @40 Hz, domain 1' centers on τf =3.87±0.02 ns, r=0.088±0.007 and τr =
1.47±0.16 ns, domain 2’ centers on τf =3.71±0.02 ns, r=0.112±0.007 and τr = 2.03±0.19 ns and
domain 3’ centers on τf =3.84±0.05ns, r=0.138±0.018 and τr = 2.92±0.69 ns. In case of the
manipulation @50 Hz, domain 1” centers on τf =3.75±0.03 ns, r=0.04±0.009 and τr = 0.54±0.16
ns, domain 2” centers on τf =3.64±0.06 ns, r=0.092±0.021 and τr = 1.48±0.52 ns and domain 3”
centers on τf =3.75±0.06ns, r=0.144±0.021 and τr = 3.10±1.10 ns. We calculated the contribution percentage of conformations populated in case of 0 Hz, 40 Hz, and 50Hz (Table 4.1). The three domains populated in the native enzyme (0Hz) share the same contribution. However, the contribution of the three domains populated under 40 Hz and 50Hz oscillation force manipulation are both distinct with the unperturbed enzyme, with a preferential conformational state (domain 3' (67%), domain 3" (57%)) dominated. The enzymatic dynamics in high oscillation frequency manipulation can be explained in the following two situations: the three main domains of the native enzyme active site in Figure 7A is the same as with those under manipulation in Figure 7E and 7F. The contribution of the domain 3 increases because the high frequency of oscillation force manipulation matches the thermal fluctuation of the domain. The thermal fluctuation of the protein domains and residues depends on their mass, matrix, and dimensions, have typically broad time scale from µs to s. The millisecond scale protein domain
fluctuation reported in this work only represents part of the complex protein fluctuation
dynamics; The other explanation is that the three main conformational states of the native
enzyme active sites are slightly different with those under manipulation @40 Hz and 50Hz,
which might be higher energy conformational states and less dominated in native enzyme. As 125
predicted in theoretical works, high-frequency oscillation force manipulation has the capability
of exploring new conformational states, which could be those short-lived, high energy
conformational states. Nevertheless, we consider that the preferentially dominated
conformational state with the largest anisotropy signifies a tightly bound enzyme active-site
conformational state in product release. The complex enzyme active-site conformational dynamics change due to the real-time oscillatory force manipulation can even significantly change the product releasing pathways, which ultimately impact the completion of an enzymatic turnover. 126
Table 4.1 Rotational Correlation time of the three domains present at the falling Edge under
force oscillation frequency of 0Hz (Figure. 4.12A), 40Hz (Figure. 4.12E) and 50Hz (Figure.
4.12F). (τf: Fluorescence Lifetime; r: Anisotropy; τr: Correlation Rotational time.)
Oscillation frequency Falling Edge ω Domain 1 Domain 2 Domain 3
No Force τf=3.84±0.03 ns τf=3.93±0.03 ns τf=3.84±0.03 ns ω (0 Hz) r=0.099±0.007 r=0.122±0.007 r=0.146±0.008
τr=1.73±0.21 ns τr=2.45±0.28 ns τr=3.26±0.40 ns (34%) (35%) (31%)
ω (40 Hz) τf=3.87±0.02 ns τf=3.71±0.02 ns τf=3.84±0.05 ns r=0.088±0.007 r=0.112±0.007 r=0.138±0.018
τr=1.47±0.16 ns τr=2.03±0.19 ns τr=2.92±0.69 ns (16%) (17%) (67%)
ω (50 Hz) τf=3.75±0.03 ns τf=3.64±0.03 ns τf=3.75±0.06 ns r=0.040±0.009 r=0.092± 0.021 r=0.144±0.021
τr=0.54±0.16 ns τr=1.48±0.52 ns τr=3.10±1.10 ns (15%) (28%) (57%) 127
4.4 Conclusions
We developed a new generation of magnetic tweezers, which can impose the millisecond scale oscillation force with different frequencies simply by controlling the electric input of the applied voltage. Utilizing the feature of oscillation force generation of our home-developed magnetic tweezers, we experimentally unraveled the impact of the external oscillation force on a single enzyme molecule. Our novel force-manipulating photon stamping single-molecule microscopy provides a unique approach of studying the enzyme activity and conformational changes under the enzymatic reaction condition, which enables us to investigate the enzymatic turnover activity and pathways for the deformed, partially unfolded and perturbed enzymes. Our real-time manipulating the enzyme conformations under the enzymatic reaction condition is beyond the conventional methods of unfolding the enzymes by denaturing chemical agents, temperature, or pH conditions other than the typical enzymatic reaction conditions. By time- resolved multi-parameter analysis, we carefully addressed the enzyme activity change due to oscillation force manipulation. The enzyme activity decreases with increasing oscillatory frequency. Furthermore, we followed the enzymatic conformational dynamics during the enzymatic reaction as the nascent formed product Resorufin in the enzyme active site serves as an in-situ probe to perfectly report the real-time enzyme active site conformation. Particularly, during the product releasing, the multiple conformational states disappear and a single conformation with broad distribution populated under the low oscillatory frequency @ 10 Hz and
15 Hz. However, under the high oscillatory frequency @ 40 Hz and 50 Hz, the multiple conformational states feature in the step of product release resumes, but the contribution of the multiple conformations is largely altered, which can significantly change the product release pathways. Enzymatic product releasing, involving loose-bound open up active site conformations 128
and tightly bound active site conformations, showing two pathways: a product solvation-
mediated diffusion releasing pathway and a spilling-out releasing pathway. The spilling-out
pathway, which is associated with tightly bound enzyme active-site conformation (high
anisotropy and large rotational correlation time conformational state), is significantly perturbed
under the oscillatory force manipulations. High oscillatory frequency shows the capability of
increasing the weight of a certain conformational state or exploring new conformational states,
which evidences the results from the previous theoretical studies. Not only do our experimental
studies support the previous theoretical results, but they can also provide new insights on the
understanding of enzymatic dynamics, i.e. exploring more conformational states and offer a
better understanding of the interplay between force and complex enzymatic dynamics.
