Interaction Between Fragments of Tau Protein Investigated by Force Spectroscopy Using AFM

Interaction between Fragments of Tau Protein Investigated by Force Spectroscopy Using AFM

by Anad Mohamed Afhaima

Bachelor of Science, Sirte University Master of Science, Sirte University

A Dissertation submitted to the Department of Chemistry at Florida Institute of Technology in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry

Melbourne, Florida December 2017

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Interaction between Fragments of Tau Protein Investigated by

Force Spectroscopy Using AFM

a dissertation by

Anad Mohamed Afhaima

Approved as to style and content

______Boris Akhremitchev, Ph.D. Committee Chairperson

Associate Professor, Department of Chemistry

______Nasri Nesnas, Ph.D.

Associate Professor, Department of Chemistry

______Yi Liao, Ph.D.

Associate Professor, Department of Chemistry

______David Carroll, Ph.D.

Associate Professor, Department of Biological Sciences

______Michael Freund, Ph.D. Professor and Head, Department of Chemistry IV

Abstract

Interaction between Fragments of Tau Protein Investigated by

Force Spectroscopy Using AFM

Anad Mohamed Afhaima

Major Advisor: Boris Akhremitchev, Ph.D.

Over the last couple of decades, there has been rapidly growing interest in research of natively unfolded proteins. Some proteins from this group are implicated in a number of neurodegenerative diseases. One group of neurodegenerative diseases, taupathies, is associated with tau protein. A number of biophysical and spectroscopic studies have revealed that tau protein can expand to a largely extended state and to transition rapidly between many different conformations. Although many of the techniques that elucidate structure of macromolecules have provided important information about the folded states of proteins, important information about the transition between the extended conformation states remain obscure. The goal of our work in the first part is the development of a new methodology to detect the presence of substructures in tau protein.

This methodological advance may provide new insights in understanding tau protein behavior and its tendency toward aggregation. In this research, atomic force microscopy

(AFM) has been used to study the effects of heparin polyanions on the conformational dynamics of tau protein. In AFM measurements tau protein fragment (255-441) equipped with cysteine at the C terminus was attached to the gold-coated AFM probe.

Experiments measure interaction between tau-protein equipped tip and various surfaces.

Distances and forces of sudden ruptures are extracted from the recorded approach- withdraw dependencies. The experiments were performed with using various concentrations of 11 kDa heparin solution. In addition, results obtained for heparin with molar mass of 11 kDa are compared with results obtained for heparin with molar mass of

18 kDa. Our data show that, heparin affected tau protein in concentration-dependent manner. The effect of adding heparin at concentration of 10 M and above was in apparent decrease of the rupture distances and the interaction forces. Changes in behavior of tau protein molecules after adding heparin to solution were irreversible: removal of heparin did not restore behavior of tau protein molecules. The irreversible changes were also concentration dependent. Adding 18 kDa heparin to solution shows qualitatively similar changes to those observed in 11 kDa heparin but the changes are more pronounced. We suggest that the observed irreversibility might be caused by irreversible binding of heparin molecules to tau protein molecules such that heparin molecules remains bound even after removing heparin molecules from solution. In order to determine whether accumulation of heparin on the tip caused the irreversible changes, we designed another approach where the heparin molecules were grafted to the gold- coated surface to prevent heparin deposition on tau-protein-coated tip. Comparing the results measured with two different approaches revealed that the reduction in the rupture distance is observed with heparin grafted on the surface, but this decrease is reversible: rupture distance restores on surface that does not contain heparin.

In the second part of this dissertation, a new method to analyze transitions that occur after rupture is employed. It is noted that transitions after a rupture event in a series of rupture events occurs considerably slower than the expected transition of freely moving AFM VI

probe. To model such transitions a simple viscoelastic Kevin-Voigt model is used to fit the force transients after rupture events. This approach allows to extract viscous properties of molecules that remain attached to the AFM probe, and to see how the model parameters change with changes in the environment. Results indicate that after adding and removing heparin, transitions exhibit more elastic (less viscous) behavior. This observation is consistent with picture of somewhat compact structure of tau protein with bound heparin as obtained from the rupture distance analysis.

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Table of Contents

1.1 Introduction………..……………………………………………………………….. (1)

1.1.1 Protein structure and function……………..………………………………..….… (1)

1.1.1.1 Natively folded and unfolded proteins……………………...………..……...…. (1)

1.1.1.2 Structural analysis techniques for the analysis of IDPs………...…………….... (4)

1.1.1.3 The feature of amino acid sequences of IDPs that is responsible for the lack of ordered structure…………………………………….……………………………….…. (5)

1.1.1.4 The benefits of IDPs to being disordered…………………………………….… (5)

1.1.1.5 Physiological and pathological functions of IDPs………………………...... (7)

1.1.2 Tau protein localization and function………………………………………..…… (9)

1.1.2.1 Tau protein isoforms………………………………………………………….. (10)

1.1.2.2 Primary structure and disordered prediction of tau protein…………….…..…. (14)

1.1.2.3 Tau protein aggregation under different conditions……………………….….. (17)

1.1.2.4 Methods of investigation of tau protein aggregation……………………….… (24)

1.1.3 Force spectroscopy………………………………………………………….…... (26)

1.1.3.1 Force spectroscopy techniques…………………………………………….….. (26)

1.1.3.2 Principles and applications of atomic force microscopy…...……………….. (28)

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1.1.4 Previous studies aimed at characterizing the conformational dynamic structures of

IDPs using AFM comparing to other methods ……………………………………….. (33)

1.5 Research statement…………………………...…………………………………… (42)

1.6 References …………………………………………..……………………………. (43)

2 Chemicals and instrumentation...... (60)

2.1 Materials and samples preparation...... (60)

2.1.1 Choice of substrate...... (60)

2.1.2 Cleaved mica...... (60)

2.1.3 Gold-coated substrate...... (61)

2.1.4 Choice of the probe...... (62)

2.1.5 Tau protein sample...... (62)

2.1.6 Materials...... (63)

2.1.7 Mica substrate modification...... (63)

2.1.8 Probe preparation...... (64)

2.1.8.1 Modification of the gold coated probe directly with tau protein without cleaning with solvents...... (64)

2.1.8.2 Modification of the gold coated probe with tau protein after cleaning the probe with solvents...... (65) IX

2.1.9 Modified gold-coated substrate with heparin...... (66)

2.1.9.1 Cleaning the gold-coated substrate...... (66)

2.1.9.2 Attaching of MUA and MU to the gold-coated substrate...... (67)

2.1.9.3 Activation of the carboxyl group in the MUA molecules...... (68)

2.1.9.4 Conjugation of heparin molecules to EDC cross-linker...... (69)

2.2 Force spectroscopy...... (70)

2.3 Data collection and analysis...... (70)

2.4 References...... (72)

3. Analysis of rupture forces...... (74)

3.1 The selecting sample preparation procedure...... (75)

3.1.1 Examination the cleaning of the AFM gold-coated tip...... (76)

3.1.2 Examining the effect of the period of the soaking time of the AFM gold-coated tip with tau protein solution on the extracting separation distance...... (79)

3.2 Studying the interaction of tau protein with a freshly cleaved mica surface...... (80)

3.2.1 Using different concentrations of heparin solution (1 μM, 10 μM and 20

μM)...... (80)

3.2.2 Using high molecular weight of heparin solution (18 kDa)……………...... (96)

3.3 Characterizing the conformational dynamics of tau protein molecules using heparin grafted to a gold-coated surface...... (99)

3.4 Conclusion………………………..………...…………………………………… (113)

3.5 References...... (115)

4 Analysis of rate of rupture transitions ………………………………….…………. (117)

4. 1 Introduction...... (117)

4.1.1 Previous studies that have been performed to characterize the mechanical properties of proteins...... (117)

4.2 Using a novel method to characterize the viscous elements of tau protein by analyzing the collected force curves...... (125)

4.2.1 Kelvin-Voigt model ...... (127)

4.2.1.1 Characteristic points and features in F (t) and x (t) dependencies...... (129)

4.2.1.2 Estimation of model parameters from the data...... (131)

4.2.1.3 Fitting the model to the data...... (132)

4.2.1.4 The values of the number of parameters that have been estimated by fitting KV model……………………………………………………………………………….... (134)

4.3 Conclusion…………………………………………………..…………………… (145)

4.4 References…………………………………………………………………..…… (147)

List of Figures

Figure 1.1 Model of the most common secondary structural elements (α-helices and β- sheets) present in a variety of protein structures……………………………………….. (2)

Figure 1.2 Structure–function paradigms of folded protein and disorder–function paradigm of unfolded protein ………………………………………………...………... (3)

Figure 1.3 Diagram illustrating functional classification of intrinsically disordered proteins (IDPs)………………………………………………………….……………… (6)

Figure 1.4 The flexibility of IDPs allows them to wrap around their target……..……. (6)

Figure 1.5 Diagram of the distribution of intrinsically disordered proteins (IDPs) implicated in the biological processes of neurodegenerative diseases (HD, PD and

AD)…………………………………………………………………………..…………. (8)

Figure 1.6 Scheme of the essential role of tau in promoting the self-assembly of tubulin subunits………………………………………………………………………………... (10)

Figure 1.7 Illustrates six different isoforms of tau protein existing in the adult human brain with a range from 352 (the shortest 3RS isoform) to 441 residues (the longest

4RL)……………………………………………………………………………….….. (11)

Figure 1.8 Amino acid sequence of the longest tau isoform (441 amino acids) …..… (12)

Figure 1.9 Schematic illustrates the domain organization of the longest tau isoform of

441, which consists of 441 residues.…….…...……………………………………..… (14)

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Figure 1.10 Analysis of tau protein sequence by prediction algorithms (PONDR and

FoldIndex)…………………………………………………………………………….. (16)

Figure 1.11 Pathological hallmarks of AD………………………………………...… (19)