Oscillation force could also be generated in vivo by some motor proteins,30 which are direct to
the biological functions.
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CHAPTER 5. MANIPULATING MOTIONS OF TARGETED SINGLE CELLS IN SOLUTION
BY AN INTEGRATED DOUBLE-RING MAGNETIC TWEEZERS IMAGING
MICROSCOPY
Controlling and manipulating living cell motions in solution holds a high promise in developing new biotechnology and biological science. Here, we developed our 3rd generation of magnetic tweezers, which employs a combination of two permanent ring magnets, allowing a picoNewton (pN) bidirectional force and motion control in the up-down z-coordinate beyond a conventional single upward pulling. Double-ring magnets serve a perfect fit for the microscopic imaging system, allowing the lower magnet to fit with the inverted microscopy and upper magnet to facilitate a light illumination. The experimental force calibration and magnetic field simulation using Finite Element Method Magnetics (FEMM) both indicate that double-ring magnetic tweezers cover a linear-combined pN force with positive-negative polarization changes in a tenability of sub-pN scale, which can be utilized to further achieve upward and downward motion manipulation by shifting the force balance. We have also validated the feasibility of our up-down configured double-ring magnetic tweezers for bidirectional motion manipulation on an aqueous solution of paramagnetic beads. The up-down configured magnetic tweezers setup is applicable for single cell manipulation, showing that the cells with internalized paramagnetic beads can be selectively picked up and guided in a controlled fine motion. Our approach can significantly expand the application of magnetic tweezers and provide a path forward on more sophisticated designs of the magnetic tweezers capable of more complex spatial and temporal field distributions and manipulations. 133
5.1 Introduction
Motion manipulation of living cells and other biological entities has a significant implication in the fields of biological, medical research and tissue engineering. Particularly, quantitative studies of the living cell manipulation are necessary for the development and maintenance of organisms as well as in the advancement for various types of diseases such as cancer metastasis.1 Mechanical force has been known as an external factors acted on the cell for controlling the cell motility.2-4 Several methods have been developed for motion manipulation of small biological entities and single cell, such as optical and magnetic tweezers,5-7 acoustic waves,8,9 electrophoresis10,11 based approaches. Nevertheless, each method exhibits distinct advantages and features. For instance, Jager and coworkers have developed microrobots based on poly-pyrrole gold micro-actuators, can pick up, lift, move, and place micrometer-size objects within an area of about 250 micrometers, which makes microrobot an excellent potential tool for single-cell manipulation.12 Recently, Huang and coworkers have developed a method based on acoustic waves for the manipulation of single cell, as well as single microparticles and organisms.8 In comparison to the other approaches for manipulation of the biological entities, such as optical tweezers, the magnetic tweezers feature a number of unique advantages: 1) force is applied in noncontact mode, meaning no physical or chemical contact required; 2) no photon damage, thermal damage and optical background is induced; 3) force is adjustable in magnitude and direction, static or oscillatory; 4) a large amount of targets can be manipulated under the same magnetic field simultaneously.13-15
Over the years, single-molecule magnetic tweezers have been developed and demonstrated as a capable approach to manipulate biological systems using micron sized paramagnetic beads.16-27 Besides those unique advantages, it is easy to handle, low cost and 134
allows a direct investigation of the dynamics and mechanical properties of important
macromolecules.28,29 Direct investigation of single molecules is useful to address many
fundamental questions, such as the relationship between the structure of molecules and
functions.30-35 A range of applications have been demonstrated in investigating the elastic properties of DNA,36-41 proteins conformational dynamics,20,42-45 molecular interaction force
analysis and mechanical properties of a living cell.46-48 In a typical experiment by magnetic
tweezers, a stretching force is applied to deform a biomolecule by magnetic field gradient from a
permanent or electromagnet in a particular direction (preferably upward), whereas the downward
motion is driven only by the gravity. However, employing a single pulling force limits the
manipulation of the particle in multiple directions in a controlled approach. Additionally, using a
single pulling force, the stretch in a bimolecular segment is essentially not actively controllable.
This is because once a paramagnetic bead enters a magnetic field gradient region, the bead will
undergo maximum possible displacement depending on the strength of the magnetic field.
Although, over the times, variety of magnetic tweezers have been developed, including
permanent49,50 and electro-magnet based apparatus25,51,52 to achieve sophisticated control of
paramagnetic beads in the focal plane or single upward pulling and stretching control, the studies
involve a downward magnetic force are barely reported.53 This definitely limits the advanced
application of magnetic tweezers.54
In this report we demonstrate an advanced method of up-down configured magnetic
tweezers, which is capable for the targeted single cell manipulation. The advancement of our
magnetic tweezers relies on the combination of two permanent magnets, which allow a tunable
bidirectional force and motion control on the paramagnetic beads in the z-direction, i.e.,
controlling a paramagnetic bead in any horizontal plane of interest where a polarized linear sum- 135 over magnetic force is practically achieved.55,56 This approach will give us the edge of pausing the deformation of a biomolecule at any point without adding any chemical denaturant as we have demonstrated in other study.44 Bidirectional force and motion manipulation is also supported by the force calibration and magnetic field distribution simulation analysis using Finite
Element Method Magnetics (FEMM).57,58 In addition, the magnetic force is calibrated with distance in the z-direction by analyzing the diffraction pattern of the paramagnetic beads under white light illumination.18,54 Further in this report, we have demonstrated that the bidirectional manipulation can be applicable to study living cell adhesion, motion, interaction, and other force- related functions. Besides selectively calling out the bead-targeting cells through the paramagnetic beads inside the cells, the cells motion can also be regulated, controlled, and monitored by a piconewton mechanical force through the force manipulation of our magnetic tweezers. From that perspective, our work is unique and creates an important stage in the application of magnetic tweezers.
5.2 Experimental Sections
5.2.1 Sample Preparation
We have conducted living cell motion manipulation experiments on HEK-293 and HT22
Cells: applying the pN mechanical force manipulation to control/manipulate the single cell motions and to selectively pick up a targeted living cell.
Both HEK-293 and HT22 cell samples are prepared in the same procedure. Cell culture was performed in an incubator with an atmosphere of 5% (v/v) enriched CO2 air 37 °C. Firstly, Cells were cultured in a T-75 flask in DMEM (Dulbecco's Modified Eagle Medium, 11995, Gibco/Life
Technologies) supplemented with 10% fetal bovine serum (Sigma-Aldrich) and 1% penicillin- 136 streptomycin (Gibco/Life Technologies). The cell population reached ~75% coverage on the surface of T-75 flask in 2-3 days, then the cells were sub-cultured in a new T-75 flask for future use and in 35 mm petri-dish containing a 25 mm roundish glass slide, which is further used for our experiment. Meanwhile, the paramagnetic beads (Dynabeads® M-280 Streptavidin) were dispersed in the same culture medium before use. When the cell population in petri-dish reached
~50%, the previous prepared paramagnetic beads suspension was added into the cell culture 2 days prior to the experiment to allow for phagocytosis. Finally, the adherent cell sample was trypsinized by Trypsin EDTA and then transferred to the cell chamber for live cell imaging and further measurement.
5.2.2 Integrated Double Ring Magnetic Tweezers
Figure 5.1 shows the conceptual scheme of the designed up-down configured double- ring magnetic tweezers. Our magnetic tweezers are based on the concept of addition of two vectors. As shown in Figure 5.1, ring magnet α is placed at a fixed position underneath the sample chamber whereas magnet β placed right on top of the sample. Magnet β is placed on a translational stage coupled with a 2D rotational stage. The paramagnetic beads are typical polystyrene beads loaded with ferrite content. In the presence of external magnetic field, the paramagnetic beads are magnetized and move towards the higher magnetic flux. Thus, a constant downward pulling magnetic field is generated by the magnet α while a tunable upward magnetic field is generated by the magnet β as we translate it vertically. By tuning the upward magnetic field, the resultant force vector can be achieved either upward or downward. Moreover, a zero- force state can be achieved when the upward force is equal and opposite to the downward force.
Consequently, we can essentially control the paramagnetic beads in a particular plane within experimental time scale as a result of a zero force practically achieved.55,56 137
Figure 5.1 Conceptual scheme of the experimental set-up. Magnetic tweezers are made up of a combination of two permanent magnets. Magnet α (R1106, Super Magnet Man) is placed underneath the sample, which generates a constant downward force. However, Magnet β (R828,
K&J Magnetics) is held above the sample by a home-built stage which can generate a tunable upward force in the vertical direction and translational movement. 138
In our experimental setup, magnet α is placed 2 mm underneath the sample stage and magnet β is mounted on the positioning stage. The experimental set up is based on an inverted confocal microscope (Axiovert 200M, Carl Zeiss) equipped with a 63×oil-immersion objective
(NA 1.4, plan-APOCHROMAT, Zeiss). The sample is illuminated by a white light from the top and the images are collected by an Electron Multiplying Charged Coupled Device (EMCCD)
(Photomax512B, Princeton Instruments) with exposure time 50ms.