Figure 1.12 Model of stages leading to tau pathology and factors that may contribute to the generation of tau oligomers……………………………………………………….. (20)

Figure 1.13 Amyloidogenic propensities of some tau sequences to form fiber states……………………………………………………………………………..……. (22)

Figure 1.14 Some applications of AFM to study conformations of molecules and intermolecular interactions ………………………………………………………….... (29)

Figure 1.15 The calculation of tip-sample distance (x)………………………..……... (30)

Figure 1.16 Schematic of the basic of measurements of an AFM instrument……….. (31)

Figure 1.17 Representation of the conformations of htau40…………….…………… (35)

Figure 1.18 (a) Schematic shows the experimental procedure of creating gas phase ions of several charge stats…………………………………………………………...……. (36)

Figure 1.19 CD spectra of 4RMBD in a buffer of Tris-HCL with different contents of trifluoroethanol (TFE) ………………………………………………………………… (37)

Figure 1.20 The experiment for probing the unstructured conformation of α-synuclein, misfolded and aggregation-prone conformation of α-synuclein and the interaction between monomeric α-synuclein molecules at pH 7.0 in the presence of Zn2+…....…. (39)

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Figure 1.21 A representative interactions between α-synuclein monomers ……...….. (40)

Figure 1.22 Histograms of the contour length distribution for (a) Ab40, (b) [VPV] Ab40,

Figure 2.1 Inside view of mica structure and top view of the cross-section top view of mica made in the plane with K atoms………………………………………………… (61)

Figure 2.2 Self-assembled monolayers (SAMs) of organic alkanethiols on gold substrate……………………………………………………………………………..… (62)

Figure 2.3 Sequences of tau 441 isoform and the mutant fragment (255-441)………. (63)

Figure 2.4 Modification of gold-coated tip directly with tau-mutant protein……..…. (65)

Figure 2.5 Modification steps of gold-coated tip with tau-mutant protein………..…. (66)

Figure 2.6 Cleaning of gold-coated substrate using solvents on shaker………...…… (67)

Figure 2.7 Modification of the gold-coated substrate with sample…………………... (68)

Figure 2.8 Modification of gold-coated substrate with mixture of MUA and MU molecules………………………………………………………………………..…….. (68)

Figure 2.9 Activation of the carboxyl groups (-COOH) of MUA molecules using EDC

………………………………………………………………………………...... … (69)

Figure 2.9 Conjugation of heparin molecules to EDC cross-linker………………….. (68)

Figure 2.10 Tuning the laser beam of AFM…………………………….……………. (71)

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Figure 3.1 Typical force curve with multiple rupture events obtained from the interaction of tau protein molecules with mica………………………………………...... … (74)

Figure 3.2 The force-distance curve of the single molecule approach and the multiple molecules approach….………………………………………………………………... (76)

Figure 3.3 The force distance curve and the Gaussian distribution of the interaction of the tau protein and the mica surface before the cleaning……………………………... (77)

Figure 3.4 The force - distance curve of the interaction of the free gold-coated tip with mica before and after cleaning…………………………………………………..……. (79)

Figure 3.4 Scheme of experiment setup to the interaction of the tau protein and mica surface in PBS solution and 11 kDa heparin solution…………….…………...……… (81)

Figure 3.6 Histograms of separation distances of the interaction between the tau protein and freshly cleaved mica with in PBS (pH 6.9) and an 11 kDa heparin solution (1 μM)

………………………………………………………………………………..……….. (82)

Figure 3.7 Histograms of interaction forces of the interaction between the tau protein and freshly cleaved mica in PBS (pH 6.9) (A,C) and an 11 kDa heparin solution (1 μM) (B) with1 s dwell time………………………..…………………..……………...…………(83)

Figure 3.8 Histograms of spring constant of the interaction between the tau protein and freshly cleaved mica in PBS (pH 6.9) and an 11 kDa heparin solution (1 μM) with1 s dwell time……………………………………………………..………………….…… (84)

Figure 3.9 The plotting of dwell time vs. separation distance (nm), force (pN) and spring constant o of the interaction between the tau protein and freshly cleaved mica in PBS (pH

6.9) and an 11 kDa heparin solution (1 μM)……………………………………..…… (85)

Figure 3.10 Histograms of the separation distance of the interaction between the tau protein and freshly cleaved mica in PBS (pH 6.9) and with an 11 kDa heparin solution

(10 μM)…………………………………………………………………….…….…… (87)

Figure 3.11 Histograms of interaction forces of the interaction between the tau protein and freshly cleaved mica in PBS (pH 6.9) and an 11 kDa heparin solution (10

μM)……………………………………………………...…………………………….. (88)

Figure 3.12 Histograms of spring constant of the interaction between the tau protein and freshly cleaved mica in PBS (pH 6.9) and an 11 kDa heparin solution (10 μM)

……………………………………………………………………………………....… (90)

Figure 3.13 The plotting of dwell time (s) vs. separation distance (nm), force (pN) and spring constant of the interaction between the tau protein and freshly cleaved mica in

PBS (pH 6.9) and an 11 kDa heparin solution (10 μM)………...……...……..………..(91)

Figure 3.14 Histograms of separation distances of the interaction between the tau protein and freshly cleaved mica in PBS (pH 6.9) and an 11 kDa heparin solution (20

μM)……………………………………...…………………………………………….. (92)

Figure 3.15 Histograms of the interaction forces of the interaction between the tau protein and freshly cleaved mica in PBS (pH 6.9) and an 11 k Da heparin solution (20

μM)……………………………………………………………………………….…….(94) XVI

Figure 3.16 Histograms of spring constant of the interaction between the tau protein and freshly cleaved mica in PBS (pH 6.9) and an 11 kDa heparin solution (20 μM) ….… (95)

Figure 3.17 The plotting of dwell time vs. separation distance , force (pN) and spring constant of the interaction between the tau protein and freshly cleaved mica in PBS (pH

6.9) and an 11 kDa heparin solution (20 μM)……………………………..………..… (96)

Figure 3.18 Histograms of the separation distance of the interaction between the tau protein and freshly cleaved mica with a PBS buffer (pH 6.9) (A,C) and an 18 kDa heparin solution (10 μM) (B) with 1 s dwell time. …………………………..……………..… (97)

Figure 3.19 Histograms of the interaction forces of the interaction between the tau protein and freshly cleaved mica with a PBS buffer (pH 6.9) (A, C) and an 18 kDa heparin solution (10 μM) (B) with 1 s dwell time. ………………………………….... (98)

Figure 3.20 Schemes of experiment setup to the interaction of the tau protein vs. free gold-coated substrate and the tau protein vs. heparin grafted to gold-coated substrate…………………………………………………………………………….. (100)

Figure 3.21 Histograms of the separation distances of the interaction between the tau protein and free gold-coated substrate in PBS (pH 6.9) and an 11 kDa heparin molecules grafted to gold-coated substrate with1 s dwell time………………………...…….…. (101)

Figure 3.22 Histograms of interaction forces of the interaction between the tau protein and free gold-coated substrate in PBS (pH 6.9) and an 11 kDa heparin molecules grafted to gold-coated substrate………………………………………………………..…….. (102)

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Figure 3.23 Histograms of spring constant of the interaction between the tau protein and free gold-coated substrate in PBS (pH 6.9) and an 11 kDa heparin molecules grafted to gold-coated substrate with1 s dwell time……………………….……………..…….. (103)

Figure 3.24 The plotting of dwell time vs. separation distance , force and spring constant of the interaction between the tau protein and free gold-coated substrate in PBS (pH 6.9) and 11 kDa heparin molecules grafted to gold-coated substrate…………………..….(105)

Figure 3.25 Force- distance curves of unzipping of Aβ1-42 and the force plateaus of gradually unzipping of the β sheets……………………………………..…...………..(106)

Figure 3.26 The plateau force histograms of unzipping of β1-42 and Aβ1-42 and the

Gaussian distribution of the interaction force of the tau protein with mica substrate (107)

Figure 3.27 Repeatability of the force plateau of plateau force of unzipping of Aβ1-42 and Aβ1-42 and repeatability of interaction forces of the tau protein with mica surface……………………………………………………………………………….. (108)

Figure 3.28 Off-normal pulling geometry of tether attachment and off-apex geometry of tether attachment………………………………………………………………….…. (110)

Figure 3.29 The distribution of contour length of Aβ40 and the distribution of contour length of tau protein sequence……………………………………………….………. (111)

Figure 3.30 The histogram distribution of unbinding forces of antilysozyme antibodies and the jump forces of the interactions of tau protein sequence with mica surface………………………………………………………………………………...(113)

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Figure 4.1 Scheme of the experimental setup including the protein (Guanylate Kinase,

GK) attached by the 171 and 75 sites to a gold nanoparticle (GNP)………………... (118)

Figure 4.2 Shows two different curves: one for a low driving force has a characteristic linear elasticity response (a simple spring), and the other, for a higher driving force, is characteristic of a viscoelastic response. ………………………………………….… (119)

Figure 4.3 Experimental force–extension curve vs. deformation measured at 10 Hz indicates that the protein GK exhibiting a yield deformation of 1.1 Å and direct and indirect measured of the storage modulus vs. applied force at 10 Hz …………….… (120)

Figure 4.4 Amplitude of the frequency response of the protein Guanylate Kinase (GK) vs. the driving frequencies in the absence and presence guanosine monophosphate (GMP) substrate…………………………………………………………………...…...……...(121)

Figure 4.5 Kelvin-Voigt model of viscoelastic system………………………….….. (122)

Figure 4.6 The associated restoring force F(x), solid (blue) and the potential energy,

V(x), dashed (red), where the force reaches ±F0 for large displacement x…………....(123)

Figure 4.7 Force curve on mica, force curve on individual lysozyme enzyme and Force curve on aggregate of lysozyme……………………………………………………... (124)

Figure 4.8 The cartoons show the rupture process of the tau protein from the surface during retraction process and force –distance curve with multiple rupture events..……………………………………………………………………………….. (126)

Figure 4.9 Force-distance curve with the number of stretching slopes……..….....… (127)

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Figure 4.10 The cartoon of the interaction between the attached molecules and surface and the Kelvin-Voigt model…………………………………………………………. (128)

Figure 4.11 Examples of the fitting of two extracting fore-distance curves with KV model………………………………………………………………………...…….… (133)

Figure 4.12 The plots of remaining spring constant (k1) values vs. z scan rate. The magnitudes of the remaining spring constant (k1) were extracted from fitting the data with

KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 1 μM of 11 kDa heparin solution.….....…………… (135)

Figure 4.13 The plots of decay time (τ) values vs. rate. The magnitudes of the remaining spring constant (k1) were extracted from fitting data with KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH

6.9) and 1 μM of 11 kDa heparin solution....……………………………………...… (136)

Figure 4.14 The plots of friction coefficient (β) values vs. rate. The magnitudes of the remaining spring constant (k1) were extracted from fitting data with KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer

(pH 6.9) and 1 μM of 11 kDa heparin solution…………………………………….... (137)

Figure 4.15 The plots of the remaining spring constant values vs. rate. The magnitudes of the remaining spring constant (k1) were extracted from fitting data with KV model.

The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 10 μM of 11 kDa heparin solution…………………...………… (138)

Figure 4.16 The plots of the decay time (τ) values vs. rate. The magnitudes of the remaining spring constant (k1) were extracted from fitting the data with KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 10 μM of 11 kDa heparin solution…………………...………… (139)

Figure 4.17 The plots of the friction coefficient (β) values vs. rate. The magnitudes of the remaining spring constant (k1) were extracted from fitting data with KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 10 μM of 11 kDa heparin solution………………………..……. (140)

Figure 4.18 The plots of the remaining spring constant (k1) values vs. rate. The magnitudes of the remaining spring constant (k1) were extracted from fitting data with

KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 20 μM of 11 kDa heparin solution………………… (142)

Figure 4.19 The plots of the Decay time (τ) values vs. rate. The magnitudes of the remaining spring constant (k1) were extracted from fitting the data with KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 20 μM of 11 kDa heparin solution. ……………………….…… (143)

Figure 4.20 The plots of the friction coefficient (β) values vs. rate. The magnitudes of the remaining spring constant (k1) were extracted from fitting data with KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 20 μM of 11 kDa heparin solution……………………….…….. (144)

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List of Tables

Table 1.1 Comparison of numbers of single-force spectroscopy techniques (SMTs)

………………………………………………………………………………………… (26)

Table 4.1 Shows the values of the remaining spring constant (k1) that was extracted from fitting data with the KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 1 μM of 11 kDa heparin solution………………………………………………...…………………………….. (134)

Table 4.2 Shows the values of the decay time (τ) that were extracted from fitting the data with the KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 1 μM of 11 kDa heparin solution………………………………………………………………………………. (135)

Table 4.3 Shows the values of the friction coefficient (β) that was extracted from fitting data with the KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 1 μM of 11 kDa heparin solution……………………………………………………………………………… (136)

Table 4.4 Shows the values of the remaining spring constant (k1) that was extracted from fitting data with the KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 10 μM of 11 kDa heparin solution...... (138)

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Table 4.5 Shows the values of the decay time (τ) that was extracted from fitting data with the KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 10 μM of 11 kDa heparin solution………….... (139)

Table 4.6 Shows the values of the friction coefficient (β) that were extracted from fitting the data with the KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 10 μM of 11 kDa heparin solution……………………………………………………….…………...…………. (140)

Table 4.7 Shows the values of the remaining spring constant (k1) that were extracted from the fitting data with the KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 20 μM of 11 kDa heparin solution…………………………………………………….………………………… (141)

Table 4.8 Shows the values of the decay time (τ) that were extracted from fitting data with the KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 20 μM of 11 kDa heparin solution...... (142)

Table 4.9 Shows the values of friction coefficient (β) that were extracted from fitting data with the KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 20 μM of 11 kDa heparin solution...... (143)

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List of Abbreviations

Abbreviations Meaning

IDPs Intrinsically disordered proteins

NMR Nuclear magnetic resonance spectroscopy

CD Circular dichroism spectroscopy

ROA Raman optical activity

FTIR Fourier-transformed infrared spectroscopy

MTs Microtubules

MAPs Microtubule associated proteins

AD Alzheimer's disease

PONDOR Prediction of natural disordered regions

NFT Neurofibrillary tangles

MTBD Microtubulebinding domain

PHF Paired helical filament

TFE Trifluoroethanol

CytC Cytochrome

ESI-MS Electrospray mass spectrometry

STM Scanning tunneling microscopy

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Abbreviations Meaning

SMFS Single-molecule atomic force microscopy

AFM Atomic force microscopy

SAMs Self-assembled monolayers

PBS Phosphate buffer saline

EDC (N-3-dimethyl-aminopropyl) -N-ethylcarbodiimidehydrochoride)

MUA 11-mercaptoundcanoic acid

MU 11-mercapto-1-undecanol

UV Ultraviolet

Aβ Amyloid β protein

GK Guanylate Kinase

GNPs Gold nanoparticles

GMP Guanosine monophosphate

KV Kelvin-Voigt model

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Acknowledgement

I thank Allah for providing me the chance and granting me the strength, patience, and knowledge to complete this work.

I would like to express my deep gratitude to my advisor, Dr. Akhremitchev, for all his support, guidance, help and encouragements throughout my Ph.D. study. He has massive knowledge and experience in his field.

I would like to thank my committee members Dr. Nasri Nesnas, Dr. Yi Liao and Dr. David Carroll for serving on my dissertation committee and for their helpful suggestions.

I would like to thank all faculty and support staff within the Chemistry

Department at FIT, especially Dr. Michael Freund, Dr. Mary Sohn and Christen

Gates.

I would like express my deep gratitude to my parents, my sisters, brothers, whose love and support have enabled me to accomplish my goals.

I would like to express my most sincere gratitude to Dr. Hamid Rassoul for his support and help.

My appreciation is also given to Professor Han’s group at university of

California, Santa Barbara for providing us with the tau protein sample.

I would like to thank my friends, Faieza, Asma, Rajaa, Maisa, Samah and

Manal for their love, support and continuous encouragement. I love you all!

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Dedication

To:

My father and the memory of my beloved mother for their endless love,

prayers and support.

My brothers, sisters, and friends for their love and encouragement.

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1.1 Introduction

1.1.1 Protein structure and function

Proteins are biological macromolecules that play essential roles in biological systems.

Each type of protein possesses its unique sequence of amino acids and they perform remarkable dynamic processes in living cells. Proteins have the ability to couple to other selected molecules. This binding enables proteins to acts as receptors, motors, and regulators. Proteins catalyze or inhibit chemical reactions. They are both transport and storage materials [1,2]. Indeed, proteins support cell and body structure. Understanding of protein conformation is the key to realizing how proteins participate in their various roles and carry out a broad range of biological functions. Each sequence of amino acids has a different level of flexibility, allowing proteins to fold into a variety of structures that differ in their abilities to undergo conformational changes related to function.

Intermolecular forces are responsible for formation of particular conformation of the peptide sequence and preservation of its unique structure [3]. A change in protein shape is constrained by four types of weak bonds or forces (hydrogen bond, ionic bond, van der

Waals attractions, and attractive interactions arising from the hydrophobic effect). These bonds form between different parts of the protein sequence to form compact domains with a specific three-dimensional structure (3D), which depends on the order of the amino acids in the protein chain [3,4].

1.1.1.1 Natively folded and unfolded proteins

There are two major folding structures known as the α-helix and β-pleated sheet are present in a variety of protein structures, formed through regular hydrogen bonding 1

interaction between N-H and C=O groups in the polypeptide chain (Figure 1.1). The amino acid sequence composition of proteins determines how the protein folds into the secondary structures. Most proteins in order to be biologically active adopt a specific folded shape which consists of compact conformational elements of the polypeptide chains. These substructures play a critical role in determining the protein structures and, as a consequence, the functionality of a protein relies mainly on the final protein structure

[3].

Figure 1.1 Model illustrates the most common secondary structural elements (α-helices and β- sheets) present in a variety of protein structures. Adapted from “Mechanics of Proteins with a

Focus on Atomic Force Microscopy,” by Felix, R.; Annafrancesca, R.et al, Nanophysics for

Health, 2012, 11, p. 4 [3].

Besides the natively folded proteins, a new group of proteins known as “natively unfolded proteins” or “intrinsically disordered proteins” (IDPs) has recently been investigated. The particular term IDPs has been used to introduce proteins or protein domains that don't form compact globular structure either entirely or in part in their native state [5,6,7]. Since discovery of IDPs abundance development the relationship between protein structure and molecular function which is known as the protein structure-

function paradigm becomes not universal property [8]. The structure-function paradigm was proposed by Christian Anfinsen and colleagues in 1973. They suggested that the sequence of a protein is the principle determinant of its a compact conformation, and this structure is required for molecular function [9,10,11]. A large number of protein sequences that don't fold into specific tertiary structures have been discovered over the last couple of decades [12]. Instead of folded proteins, these proteins and polypeptide segments adopt a broad range of different conformations and can still achieve their function in an unstructured/disordered state (Figure 1.2) [13].

Figure 1.2 (A) Structure–function paradigm of folded protein. (B) Disorder–function paradigm of unfolded protein. Adapted from “Classification of Intrinsically Disordered Regions and

Proteins,” by van der et al, Journal of the American Chemical Society, 2014, 114, p. 6590 [13].

One of the striking characteristics of IDPs is their ability to take up various conformation structures upon binding to different partners or due to change in the surrounding solution.