5.2.3 Force Calibration
As described in Chapter 2, a number of experimental methods have been reported for force calibration of magnetic tweezers.43,59 In our experiment, we estimated the magnetic force experienced by the paramagnetic beads by measuring its magnetization and the magnetic field at the sample plane.43,44,59 Typically, a magnetic field as a function of the distance is plotted, from which the magnetic field gradients at different positions are determined. Finally, for a given magnetic bead, magnetic force can be calculated by the following equation:
= × = × = Equation 5.1 𝝏𝝏𝑩𝑩��⃑ �𝑭𝑭�⃑ 𝜵𝜵�𝒎𝒎���⃑ �𝑩𝑩�⃑� �𝑴𝑴��⃑𝑽𝑽 𝜵𝜵𝑩𝑩��⃑ �𝑴𝑴��⃑𝑽𝑽 𝝏𝝏𝝏𝝏 where, m is the induced magnetic moment of the magnetic beads by the external magnetic field and B is the applied magnetic field. The magnetization (M) of the paramagnetic beads is almost saturated when the applied magnetic field (B) reaches ~2000G. Therefore, the saturation magnetization (M) is used for force calculation, which may cause a small error. For the paramagnetic beads used in our experiment (Dynabeads® MyOne™ Streptavidin T1 and
Dynabeads® M-280 Streptavidin, Invitrogen), the volume saturation magnetization (M) and the volume (V) are known values according to the product specification from Invitrogen. We 139 acquired the overall magnetic field gradients (dB/dz) (Figure 5.2), from which the magnetic forces are calculated using Equation 5.1. According to the force calibration, we can apply a resultant force ranges from ~16pN to -2pN on Dynabeads® M-280 Streptavidin, which can be utilized to manipulate the mammalian cell with few ng. 140
Figure 5.2 The experimental measured magnetic field gradients (dB/dz) when the upper magnet at different distances away from the sample plane. 141
5.3 Results and Discussions
5.3.1 Finite Element Magnetics Method Simulation
To characterize the magnetic field of the up-down configured magnetic tweezers, we have used Finite Element Method Magnetics (FEMM) to simulate the magnetic field distribution. FEMM is a widely available software package used for 3D axisymmetric magnetic field simulation in low frequency. We simulated the magnetic field of our magnetic tweezers as
3D axisymmetric problem using FEMM software.57 The materials of the two magnets are
specified accordingly, while the surrounding is specified as air. Then, mesh is generated for each
configuration and analyzed using Newton AC solver with 10-8 precision. In details, bottom cross
section represents fixed magnet α with inner radius R1 = 12.5mm, outer radius R2 = 17.5mm, and height l = 5mm. Upper cross section represents upper magnet β with inner radius R1 = 1.6mm,
outer radius R2 = 6.35mm, and height l = 12.7mm. The dimensions of the simulated magnets are
consistent with our experimental set up. 142
Figure 5.3 (A) Model construction and 3D finite mesh generation of the up-down configured magnetic tweezers using FEMM. Cross-section denotes the magnet and the open square denotes the sample plane. FEMM simulation results when upper magnet β is (B) 2mm, (C) 12mm, and
(D) 30mm away from the sample plane. The two magnets are placed in attraction configuration.
Sample plane is at ~2mm from upper surface of the magnet α indicated as open square in
Figure 5.3A. We simulated the magnetic field distributions for this system by keeping the lower magnet α at fixed position while moving the upper magnet β at different distance away from the sample plane. Specifically, when upper magnet β is close to sample plane, for instance, at 2mm
(Figure 5.3B), the resultant magnetic field is directed upward, dominated by the stronger upper magnet β. While moving the upper magnet β away to a distance ~12mm (Figure 5.3C), the upward magnetic field equals to the downward magnetic field and the resultant magnetic field at the sample plane becomes zero, which corresponds to a zero-force state. When upper magnet β is moved further away from the sample plane, e.g., at 30mm (Figure 5.3D), the downward 143 magnetic field is dominant at the sample plane due to the lower magnet. Moreover, Figure 5.4A shows the simulated vector representation of magnetic field B experienced by the paramagnetic beads. When magnet β is 12mm away from the sample plane, the addition of upward and downward magnetic field with the same magnitude gives rise to a zero magnetic field and force.
We have further acquired the simulated magnetic field that exerted on the sample plane when moving the upper magnet β up and down (Figure 5.4B), which matches well with the experimental measured values.
Figure 5.4 (A) FEMM simulated vector representation of magnetic flux density B when upper magnet β is 12mm away from the sample plane where a zero magnetic field/force is achieved.
(B) FEMM simulated and experimental measured magnetic field at the sample plane when moving the upper magnet β up and down. 144
5.3.2 Bidirectional Manipulation on Single Paramagnetic Beads
Figure 5.5 Representative bright field images of paramagnetic beads in solution under force manipulation (A1) and its correlated calibration images (A2). Intensity profiles of the images are plotted in (B). Displacements of paramagnetic beads in z-direction under force manipulation recorded from ~2000 frames (50ms exposure time) (C).