However, the interaction of the IDPs polypeptide sequence with their interacting partners

will not lead to the formation of a complete well-defined structure; distinct segments can still remain unfolded [14,15,16,17]. Owing to their conformational flexibility, IDPs exhibit multiple conformational states, which gives them functional flexibility

(multitasking) through the formation of fuzzy complexes [18,19].

1.1.1.2 Structural analysis techniques for the analysis of IDPs

The lack of a unique, well-defined folded structure (3D) within an intrinsically unstructured protein group has been investigated by using various types of structural analysis techniques such as multidimensional magnetic resonance (NMR), circular dichroism (CD) spectroscopy, and X-ray crystallography. In some studies these techniques are coupled with other techniques, such as Raman optical activity (ROA) and

Fourier-transformed infrared (FTIR) spectroscopy and a hydrodynamic technique (small angle X-ray scattering, ultracentrifugation, and gel-filtration) [20,21,22]. In fact, since

IDPs are very sensitive to the environment and tend to form broad heterogeneous ensembles of structures due to the conformational fluctuations of their amino acid sequences on multiple timescales, until now there have been no appropriate methods for the characterization of conformation ensembles of IDPs [8]. Indeed, all the methods that have been used to describe the conformation ensemble of IDPs give rise to a snapshot of conformations and only provide an ensemble-average for the protein monomers.

1.1.1.3 The feature of amino acid sequences of IDPs that is responsible for the lack of ordered structure

The inability of IDPs to adopt a regular 3D structure under physiological conditions is reflected in particular features of their amino acid sequences [11,23,24]. Several structural analyses that have been performed on IDPs have shown that there are some common features shared between disordered regions in IDPs. In comparison with natively folded proteins, IDPs under physiological conditions are enriched in unfolded conformation promoting amino acid residues (A, G, R, Q, P, S, K and E) and lowered in folded conformation promoting amino acid residues (F, W, C, Y, I, L, N and V) [25,26].

In addition, IDPs are characterized by a high net charge of polypeptide chain (often negative) and a low mean of hydrophobicity that lead to the decrease in the probability of

IPD sequences to form compact structures [16,27,28].

1.1.1.4 The benefits of IDPs to being disordered

The flexibility of intrinsically disordered proteins and their existence in a wide range of dynamic ensembles of conformation give this group multiple functional advantages

(Figure 3 and 4). First, IDPs have the ability to bind broad structurally distinct targets.

Second, the conformational flexibility of IDPs gives them the ability to interact with targets and induces disorder-to-order transition as consequence forming stable structures allowing for high specificity and low affinity interactions. Third, the conformational flexibility of IDPs allows them to overcome steric restrictions because the flexibility of these proteins provides considerable surfaces of interaction in the protein complexes compared to the surfaces obtained with rigid partners (Figures 1.3 and 1.4). In 5

conclusion, IDPs can't accomplish their function without having this ability of the polypeptide chain to rapidly fluctuate among alternative conformational states [29,30,31].

IPUs

Entropic chains Recognition Directly function due to disorder

Transient binding Permanent binding

Display sites Chaperones Effectors Assemblers Scavengers (tau / casein) (α- synuclein) (securin ) (Bob1 / p53) (casein)

Figure 1.3 Diagram illustrating functional classification of intrinsically disordered proteins

(IDPs) [31].

Figure 1.4 The flexibility of IDPs allows them to wrap around their target. (a) SNAP-25 (red) bound to BoNT/A(1XTG). (B) HIF-1α interaction domain (red and yellow) bound to the TAZ1 domain. Adapted from “The Interplay between Structure and Function in Intrinsically

Unstructured Proteins,” by Tompa, P., FEBS Letters, 2005, 579, p. 3349 [31]. 6

1.1.1.5 Physiological and pathological functions of IDPs

Intrinsically disordered proteins (IDPs) are found to participate in a wide range of biological processes such as transcriptional and translational regulation, signal transduction, protein phosphorylation, and the folding of RNA and other proteins

[16,31,32,33]. Recently, several studies indicated that the conformational heterogeneity of IDPs allows them to adopt conformations that promote aggregation and contribute to etiology of the most widespread neurodegenerative diseases, such Parkinson's disease

(PD), Huntington's disease (HD), and Alzheimer's disease (AD) [34,35]. Nowadays, there is multiple evidence that has been derived from traditional bulk experiments and spectroscopies revealing that these diseases are caused by the aggregation of α-synuclein,

Huntington (Htt) protein, and tau protein, respectively (Figure 1.5) [34,36]. Despite intensive research, the correlation between intrinsically disordered proteins and neurodegenerative diseases, and the key step in the aggregation the formation of misfolded intermediates remain ambiguous.

For more than twenty five years, neurodegenerative diseases have been gaining significant attention from scientists. However, detecting the underlying causes, clarifying the molecular mechanisms, and developing medical products to cure these diseases require investment of time, resources, and hard work. For that reason, a new methodology is needed to characterize and define adequately the conformational diversity of IDPs. Advancing measurement methodology to characterize large-scale dynamics of unfolded or partially-structured macromolecules and using this methodology to describe conformational dynamics of tau protein is the major goal of this research project.

Figure 1.5 Diagram shows the distributions of the biological processes of proteins implicated in

HD, PD and AD diseases. (A) Proteins cause HD involve in all important biological process except carbohydrate metabolism and electron transport. (B) Most the biological process perform by unstructured proteins that involved in PD diseases (C) Proteins cause AD involve in all important biological process excepts like coenzyme and prosthetic group metabolism and immunity and defense, proteins involved in all other important processes in AD. Adapted from

“The Role of Intrinsically Unstructured Proteins in Neurodegenerative Diseases,” by

Raychaudhuri, Set al, PLOS one, 2009, 4, p. 6. [34]

1.1.2 Tau protein localization and function

Neurons are structural units of the nervous system with a very complex morphology consisting of the cell body, axons, and dendrites. Neural transmission occurs through parts of neurons, and therefore, any changes in neuronal morphology may affect their function and even produce pathological events. Microtubules (MTs) are one of the major cytoskeletal components of neurons. They have highly charged hollow cylinders assembled of tubulin dimers. MTs are very dynamic structures which participate in maintaining neuron architecture shape, cell division, receptor activity, and axonal transport which serve as the "tracks" upon which molecular motors and cellular transport occur [37]. The microtubule associated proteins (MAPs) are believed to be important for

MT formation and stabilization (Figure 1.6). Tau protein is one of the microtubule associated proteins that is strongly expressed in the axons of neurons. Since the initial isolation of tau protein, several attempts have been made to clearly realize tau’s role in the neuron. Because tau protein is associated with many proteins, it can be thought of as a sticky protein. One of the best known and studied associated tau proteins is tubulin.

Likewise, tau protein interacts also with other proteins such as spectrin, actin, kinases, and ferritin. Several key in vitro experiments indicated an essential role for tau in promoting the self-assembly of tubulin subunits and in stabilizing microtubules by interacting with tubulin and binding to their surfaces [38,39,40,41]. The normal function of tau proteins is to serve as links between microtubules and other cytoskeletal elements or proteins such as plasma membrane phosphatases. Further, some studies have shown that tau has an important role in cellular response to heat shock, where it binds to DNA and protects it against heat and oxidative stress [42]. 9

Figure 1.6 Scheme showing the essential role of tau in promoting the self-assembly of tubulin subunits and stabilizing of microtubules by interacting with tubulin and binding to their surfaces.

Adapted from “Structure and Pathology of Tau Protein in Alzheimer Disease,” by Kolarova, M et al, International Journal of Alzheimer’s Disease, 2012, 2012, p. 3.

1.1.2.1 Tau protein isoforms

Tau is a neuronal microtubule-associated protein occurring mainly in the axons of neurons. In the adult human brain, there are six isoforms of tau protein with a range of molecular masses from 48 to 67 kDa and lengths ranging from 352 (the shortest 3RS isoform) to 441 residues (the longest 4RL) (Figure 1.7) [43]. Tau comprises four semi- conserved sequences of 31 or 32 residues, known as "repeats". Tau isomers differ from each other by the length of the N-terminal part. Also, they vary from each other by the presence of either three or four repeat-regions in the carboxyl-terminal (C-terminal) part of the molecule and the absence or existence of one or two inserts (29 or 58 amino acids long) in the amino-terminal (N-terminal) part of the protein [44,45,46]. There are 3 (3R)

or 4 (4R) repeat domains in each isoform in the MT-binding domain (MTBD) which are responsible for the interaction with microtubules [47].

Figure 1.7 Illustrates six different isoforms of tau protein existing in the adult human brain with a range from 352 (the shortest 3RS isoform) to 441 residues (the longest 4RL). Adapted from

“Characterization of Tau in Cerebrospinal Fluid Using Mass Spectrometry,” by Portelius, E. et al,

Journal of Proteome Research, 2008, 5, p. 2114.

In physiological states, the native tau protein has negatively (D, E) and positively (K, R) charged amino acid residues [48]. In tau protein, half of the sequence consists of only five types of amino acids (G, K, P, S, and T). Polar amino acids are predominant in tau protein structure, and it has an overall basic character (Figure 1.8) [39].

Figure 1.8 Amino acid sequence of the longest tau isoform (441 amino acids) and the distribution of basic, polar, nonpolar, and acidic amino acids along the tau protein sequence. Adapted from

“Structure and Pathology of Tau Protein in Alzheimer Disease,” by Kolarova, M et al,

International Journal of Alzheimer’s Disease, 2012, 2012, p. 2.

Because it is rich in hydrophilic side-chain composition, tau protein is highly soluble, natively unfolded, and highly flexible [50,48]. Hypothetically, the deficiency of structure in natively unfolded tau protein is due to its low content of hydrophobic amino acids and a high net charge near the physiological pH [51]. Both of these characteristics tend to counteract a well-folded globular state of tau protein. The minimization of the net charge of tau protein increases the aggregation propensity especially at its isoelectric point by 12

permitting the hydrophobicity-driven collapse [52]. Furthermore, all of the tau isoforms are flexible macromolecules, characterized by low content of secondary structure, and are classified as naturally unfolded proteins. In solution, they are highly water-soluble so they polymerize weakly at physiological concentration and exhibit a random coil behavior.

The presence of three major domains in the sequence of the tau molecule (an acidic N- terminal part; a proline-rich region, and a basic C-terminal domain) demonstrates that tau protein is a dipole, and each domain carries a different high net charge (Figure 1.8) [53].

The C-terminal domain is known as the “assembly domain” because it binds to the microtubules and enhances their assembly, while the N-terminal (the acidic part) in tau protein is known as the “projection domain” because it does not bind to the microtubules but projects away from the microtubule surface [54,55]. There are two basic regions in the projection domain: the acidic region (negative charge) and the proline-rich region

(with net positive charge) [49]. The projection domain is responsible for interaction with mitochondria, components of neuronal plasma, or with other cytoskeletal elements and also regulates spacing between microtubules in the axon [56,57,58]. Figure 1.9 represents the domain organization of the longest tau isoform of 441 [59].

Figure 1.9 Schematic illustrates the domain organization of the longest tau isoform of 441, which consists of 441 residues. Adapted from “The Role of Tau Oligomers in the Onset of Alzheimer's

Disease Neuropathology,” by Maria, C. C.et al, American Chemical Society, 2014, 5, p. 1179

[59].

1.1.2.2 Primary structure and disordered prediction of tau protein

Many proteins acquire their native function by adopting a specific three-dimensional structure which is energetically stable. A number of biophysical and spectroscopic studies have revealed that, in solution, tau protein is highly dynamic and behaves as an intrinsically disordered protein (IDP) [60]. As stated earlier, this is caused primarily due to tau protein containing very low content of hydrophobic amino acid and the high net charge causes electrostatic repulsion, which inhibits the molecule from forming close intermolecular contacts [61].

A number of studies that have been done on tau protein from brain tissue indicated that the protein is devoid of secondary structure and maintains its biological function even after harsh treatment [62,63]. In aqueous solution, tau protein exhibits a mostly random coil structure with little α-helix and β -sheet, as shown by sequence analysis such as circular dichroism (CD), Fourier transform infrared, X-ray scattering and biochemical

assays. This means that the polypeptide chain of tau protein is highly flexible and mobile. By its nature, tau shows unusual properties such as the ability to expand to a largely extended state and to transition rapidly between many different conformations

[41]. As a monomeric protein, tau has random coil features, although some regions demonstrate a preference for helical or β-strand conformations [64,65]. Solution NMR measurements have indicated that tau protein has a variety of structures and a complicated network of transient long-range contacts [66]. Single molecule fluorescence resonance energy transfer (FRET) measurements detected that each tau domain (a featured part of the tau protein responsible for the interaction with microtubules) has distinct conformational properties which are strongly correlated with its degree of disorder [67,68]. Small-angle X-ray scattering measurements have revealed that each individual domain of tau protein appears as a random polymer [65]. There are two types of algorithms that have been used to predict the primary structure of tau protein and as a comparison of α- tubulin (a well-folded protein). The first approach is PONDOR (the prediction of natural disordered regions). This algorithm is based on the statistical distribution of amino acids in sequences of proteins [69,70]. The standard output ranges from -0.5 to 0.5. Positive values mean a high probability for ordered conformation and negative values indicate a high likelihood for disordered conformation.

In Figure 1.10, the black line represents the PONDOR prediction for tau protein.

Prediction by PONDOR indicated that the whole sequence of tau protein shows low probability for order structure except for the repeat domain. This method shows a good agreement with the experimental data [71]. On the other hand, the analysis of α-tubulin

by PONDOR gave values between 0.0 and 0.5. These values indicating that α-tubulin has a higher degree of foldability with the exception of the C-terminal 30 residues [65].

Figure 1.10 Analysis of tau protein sequence (b, d) by prediction algorithms (PONDR and

FoldIndex) compared with α- tubulin (a well-folded protein) (a, c). Adapted from “Spectroscopic

Approaches to the Conformation of Tau Protein in Solution and in Paired Helical Filaments,” by

Bergen, M.et al, Neurodegenerative Disease, 2006, 3, p. 199.

A second algorithm used in the prediction of unfolded protein is FoldIndex. This approach takes into consideration the hydrophobicity and net charge of the analyzing sequence [72,73]. The equation used in this calculation is:

퐼 = 2.785 < 퐻 > −∣< 푅 >∣ −1.151 (1.1)

Where I represents the FoldIndex < 퐻 > indicates the mean hydrophobicity and ∣< 푅 >∣ represents the mean net charge (the absolute value of difference between positive and negative charges divided by the number of amino acid residues). The analysis of the tau protein sequence by the FoldIndex algorithm predicts that tau protein will possess a disordered conformation for the whole sequence except for repeat 2 in the middle part to repeat 3 at the end of sequence (Figure 1.10). This prediction shows a general agreement with spectroscopic data of tau by NMR [71]. In contrast, the whole sequence of α-tubulin shows a higher probability of the tau to be folded with the exception of the C-terminal tail

[61]. Even though, all previous methods and studies indicated that tau protein is dominated by a random coil structure, compatible with the remarkable high solubility and hydrophilic composition of tau, all these information didn’t give obvious clue about aggregation process ways and reasons.

1.1.2.3 Tau protein aggregation under different conditions

Tau protein under normal physiological condition is highly soluble and natively unfolded with limited secondary structure. Under non-physiological conditions, the tau monomer associates with itself to form prefibrillar oligomeric aggregates (2 or more molecules in a multimeric structure) and assembles into paired helical filaments (PHFs) [47].

Abnormally aggregates of tau protein are found in several neurodegenerative diseases termed 'tauopathies'.Most neurodegenerative diseases are characterized by abnormal aggregation and deposition of proteins in and around neurons in brain [75]. The self- assembly of proteins into aggregates pertinent to the neurodegenerative disease often requires significant change in protein conformation. However, the molecular mechanism(s) by which the aggregation process happens are still far from being fully understood [76]. Pathologically, neurodegenerative disorders are characterized by neuronal loss and intraneuronal fibrillary materials accumulation [77]. During postmortem examination of the brain tissues, it has been noted that Alzheimer’s disease

(AD) is characterized by the presence of extracellular amyloid plaques which produce from deposit of Aβ protein and intracellular neurofibrillary tangles (NFT) which produce from the aggregation of microtubule associated protein-tau (Figure 1.11). Neurofibrillary tangles are essentially composed of bundles of filaments formed by the microtubule- associated protein tau [78,79,80].

Figure 1.11 Pathological hallmarks of AD (extracellular amyloid plaques and intracellular neurofibrillary tangles (NFT). (This figure is copied from www.ahaf.org/alzdis/about/AmyloidPlaques.htm).

The stages that lead to tau protein pathology are shown in Figure 1.12 [59]. The repeat units in the MT-binding domain (MTBD) play an essential role in PHF assembly [78].

Figure 1.12 Model of stages leading to tau pathology and factors that may contribute to the generation of tau oligomers. Probably oligomers of tau are intermediate aggregates and can cause neuronal damage before PHFs and NFTs are formed. Adapted from “The Role of Tau Oligomers in the Onset of Alzheimer's Disease Neuropathology,” by Maria, C. C.et al, American Chemical

Society, 2014, 5, p. 1180.

Due to the much greater solubility of tau in vitro compared to the concentration of tau in cells, inducers such as polyanions and fatty acids have been used to enhance and accelerate tau aggregation in vitro [81,82]. Substitution of the charge of the basic middle part of tau protein by polyanions which is achieved by sulfated glycosaminoglycans (e.g., heparin, heparin sulfate, etc.) helps to overcome the nucleation barrier [48]. For example using sulfated glycosaminoglycans promotes the hyperphosphorylation of tau protein

(thus indicating change in tau protein conformation) and induces assemble of PHFs.

Under physiological conditions, the interaction between sulfated glycosaminoglycans and 20

full-length tau triggers formation of filaments similar to those fibers isolated from the brains of AD patients. In this condition, three-repeat-containing tau isoforms form paired helical-like filaments, while four-repeat-containing tau gives straight filaments. On the other hand, under non-physiological conditions, only paired helical-like filaments are present which are formed from the fragments of tau consisting of three microtubulebinding repeats [83]. In addition, fatty acids are especially effective in inducing the fibrillization of tau protein at a near physiological pH, temperature, reducing environment, ionic strength, and tau protein concentration. Arachidonic acid (AA) is one of the important fatty acids that exist in the brain. Arachidonic acid micelles can be considered effectively as polyanions due to the burying of the hydrocarbon chains inside, with the negatively charged carboxyl groups exposed on the surface [84]. Arachidonic acid promotes fibrillization by binding tau on their anionic surface, which provided negatively charged surfaces that enhance tau protein aggregation. Therefore, studying the aggregation of tau protein at physiological conditions in vitro may require using the chemical inducers to reduce the time and the concentration of tau protein, and initiate and accelerate the aggregation process.

The prediction of how tau protein undergoes structural changes from its native structure into a beta sheet is an important task in studying molecular mechanisms of neurodegenerative diseases. Christopher and et al. have examined the amyloidogenic propensities (the potential to aggregate into toxic structures) of some tau peptide sequences to form fiber states. In this study, transmission electron microscopy (TEM) has been used to investigate if Ac10VME, Ac375KLTFR and Ac393VYK sequences seed filaments or interact with nucleating sequences in tau protein (PHF6 and PHF6*). PHF6* 21

(VQIINK) in R2 and PHF6 (VQIVYK) in R3 are essentially hexapeptides in tau protein that play an important role in aggregation. They incubated these peptides in a MOPS-Cl buffer with high molecular weight heparin to induce tau protein aggregation. These peptides were monitored overnight and fibers were observed by using TEM. The kinetics of the aggregation of tau peptides have been studied by following the time dependence of

Thioflavin S (ThS) fluorescence at 490 nm. They found that Ac10VME, AcPHF6*,

Ac375KLTFR, and Ac393VYK enhance the fraction of the β-structure of AcPHF6 whereas

Ac375KLTFR inhibits AcPHF6 and AcPHF6* (Figure 1.13) [85].