To validate the feasibility of our up-down configured magnetic tweezers for bidirectional motion manipulation, we have carried the experiment on an aqueous solution of paramagnetic beads (Dynabeads® MyOne™ Streptavidin T1). Gravitational force on a single paramagnetic bead is calculated as ~0.01pN, which is not comparable to the mechanical force applied in the experiment. The z displacement is determined by analyzing the diffraction pattern of the paramagnetic beads.54 The micron sized paramagnetic beads exhibit diffraction pattern of images under the white light illumination. In a different scenario, the diffraction patterns of the micron 145
sized beads can also be altered as we change the distance between the beads and the objective
focus plane.54 Thus, by comparing these two types of diffraction patterns, we can quantitatively
determine the z-displacement of the paramagnetic beads under magnetic force manipulation.
Therefore, we acquired a series of calibration images of the paramagnetic beads by precisely
moving the objective focal plane. By comparing the diffraction pattern of images (Figure 5.5A1)
with the corresponding calibration images (Figure 5.5A2) and further comparing the intensity
profiles of two types of images (Figure 5.5B), we quantified the displacement of the
paramagnetic beads in the z-direction. We recorded the bidirectional motion of the paramagnetic
beads in solution and quantified the z displacement during the manipulation in Figure 5.5C.
When the upward pulling force gradually increases from -0.2pN to ~1pN, the beads move
upward gradually, while the beads move downward to its original position when the upward
pulling force gradually decreases and eventually the force balance shifts to the negative (from
~1pN to ~0.2pN).
5.3.3 Targeted Single Cell Motion Manipulation in Solution
We further applied the pN mechanical force manipulation on (1) controlling and
manipulating the single cell motions and (2) selectively picking up a targeted living cell. We
have conducted experiments on HEK-293 and HT22 Cells.
The microscale paramagnetic beads are uptake/internalized by the cells through a typical
process of phagocytosis.60 Being wrapped by the plasma membrane, the particle behaves like a phagosome but still keeps the magnetic properties of the paramagnetic bead. In a phagocytosis process, there are typically 10% cells were found to have internalized paramagnetic beads.
Facilitated by the constant downward pulling force due to the bottom magnet α, the cells that 146 contain beads were settled down selectively on the surface of coverslip. Tuning upward pulling force Fβ from 0-16pN (according to the force calibration for Dynabeads® M-280 Streptavidin paramagnetic beads) to shift the force balance by utilizing the bidirectional manipulation of our magnetic tweezers, we can achieve full control of cell motion in the z direction, lifting it up or putting it back to its original position. 147
Figure 5.6 Representative bidirectional manipulated DIC images of HEK-293 with internalized paramagnetic beads (2.8 µm diameter) by tuning the upward pulling force. Images are recorded by an EMCCD camera. 148
When gradually increasing the upward force to a certain amount (>2pN) to against the constant downward force (~2pN) exerted on the paramagnetic bead, the paramagnetic bead starts to move upward. Evident from the diffraction pattern change, the paramagnetic bead inside the cell move upward until reaching the cell inner wall (Figure 5.6A-B). After that, the magnetic force exerted on the paramagnetic bead starts to act as an up-lifting or down-dragging force on the single cell in solution. However, at this stage, due to the constant downward force (~2 pN) and the gravity force of the cell (~10 pN), which is negligible in case of only paramagnetic bead as discussed earlier, the cell with internalized bead still rested on the cover glass surface. When the upward pulling force reached a certain amount (~12 pN), only the cells with internalized paramagnetic bead is lifted up and keep moving upward (Figure 5.65B-C). Likewise, by decreasing the upward pulling force, the cell with internalized bead is putted back to its original position while no change for those cells without internalized paramagnetic bead (Figure 5.6C-F).
Furthermore, the developed magnetic tweezers not only exhibit the capability to manipulate the interest of cell vertically by moving the upper magnet β up and down, but also is capable of translating the cell horizontally which can be achieved by translating the upper magnet β. 149
Figure 5.7 Representative DIC images of HT22 with internalized paramagnetic beads (2.8 µm diameter) by continuously increasing the upward pulling force. Images are recorded by an
EMCCD camera. 150
Besides implementing the bidirectional force manipulation on cells with internalized paramagnetic beads, we can also achieve selectively pick up or call out the bead-targeting cells through the paramagnetic beads inside the cells. As shown in Figure 5.7, the cells with internalized paramagnetic beads can be selectively picked out among all the cells by the mechanical force. In details, by gradually moving the upper magnet down to achieve more than 2 pN, the paramagnetic beads start to move upward within cell until reaches to the cell inner wall
(Figure 5.7A-B). Afterward, the cell can be lifted up when the overall resultant magnetic force acted on the cell direct upward (Figure 5.7C-D). In the current configuration, cell uptake is achieved through nonspecific phagocytosis, and we speculate that specific manipulation of a certain type of cells by modifying the cell surface with targeting ligands should also be possible.
5.4 Conclusions
In summary, we have demonstrated a concept and designed an up-down configured magnetic tweezers capable of manipulating the bidirectional motion of paramagnetic beads. The magnitude of the force experienced by a paramagnetic bead and its displacement under magnetic force is measured experimentally which is also supported by magnetic field simulation using
FEMM. Furthermore, the unique capability of this approach is not only to be able to apply upward and downward force but also to be capable of single cell manipulation. We have demonstrated the feasibility of our magnetic tweezers with the bidirectional manipulation of the live cell motion control. We have further show selectively pick up the targeted cells with internalized paramagnetic beads, which can be utilized in a broad applications of study targeted cell selection, manipulation, and interrogation. Our approach can significantly expand the application of magnetic tweezers and provide a path forward on the more sophisticated design of 151 the magnetic tweezers capable of more complex spatial and temporal field distributions and manipulations.
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CHAPTER 6. SYNCHRONIZING MAGNETIC RESPONSE BY A LOCK-IN AMPLIFIER
COUPLED ROTATING MAGNETIC TWEEZERS
We have developed a lock-in amplifier-coupled rotating magnetic tweezers, aiming to synchronize the oscillation magnetic force response with the oscillating frequency by incorporating an optical lock-in detection. We have demonstrated the lock-in detection with the rhodamine 6G stained paramagnetic beads by monitoring the tiny fluorescence change due to the magnetic response of the paramagnetic beads under oscillation force manipulation at different frequencies. The integration of the lock-in amplifier and rotating magnetic tweezers can significantly expand the application of the magnetic tweezers, such as adapting to the important implications in the detailed exploration of mechanical properties of biomolecules and studies of the conformational fluctuation dynamics.
6.1 Introduction
Magnetic tweezers have been demonstrated as a powerful approach to manipulate biological systems, such as DNA,1-3 proteins,4-7 biomolecular complexes,8,9 and living cell,10-12 which is capable of unveiling the dynamic and mechanical properties of the biological systems.
Compared to the other approaches that are commonly employed for single-molecule manipulation, such as atomic force microscopy (AFM) or optical tweezers, magnetic tweezers feature a number of unique and distinct advantages. First of all, the magnetic force is applied in noncontact mode, meaning no physical or chemical contact is required; meanwhile, there is no photon damage, thermal damage or optical background induced into the studied systems.
Moreover, a large number of targeted molecules can be manipulated under the same magnetic field simultaneously. Besides those unique advantages, most importantly, the exerted magnetic 156
force can be adjustable in magnitude and direction, and it can be static or oscillatory.13-15
Particularly, oscillatory force manipulation has been elucidated by the theoreticians to be able to
study the biological systems beyond the equilibrium and shows a profound impact on selecting a
frequency-favored conformational state.16-22 Recently, for the first time, we have experimentally
demonstrated an oscillation force manipulation study on enzymatic reactions and provided
experimental evidence of oscillation force manipulation on single-enzyme reaction dynamics and
conformational dynamics.23 We have found that the enzymatic conformation dynamic profiles
are distinct under various oscillatory frequencies, including both the dominant conformational
states as well as their populations. Moreover, high oscillatory frequency allows the ability to
select a conformation, which matches well with the theoretical works.
Motivated by oscillatory force manipulation, a correlated measurement of the magnetic
force peak and the enzyme behavior change is needed to acquire deep insight into the oscillation
force manipulation impact on biological systems. However, to pinpoint the real-time signal change is challenging due to the following reasons: 1) force manipulation induced signal is too tiny to be measurable directly due to the sharp duration of the oscillation force; 2) the tiny signal change is often buried within the intensive background signals; 3) uncovering the tiny signal change at each peak force is difficult in terms of data processing. Lock-in amplifier is commonly used as a phase-sensitive instrument which singles out the signal at a specific reference frequency but rejects the noise signals at the other frequencies. The reference signal has the same frequency as the input signal, therefore the mixing of the two signals pulls out the weak dc signal from the intensive background noise. This incorporates ideally with our rotating magnetic tweezers, allowing a real-time measurement of the magnetic force response under the oscillation force. In fact, lock-in amplifier has been employed in many optical detections to enhance the 157
signal readout and ultimately resolving weak signal from a large noise power and achieving high
sensitivity.24-30 It has been demonstrated as a low cost efficient device to accurately measure
ultrafast time resolved fluorescence properties, such as lifetime.24,25,27,30 Kobayashi and coworkers have utilized the lock-in amplifier to enhance the two photon absorption measurement for laser dyes.29
In this work, we have developed a lock-in coupled rotating magnetic tweezers, aiming to monitor the magnetic force response under oscillation force manipulation by measuring the fluorescence change. The lock-in detection mode enables a highly sensitive measurement of differentiating the tiny optical signal change upon oscillation force manipulation. The reference signal rooted from the rotation of the tweezers represents the exact frequency of the rotating magnets. The novelty and advancement of the designed device is that the acquisition of the reference signal is totally independent to the fluorescence signal but perfectly reports the frequency of the oscillating magnets, which enable an accurate and sensitive lock-in detection
and revealing the exact magnetic response under the oscillation force manipulation. On one
hand, the oscillation magnetic field is achieved by the rotation of the disk and the frequency is
adjusted by changing the rotation speed. On the other hand, the rotation disk works in the same
fashion as an optical chopper to generate a reference signal which has the exact frequency as the
oscillatory frequency that induced the magnetic response of the targeted molecules.