Figure 1.13 Amyloidogenic propensities of some tau sequences to form fiber states [85].

Adapted from “Secondary Nucleating Sequences Affect Kinetics and Thermodynamics of Tau

Aggregation,” by Moore, C. L. et al, Biochemistry, 2011, 50, p. 10878.

Also, interactions between amyloid-β and tau fragments have been studied by Thanhand et al. In this study, the interaction between an Aβ (25−35) and tau (273−284) fragment 22

has been investigated by using a combination of ion-mobility mass spectrometry (IM-

MS), atomic force microscopy (AFM), and computational modeling by probing the conformations of early hetero oligomers and the macroscopic morphologies of the aggregates. As a result, tau (273−284) showed a high affinity to associate with

Aβ(25−35) monomers rather than other tau monomers to form hetero oligomers. The hetero-tau oligomers can produce granular aggregates where they can be trapped at relatively small size while hetero-Aβ oligomers containing β-rich content can form heterofibrils. This study predicts that the ability of Aβ oligomers to interact with tau monomers may be the possible source of toxicity [86].

In another study, Bader and et al have studied the influence of bivalent ions (such as

Cu2+, Zn2+, Mn2+, Ca2+, Mg2+), trivalent metal ions ( Fe3+ and Al3+), and organic solvents like DMSO on tau aggregation [87]. Fluorescently labeled aggregates have been detected by employing fluorescence correlation spectroscopy (FCS) and scanning for intensely fluorescent targets (SIFT). This study demonstrated that trivalent metal ions and organic solvents like DMSO induced distinct tau oligomers, while bivalent ions had no effect. It also indicated that tau oligomers induced in DMSO are small and relatively stable [87].

Additionally, Barrantes and et al have followed tau-tau interaction in the absence of any aggregation inducer [88]. Following tau aggregation using AFM indicated that under physiological conditions, no fibrillar tau could be clearly observed earlier than two months. Also, the size of the filaments that were induced by the incubation of tau was smaller than that of those which are observed for PHFs purified from AD brains or that of those which are obtained from the incubation of tau in the presence of inducers such as heparin [88]. 23

All previous studies indicated that tau protein can aggregate and exhibit compact structure under various environmental conditions and the molecular mechanism by which this process happen might be different such as conformational changes, formation of intermolecular bonds and growth of aggregates.

1.1.2.4 Methods of investigation of tau protein aggregation

In the previous subsection, we have indicated different conditions where tau protein aggregates and adopts compact structures. These compact structures might differ from each other, and it is likely that in each particular case different molecular steps are involved, such as changing the conformation and structure of the tau protein. To understand this process, it is important to characterize how these steps occur. There are various techniques that are used to characterize the conformation of tau proteins. In this subsection we describe some of these techniques that are often used to study tau protein aggregation and indicate their limitations. A high-resolution analysis of the beta structure of PHFs that are made from entire tau protein is difficult to be achieved by X-ray or spectroscopic methods. After PHF assembly, only a small portion of the tau sequence adapts β-sheet structure whereas the rest of the sequence stays in a random coil conformation, so it can't be detected by X-ray [89,90,91,92]. PHFs extracted from AD brains and in vitro assembled tau fibrils have been studied by using scanning transmission electron microscopy (STEM). STEM is able to visualize the arrangement of tau within PHFs and measure the mass per length of the structure; both core and fuzzy coat will be taken into account [93]. Fibrils assembled from the three-repeat domain of tau also have been examined by using solid-state NMR (ssNMR) spectroscopy, and it

showed that tau fibrils consist of three major β-strands in repeats R1, R3, and R4, arranged in parallel [94,95]. Although these methods provided important structural details about tau aggregates, they haven't provided information about the conformational dynamics of tau protein molecules during aggregation.

Using STEM and ssNMR in studying the structure fibrils assembled of tau proteins has some disadvantages. STEM can cause damage to the sample due to the use of an electron beam. Also, they can't be used to study the sample under biological conditions. The measurements on a sample cannot be repeated many times using various conditions.

These techniques operate an ensemble averaging; they are not able to illustrate a variety of aspects implicit to individual molecules such as rare events, transient phenomena, crowding effects, population heterogeneity, etc. Also, these techniques can't be used in studying the dynamic properties of biological molecules [96]. These methods measure final structure and do not provide information about how these structures form. The collapsed structures (aggregates) can be formed by a variety of pathways. Detecting the resultant structure does not tell which pathways molecules follow and does not inform about properties of protein molecules on the path towards the aggregate. Our goal is to understand dynamic properties of aggregating molecules and characterize variability in intermolecular interactions. Consequently, we employ techniques that characterize transient interactions between molecules on a nanometer length scale.

1.1.3 Force spectroscopy

1.1.3.1 Force spectroscopy techniques.

Even though force plays an essential role in biological systems, controlling this vector quantity in ensemble experiments is hard and not accurate. Ensemble experiments consist of a large number of molecules, making the separation of relevant data from possible artifacts unattainable [97]. Most of the applied previous methods and techniques such as X-ray crystallography and solid-state NMR don't give information about molecular scale forces; instead of that, they depend on the population of the ensemble measurements. Today, a number of techniques known as single-force spectroscopy techniques (SMTs), which vary in force and dynamical ranges, are available (Table 1.1)

[98].

Table 1.1 Comparison of numbers of single-force spectroscopy techniques (SMTs) [98].

Method Force range (pN) Dynamical range Typical application

Stretching and Magnetic tweezers 0.01 - 100 ≥ 1 s twisting DNA

Action, DNA, Optical tweezers > 0.10 ≥ 10 ms molecular motors, proteins

Action, stretching, Microneedles 0.10 ≥ 100 ms unzipping and twisting DNA

DNA, proteins, AFM > 1 ≥ 10 µs ligand-receptor pairs

Single-force spectroscopy techniques (SMTs) became popular techniques in biophysical research and provide information at the level of individual molecules. Even though there are many types of single molecule manipulation techniques (SMTs), the most prominently used techniques are optical tweezers, magnetic tweezers, and atomic force microscopy [97]. Even though optical tweezers have the advantage of three-dimensional manipulation, thus performing specific interactions between a trapped object and a fixed molecule, this advantage is accompanied with a number of limitations. The main drawback and limitation of optical tweezers is its spatial resolution due to instrumental noise. Secondly, optical tweezers are limited to extremely purified sample and optically homogeneous preparation. Even though magnetic tweezers have the ability to probe torsion, they also have a number of limitations [97]. The high current required to produce the large magnetic field can generate significant heat that has a negative effect on the constant-force benefit of magnetic tweezers. Also, magnetic tweezers have limited spatial and temporal resolution.

In contrast, due to the small size of the AFM tip, the AFM performs high resolution imaging, relatively high-force pulling, and interaction assays [97]. AFMs have the high signal-to-noise ratio that is important in single molecule studies. An AFM also has the ability to measure inter- and intramolecular interaction force with piconewton resolution

[99]. In contrast to optical and magnetic tweezers it allows to localize interactions with much higher accuracy and perform measurements on sub-millisecond time scale.

1.1.3.2 Principles and applications of atomic force microscopy

The method that we develop to study the conformational dynamics of unfolded proteins uses an atomic force microscope (AFM) in the force measurement modality as the main tool. The AFM is considered to be a powerful structural analysis tool that can follow the most interesting features of biological molecules such as their assembly patterns, dynamic behavior or ability to interact with other molecules [100]. It also measures surface forces with very high spatial, force, and chemical resolution [101]. AFM is not only a powerful tool for mapping the surface topography of a sample but also for studying the elasticity, viscosity, hydrophobicity, and the strength of chemical bonds of macromolecules [102,103]. The AFM can be used to study biologically functional complexes without significant chemical modification or mechanical damage of bioparticles [100]. The AFM offers several advantages including: a small amount of required sample, the sample preparation process is often simple, and the measurements can be performed under physiological conditions [104]. Measurements on the same sample can be repeated many times under different conditions.

The AFM has been used to study the morphology of fibrils, their growth and assembly, and the mechanism of the aggregation process. So far, the aggregation of numerous amyloidogenic proteins such as α-synuclein, Ab-peptides, amylin, IgG light chain and insulin have been studied by using AFM techniques [100]. Also, AFM can be used to measure the forces within as well as between molecules. Figure 1.14 illustrates some applications of AFM including protein-surface interaction [105], investigate the strength of receptor-ligand binding [106], mechanical properties related to unfolded of

multidomain proteins such as titan [3], and study the interactions between different functional groups such (CH3-COOH, CH3-CH3 , COOH -COOH, etc.) [107].

Figure 1.14 Some applications of AFM. a) The interaction force between tethered fibrinogen molecules and the glass surface [105]. b) Investigating binding between enzyme-dockerin and cohesin [106]. c) The process of unfolded titin protein domain [3]. d) The interactions between different functional groups [107].

The AFM operates by allowing a tiny tip with radius of curvature on a nanometer scale attached to the free end of a microfabricated cantilever to move over the sample of interest. Most AFMs use a laser beam which is aimed at the end of the cantilever and

reflected onto a split photodiode as a method to detect tip movement (optical lever technique). As a result of the force acting on the tip, the cantilever bends, and the laser beam deflects [108,109]. The curve of deflection of the cantilever (d) versus the scanner displacement (z) is measured. Then it is transformed into a force–distance curve where the cantilever deflection converts into a force (F) using Hooke’s law.

퐹 = 푘c푑 (1.2)

where kc is the cantilever spring constant [110]. The tip-sample distance (x) is obtained by using the following equation (illustrated in Figure 1.15):

푥 = 푧 − 푑 (1.3) where z is the displacement of the AFM probe (measured by AFM z-scanner) and d is the deflection of the AFM probe (measured using optical lever technique).