This approach can be utilized to obtain a deep understanding on how the biological
chemicals fluctuates under oscillation force manipulation, i. e. fluctuation of the protein
conformational dynamics, by removing extensive nonrelevant background noise but only picking
up the signals under specific stimulus. We have validated the concept using a dye stained
superparamagnetic beads and demonstrated the feasibility of lock-in detection of an optical 158
fluorescence signal of the stained paramagnetic bead under the oscillation force manipulation.
We anticipate that the application of this newly designed device would have great use in directly
exploring the mechanical force impact on the important biological macromolecules and gaining
deep insight of understanding of structure to function in single-molecule perspective.
6.2 Experimental Sections
6.2.1 Sample Preparation
A concentration of 200 ppm rhodamine 6G (R6G) was used for the staining procedure
just before the experiment. The paramagnetic beads are incubated in the R6G solution for 12
hours and then withdraw by permanent magnet. Then, three consecutive wash with distilled
water is followed to remove the excess unbound R6G. An aqueous solution of R6G stained
paramagnetic bead was prepared just prior to the experiment. A homemade reaction chamber is
assembled by attaching the precleaned cover glass with high vacuum grease and about 200 µl of
solution was filled in the chamber. The paramagnetic beads rested on the cover glass soon by
nonspecific binding. To avoid any thermal and electrical/optical disturbance on the system, we
carefully sealed our reaction chamber with a special black lid, which is prepared by taping a
black foil on a precleaned cover glass.
6.2.2 Lock-In Amplifier Coupled Rotating Magnetic Tweezers
Figure 6.1 shows the schematic diagram of the designed lock-in amplifier coupled rotating magnetic tweezers. The home-developed rotating magnetic tweezers are capable of
generating an oscillation force by permanent magnets incorporating rotating disk. As described
in earlier publication,23 8 cylinder-permanent magnets (D4C-N52, 1/4” Dia. × 3/4” thick, K&J 159
Magnetics) are evenly distributed on a disk ( 5” in diameter) and the rotation of the disk is controlled by an electric motor. D4C-N52 is a strong Neodymium magnet with the grade of N52, which has a surface field of 7299 Gauss. The individual magnet is separated by an angle of 45° on the rotating disk. Rotating magnetic tweezers are mounted on a home-built 3D positioning stage and the rotating disk is placed 4 mm above the sample stage during experiment. The oscillatory frequency of the external force can be tuned by the rotational speed of the disk, which is ultimately adjusted by the applied voltage. Evidenced by the magnetic field measurement and
Finite Element Magnetic Method (FEMM) simulation, the new and unique design of the rotating magnetic tweezers enables a tunable frequency of the oscillation force.23,31,32
As shown in Figure 6.1, we have drawn the same amounts of the tiny holes at the corresponding positions of the magnets on the disk. A laser source has been aligned to the hole on the disk and detected by a silicon photodiode then feed into the lock-in amplifier as the reference signal. The whole instrument is isolated from the internal and external mechanical noise and acoustic noise, which is orthogonal to the optical detection. To demonstrate the lock-in detection of the lock-in amplifier coupled rotating magnetic tweezers, we have stained the paramagnetic beads (Dynabeads® MyOne™ Streptavidin T1) with rhodamine 6G. The R6G stained paramagnetic beads respond to the external oscillation magnetic field resulting the simultaneous fluorescence change of the R6G due to the focusing and out focusing of the paramagnetic beads. The optical detection is based on an inverted confocal microscope (Axiovert
200M, Carl Zeiss) equipped with a 63×oil-immersion objective (NA 1.4, plan-APOCHROMAT,
Zeiss). The sample is illuminated by a pulsed laser at 532 nm (Chameleon Discovery, Coherent,
~100 fs fwhm) and the images are collected by a single photon avalanche diode (SPAD). The fluorescence signal from the SPAD is fed into the input signal of the lock-in amplifier (SR530, 160
Stanford Research System). Finally, after the demodulation of the reference signal, the output of the lock-in amplifier is collected by the Oscilloscope (Tektronix TDS 2001c). 161
Figure 6.1 Schematic diagrams of the home-developed lock-in amplifier coupled rotating
magnetic tweezers. We have drawn the same amount of the tiny holes as the magnets on the disk,
where a green laser is aligned through and further detected by a photodiode then feed into the
lock-in amplifier as the reference signal. The fluorescence signal from the SPAD is fed into the input signal of the lock-in amplifier (SR530, Stanford Research System). Finally, after the modulation of the reference signal, the output of the lock-in amplifier is collected by the
Oscilloscope (Tektronix TDS 2001c). 162
6.3 Results and Discussions
We have demonstrated the magnetic response synchronization of our home developed the lock-in amplifier coupled rotating magnetic tweezers using the rhodamine 6G stained paramagnetic beads. In the presence of the external magnetic field, the paramagnetic beads move towards the magnetic field while in the absence of the magnetic field, the paramagnetic field rested on the surface due to no magnetic force applied. Therefore, the R6G stained paramagnetic beads change their z positions with respect to the focal plane, which induces the fluorescence change to the oscillating magnetic field. Based on the force calibration, an oscillation force ranges from 0 to 1.5 pN can be applied to the paramagnetic beads with the diameter of 1 µm, which is enough to manipulate the enzymatic activity and conformational dynamics.6,7,23,33