Tip z x

Figure 1.15 Illustration the calculation of tip-sample distance (x).

Figure 1.16 illustrates the principle of the atomic force microscopy and force-distance curve obtained from one approach-retraction cycle of the tip from the sample [111]. The

atomic force microscopy has developed into a multi-functional toolbox that is used for imaging and quantification of mechanical properties such as adhesion, deformation,

Young’s Modulus, and energy dissipation [111].

Figure 1.16 Schematic showing the basic of measurements of an AFM instrument (a) Pixel-for- pixel force−distance curve-based AFM approaches and retracts the tip from the substrate to record interaction forces, F, over the tip−sample distance. (b) The approach (blue) - retraction

(red) cycle of the tip that is converted into a force- distance curve. Adapted from “Localizing

Chemical Groups while Imaging Single Native Proteins by High-Resolution Atomic Force

Microscopy,” by Pfreundschuh, M. et al, Nano Letters, 2014, 14, p. 2958.

The majority of physico-chemical measurements deal with huge ensembles of biomolecules. In these experiments, the interactions of smaller subpopulations with various binding properties are often neglected, so the molecular mechanisms and dynamics can't be properly followed [112]. Atomic force spectroscopy (AFM) has been applied to overcome the limitations brought by the ensemble in classical methodologies.

It is able to avoid ensemble averaging, and it can characterize properties of individual molecules without contribution from the rest of the ensemble. Single molecule force spectroscopy (SMFS) offers methodology to study mechanical properties of macromolecules such as characterizing protein functions, conformations, and activities under physiological conditions [112]. Molecules of interest can be tethered by flexible linkers onto AFM probes and substrates using specific chemical reactions or non-specific physical adsorption. The former method is preferred because it provides adequate binding strength. The length and flexibility of the linkers allow the molecules to rotate freely and, thus reduce steric hindrance [113]. This approach prevents non-specific binding interaction such as electrostatic interaction between the tip and the substrate.

Using a long linker provides extra space and establishes a repulsive barrier between the surface and the tip that prevents possible artifacts and uncertainty data. Also, this method helps to discriminate single molecule interactions from multiple interactions by removing the steric hindrance effect, that will performed by using a mixture of two different functionalized and length linkers to effectively space the molecules of interest [97]. To enable specific attaching of molecules of interest to the surface, PEG linkers are functionalized with reactive groups on both ends. For example, the OH group termini of polyethylene glycol (PEG) don't show reactivity to protein. PEG and can be chemically activated with thiol groups that allow for covalent conjugation of PEG to protein [109].

1.1.4 Previous studies aimed at characterizing the conformational dynamic structures of IDPs using AFM comparing to other methods

Due to the high flexibility and dynamic structure of IDPs, there are a limited number of experimental techniques suitable for investigating the conformation states of IDPs, so that there are many aspects of IDPs that are still far from being fully understood.

In the study that was done using solution small-angle X-ray scattering (SAXS) from the full length constructs (the largest tau protein isoform in humans, 441 residues (ht40) and the smallest tau protein isoform, 352 residues) and their deletion revealed that the largest constructs have a radius of gyration (Rg) close to or smaller than the calculated random coil values. On the other hand, the shorter constructs containing only repeat domains

(K18, K19) have Rg either slightly or extremely larger than the calculated random coil values. This result demonstrates that the repeat domain is remarkably extended in comparison with other domains and the full length of tau. This result is compatible with the result that was shown by NMR; the repeat domain of tau has a tendency to form a partial β-structure even in a solution environment. It is therefore conceivable that the β- structure is extended and rigid, with the shape of an almost straight line. Also, this study indicated that unfolded proteins are not as random as they as supposed to be [114].

Furthermore, the influence of the environmental conditions (temperature and solvent) on the dynamic behavior of tau protein has been investigated using small-angle X-ray scattering (SAXS). Interestingly, they noted that both temperature and solvent play an important role in the dynamics of tau protein. The average protein radius of gyration (Rg) of tau protein increases with a decrease in the temperature, and the average protein Rg

undergoes a significant reduction when the temperature is raised. On the other hand, using two different types of solvents (water and methanol) provides a hint that the protein-solvent interactions play a critical role in controlling protein conformation dynamics [29].

In addition, nuclear magnetic resonance (NMR) has been used to characterize the conformational dynamics of the natively unfolded nature of 441-residue tau, including the entire backbone assignment of the tau sequence [8]. Within the MRTs, 343 out of

441 residues of the tau sequence are found in a disordered conformation. Transient elements of secondary structure (β-structure) were restricted to particular regions

256VKSKIG262 (in R1), 274KVQINKKLDL284 (in R2), 305SVQIVYKPVDL315 (in R3),

336QVEVKSEKLD345 (in R4), and 351QSKIGSL357 (in R4) (Figure 1.17). This observation supports the fact that these segments with the two motifs in R2 and R3

(274Lys-Leu284 and 305Ser-Asp315) have a high propensity to form β-structures and serve as seeds of aggregation. Moreover, other types of intermediate structure have been characterized within tau 441, including anα-helical structure and the 175TPPAPKTPPS184,

216PTPPTREP223, and 232PPKSPSSA239 in the proline-rich regions P1 and P2

[115,116,117]. However, using NMR technique only gives an average view of the overall transient structures.

Figure 1.17 Representation of the conformations of htau40 illustrates the regions of transient α- helical structure (H1[114–123] and H2[428–437]) and β-structure (B2[274–284], B3[305–315] and B4[336–345]) are shown in red and yellow, respectively. Adapted from “Structural

Polymorphism of 441-Residue Tau at Single Residue Resolution,” by Mukrasch, M. D.et al,

PLOS Biology, 2009, 7, p. 406.

Moreover, the conformational dynamics of individual unfolded protein (cytochrome

(CytC), consisting of 104 amino acids) has been controlled using electrospray mass spectrometry (ESI-MS) (Figure 1.18). By adjusting the kinetic energy and charge state, unfolded proteins were found lying on the surface as evidenced by scanning tunneling microscopy (STM). The generated conformations of unfolded protein ranged from fully extended, almost straight, strands to compact and two-dimensional refolded patches

[118].

Figure 1.18 (a) Schematic shows the experimental procedure of creating gas phase ions of several charge states ranging from z = +8 to z = +19 from a solution of unfolded CytC using electrospray mass spectrometry (ESI-MS). (b) STM images unfolded protein CytC after deposition the Cu(100) surface. Adapted from “Active Conformation Control of Unfolded

Proteins by Hyperthermal Collision with a Metal Surface,” by Rinke, G. et al, Nano Letters,

2014, 14, p. 5610.

Since four-repeated microtubule-binding domain (4RMBD) is implicated in the formation of paired helical filament (PHF), several studies have been completed on this motif to clarify its role in tau aggregation process. The transition of the 4RMBD from random structure to the helical was investigated using circular dichroism (CD). They monitored the assembly propensity of 4RMBD in a Tris-HCL buffer with various 36

concentrations of trifluoroethanol (TFE) (Figure 1.19). It was indicated that a10-30% content of TFE in the buffer activates a state of 4RMBD from a random structure to a helical structure [119].

Figure 1.19 CD spectra of 4RMBD in a buffer of Tris-HCL with different contents of trifluoroethanol (TFE). Adapted from “Conformational Transition State is Responsible for

Assembly of Microtubule-binding Domain of Tau Protein,” by Hiraoka, S. et al, Biochemical and

Biophysical Research Communications, 2004, 315, p. 661.

Even though, the applied previous methods and techniques provided important information about the conformational properties of various types of IDPs, they do not provide atomic-level details on conformations like they typically do for globular folded proteins. It is likely that obtaining an atomically detailed description for IDPs is unattainable because they are natively unfolded and don't adopt a fixed and rigid 37

structure. For that reason, a new methodology is needed to characterize and define adequately the conformational diversity of IDPs at the molecular level.

Recently, conformational changes of proteins have been getting more attention as a possible early step in the pathology of neurodegenerative diseases. Yu et al. have applied a single-molecule AFM probing method to study the effects of Al+3 and Zn+2 on the early stages of α-synuclein aggregation. Also, they performed experiments under acidic conditions without and in the presence of metal cations. The forces and contour lengths were measured at neutral and acidic conditions. Figure1.20 illustrates the experiment for the interaction between monomeric α-synuclein molecules. The results of this study showed that the interaction between monomeric α-synuclein molecules increases dramatically in the presence of Al+3 and Zn+2 at pH 7.0 and then the propensity for protein aggregation increases, as well. Furthermore, Al+3 has more effect on the protein monomeric interaction than Zn+2 so that the Al+3 cation promote the aggregation of protein faster compared to Zn+2. Measuring the contour lengths provided information about the binding site of α-synuclein dimer. In the interaction of protein monomers at a neutral pH in the presence of a metal cation, the longer counter lengths were found than were found in the acidic environment. This result indicates that the acidic C-terminal region of α-synuclein contributes in the formation of α-synuclein dimerization in the presence of Al+3 and Zn+2 cations, and the conformations of protein change with the binding of the cations [120].

Figure 1.20 Illustration of the experiment for probing: (A) The unstructured conformation of α- synuclein (B) Misfolded and aggregation-prone conformation of α-synuclein. (C) The interaction between monomeric α-synuclein molecules and a rupture event at pH 7.0 in the presence of Zn2+ are shown in the curve-distance force. This figure is adapted “Nanoprobing of α-synuclein misfolding and aggregation with atomic force microscopy,” by Yu, Jet al, Nanomedicine, 2011,

7, p. 149.