Figure 6.2A shows the signal of the R6G stained paramagnetic bead, from which no significant fluorescence change has been observed. In contrast, under oscillation force manipulation, we have seen significant fluorescence change of the R6G stained paramagnetic bead. Figure 6.2B shows a typical raw data of the R6G stained paramagnetic bead under an oscillatory magnetic force at the frequency of 25Hz, indicating that the signal is mixed and buried in the background signal, which results an unresolved signal. To selectively pick up the tiny signal change from the intensive background noises when the magnets are right on top of the sample where a peak magnetic force is applied on the paramagnetic beads, the reference signal is needed for the lock- in detection which perfectly reports the frequency of the oscillation magnetic force. The reference signal is obtained by aligning a laser source through the tiny holes on the rotation disk where the same amounts of the holes have been created with respect to the magnets attached on the rotation disk (Figure 6.2C). 163
Figure 6.2 Typical raw data from the lock-in detection. (a) Signal of the R6G stained Myone paramagnetic bead; (b) Signal of the R6G stained Myone paramagnetic bead under oscillation
@25 Hz; (c) Reference signal @25 Hz; (d) Lock-in signal detection of the R6G stained Myone paramagnetic bead under oscillation @25 Hz. 164
Figure 6.3 Surface plot of fluorescence response counts, fluorescence response (Voltage) and applied oscillation force frequency. 165
The novelty and advancement of the developed device is that the rotation disk serves as two significant purposes in our design. On one hand, the oscillation magnetic field is achieved by the rotation of the disk and the frequency is adjusted by changing the rotation speed. On the other hand, the rotation disk works in the same fashion as an optical chopper to create a reference signal which has the exact frequency as the oscillatory frequency that induced the magnetic response of the targeted molecules. Moreover, the acquisition of the reference signal is completely innocent to the optical fluorescence detection, which provides the foundation of the accurate measurements. 166
Figure 6.4 Lock-in fluorescence signal response as a function of applied oscillation force
frequency.
In the lock-in detection mode, the fluorescence signal detected by the SPAD (Figure
6.2B) is fed into the signal input port of the lock-in amplifier while the reference signal (Figure
6.2C) is fed into the reference signal port of the lock-in amplifier. After the modulation, the background signal which has a different frequency with the reference signal is rejected by the lock-in amplifier and it only singles out the fluorescence signal which has the same frequency with the reference signal. As shown in Figure 6.2D, a typical example of lock-in detection under the manipulation of the oscillatory frequency @25 Hz is obtained, which exhibits the distinct resolution of the fluorescence signal in comparison to the signal before the lock-in detection. We have observed the repetitive fluorescent behavior, which greatly describes the magnetic response of the paramagnetic beads under the oscillation force manipulation. To investigate the details, we 167
have conducted the experiments of the lock-in detection of the R6G stained paramagnetic beads
under various oscillatory frequencies @ 10, 20, 30, 40 and 50 Hz. We further quantified the
relationship between the lock-in detection with the oscillatory frequency by calculating the signal
response counts and signal amplitude. Signal response counts is defined as the occurrence counts
of the signal peaks; Signal amplitude represents the amplitude of each signal peak. We further
obtained a 3D correlation plot among response counts, signal peak and the oscillation frequency
(Figure 6.3). According to the 3D correlation plot, we have found that signal response fluctuates
within the same amplitude, which agrees well with the same peak magnetic force applied. The
signal response counts are completely dependent on the oscillatory frequency, verifying that
lock-in detection is achieved. We further plotted the signal response counts as a function of the
oscillatory frequency in Figure 6.4, where the response counts are linearly proportional to the oscillatory frequency.
The results from the stained paramagnetic beads experiment suggests that the home developed lock-in amplifier coupled rotating magnetic tweezers are capable of real-time synchronizing the magnetic response by monitoring the tiny fluorescence change. The validation experiment by the dye stained paramagnetic beads elucidates the success of the synchronization of the magnetic response using a lock-in amplifier. This would be utilized as a powerful approach to study the mechanical properties of the biological macromolecules, i.e. to explore the conformational dynamics of the proteins by labeling a dye molecule.
6.4 Conclusions
We have developed a lock-in amplifier coupled rotating magnetic tweezers, where a lock-
in amplifier was incorporated directly into the fluorescence detection setup, aiming to monitor 168 the small magnetic force response under oscillation force manipulation by measuring the fluorescence change. Lock-in amplifier is commonly used as a phase-sensitive instrument that singles out the signal at a specific reference frequency but rejects the noise signals at the other frequencies. This incorporates ideally with our rotating magnetic tweezers, allowing real-time measurement of the magnetic force response under the oscillation force. The integration of the lock-in amplifier and rotating magnetic tweezers provides us a novel lock-in synchronized detection mode, where the output signals from the lock-in amplifier are used to real-time synchronize the magnetic response under oscillation force manipulation. This technique can significantly expand the application of the magnetic tweezers, such as adapting to the important implications in the detailed exploration of force impact on the conformational fluctuation dynamics.
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