Single molecule force spectroscopy has been utilized by Krasnoslobodtsev et al to investigate α-synuclein folding and the effect of single point mutation of α-synuclein

(A30P, E46K and A53T) on the propensity of α-synuclein protein to misfold. In this study, the results depend on the analysis of the distances between the AFM probe and the sample where the ruptures of molecular bonds have occurred. This distance is known as the rupture length; it is the location of a rupture peak on the force-distance curve. The analysis of rupture length gave important details on the structure of the dimer that formed with variants of α-synuclein at low pH and identified segments that are involved in the formation of the dimer. The interaction model and force-distance curve are shown in

Figure1.21. The comparison of contour lengths of mutant α-synuclein indicated that

E46K and A53T mutants increase the possibility of dimerization by establishing multiple

contacts which contribute to the stabilization of the misfolded state of the dimer, whereas the A30P segment exhibits only a single type of interaction in the dimer. They suggested that the difference in interaction patterns may offer significant details regarding the stabilization of the misfolded state of the dimer and the aggregation pathway of the protein [121].

Figure 1.21 A representative interactions between α-synuclein monomers. (A) The distance between the location of the interaction site and the C-end terminal lead to short (LC1) or long

(LC2) contour length value. (B) The positions of A30P, E46K, and A53T mutations and the interaction sites (colored arrows). (C and D) Force-distance curves indicate rupture events corresponding to LC1 and LC2. Adapted from “α-Synuclein misfolding assessed with single molecule AFM force spectroscopy: effect of pathogenic mutations,” by Krasnoslobodtsev, A. V. et al, Biochemistry, 2013, 52, p. 7383.

Lv et al have developed a single-molecule atomic force microscopy (SMFS) approach to characterize interpeptide interactions for Aβ40 and Aβ42 and corresponding mutants.

The SMFS analysis revealed that there are various interaction patterns of Aβ40 and Aβ42 within dimers. Nevertheless, the difference between Aβ40 and Aβ42 sequences is at C- termini; the N-terminal plays an essential role in the interaction and may be a potential target for intervening into Aβ aggregation. The contour length distributions of Aβ40 and

Aβ42 and corresponding mutants are shown in Figure 1.22. The dynamic force spectroscopy analysis detected those interpeptide interactions of Aβ42 is stronger than interpeptide interactions of Aβ40. The stability of the dimer is affected by the change in the peptide sequence at C-terminus. In an Aβ peptide, the N-terminal region causes its low inter-peptide interaction while the extra two amino acids in Aβ42 eliminate this effect. Additional C-terminal modification with triple substitutions (VAV) to Aβ40 modulates its aggregation propensity, causing it to be similar to Aβ42 due to the increased interaction between the center of the sequence and the C-terminal in

[VPV]Aβ40 [122].

Figure 1.22 Histograms show the contour length distribution for (a) Ab40, (b) [VPV] Ab40, (c)

Ab42 and (d) [VPV] Ab42 dimers [122]. Adapted from “Mechanism of amyloid β protein dimerization determined using single-molecule AFM force spectroscopy,” by Lv, Z. et al,

Biochemistry, 2013, 3, p. 6.

1.5 Research statement

The major goal of my work is to characterize and extract information about large-scale motion and presence of dynamic internal structures in tau protein that is directly relevant to the initiation of the aggregation process. Even though there has been a growing body of data collected during recent years on the physicochemical behavior of tau protein, most of these data give us only a general view of the overall structures of tau protein; they do not provide the a complete picture about the mechanism at the molecular level

and the conformational motions of tau protein on large scale. Therefore, characterizing tau protein dynamic structures is physiologically relevant because these dynamics determine how protein binds to microtubules and other cellular components and provides us with information about the pathway of the mechanisms involved in the transformation of tau protein into high-ordered PHF in the presence of typical aggregation inducer.

Results reported here focus on behavior of a group of protein molecules attached to the

AFM probe, not on behavior of single molecules. Clarifying conformational transition transitions of tau protein in oligomeric states is an important step in providing insights for designing inhibitors of PHF formation that may be effective in blocking the progression of Alzheimer's disease.

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2. Chemicals and instrumentation

2.1 Materials and samples preparation

2.1.1 Choice of substrate

Choosing appropriate types of substrates and probes is a key step in studying interactions using an AFM and getting reliable results. Immobilization of biological molecules is still a challenging part of AFM studies. Over the past years, a number of substrates were developed for the immobilization of biological molecules such as mica, silicon, and glass.

Surfaces coated with metals such as gold, silver, chromium and copper are also used. To perform this study, we chose to use two kinds of substrates to prepare the samples: a cleaved mica substrate and a gold-coated substrate.

2.1.2 Cleaved mica

Mica is composed of layers of negatively charged aluminosilicate. Each aluminosilicate layer is composed of tetrahedral silica sheets sandwiching octahedrally coordinated aluminum atoms (Figure 2.1). The tetrahedral coordinated sheets are separated by an interlayer of potassium cations, and they are held together by electrostatic and vander

Waal forces. Those crystals exhibit a large degree of basal cleavage, allowing them to be split into atomically flat sheets. Mica crystal shows a large degree of basal cleavage [1].

Mica can be easily cleaved using razor blades or scotch tape, where single or multiple sheets are removed. Negative charges at the mica-water interface are generated by

+ + dissolving the cations (K , Al3 ) in a liquid. The freshly cleaved surface of mica is atomically smooth and hydrophilic, and its negative charge is stable under a range of

physiological pH values. These attractive features of mica make it suitable for AFM study of various biological objects [1,2].

Figure 2.1 (a) Inside view of mica structure (b) Top view of the cross-section top view of mica made in the plane with K atoms. The unit cell of the quasi-hexagonal lattice has the dimensions: a

= 0.52 nm and b = 0.9 nm [2].

2.1.3 Gold-coated substrate

Gold is a mostly chemically inert metal; it doesn’t react with large set of chemicals, and it is not affected by ambient conditions. In particular, gold is chemically inert to oxygen and radicals. Its chemical inertness makes it one of the most used substrates for the preparation of samples for use with an AFM. On the other hand, flat gold substrate is suitable for the formation of self-assembled monolayers (SAMs) of organic alkanethiols as illustrated in Figure 2.2. It has an affinity to form stable gold-thiol bonds with proteins containing the amino acids methionine and cysteine. Then, it is accessible to chemisorption [3].

Figure 2.2 Self-assembled monolayers (SAMs) of organic alkanethiols on gold substrate.

2.1.4 Choice of the probe

The AFM probe is typically made of silicon or silicon nitride. For specific applications, the probe is required to be coated with specific types of metals, such as gold and aluminum. Coated AFM tip scan be further equipped with desired types of molecules.

Such chemically modified tips can be used to measure the strength of molecular interactions. In this approach, the gold-coated tips were used to chemically immobilize tau protein molecules.

2.1.5 Tau protein sample

In this study, a mutant tau protein fragment (tau 255-441) consisting only of (4R) repeat domains and C- terminus was used (Figure 2.3). The used fragment has the same assembly units as tau 441 but without the N-terminus and proline–rich region. In order to attach tau protein to a gold-coated tip, additional cysteine residue is added at the C- terminus. The tau protein attaches to the gold-coated tip through the thiol group by forming gold-thiol bonds. Also, the tau fragment contains an additional six histidine residues at the N-terminus region. Adding six histidine residues to the tau fragment 62

changes the overall charge 44 from (-2) to (+9) at neutral pH. The positive net charge enhances the interaction between the tau fragment and substrates and makes characterizing the conformation of the tau protein easier to detect.

N N1 N2 Proline rich R1 R2 R3 R4 C Tau 441

R1 R2 R3 R4 C Mutant tau 255 - 441

Figure 2.3 Sequences of tau 441 isoform and the mutant fragment (255-441).

2.1.6 Materials

A mutant fragment of tau441 protein and heparin have been provided by Dr. Han’s group from the University of California, Santa Barbara [4]. The following commercial materials were purchased from different companiesand utilized as received. Phosphate buffer saline (PBS) (pH 7.2) was obtained from Thermo Scientific (Rockford, Illinois), and ethyl alcohol (absolute, 99.5+ %) was obtained from Across Organics (Morris, New

Jersey).Methyl alcohol (99.8 %), potassium hydroxide (pellets, 85+ %), toluene (99.5 %),

MUA (11-mercaptoundcanoic acid), MU (11-mercapto-1-undecanol)) and EDC ((N-3- dimethyl-aminopropyl)-N-ethylcarbodiimidehydrochoride)) were purchased from Sigma-

Aldrich (St. Louis, Missouri). Isopropanol and hydrochloric acid (1 N) were obtained from Fisher Scientific (Fair Lawn, New Jersey).

2.1.7 Mica substrate modification

A 22 mm x 22 mm mica substrate (manufacturer SPI Supplies, West Chester,

Pennsylvania) was cleaved more than one time with an adhesive tape. The freshly 63

cleaved mica surface was treated with a PBS (pH 6.9) to obtain a negatively charged surface due to dissolving the cations (K+, Al3+).

2.1.8 Probe preparation

For the chemical attachment of tau protein to the tip, two types of commercially gold- coated tips were used. The first type, the CSG10 probe, was purchased from NT-MDT

Spectrum Instruments, Tempe, Arizona. The CSG10 probe (manufacturer NTEGRA

Spectra II) was made from silicon and coated with Au with a thickness of 35 nm. The second type, the RC800PB probe (manufacturer Olympus, Waltham, Massachusetts), was made from silica nitride and coated with Cr and Au with thicknesses of 5 and 40 nm, respectively. Those two types of gold-coated probes were modified with tau proteins in two different ways, depending on the method of cleaning the gold-coated probe.

2.1.8.1 Modification of the gold coated probe directly with tau protein without cleaning with solvents.

In the first method, a gold-coated probe was cleaned only with UV radiation by placing it one inch from a UV source (mercury lamp, United Visual Products (UVO), 11sc-1) for 2 h in the laminar flow hood. Next, the probe was fixed into the custom-made stainless steel probe holder and then placed into a clean 250 ml Hamilton syringe (Hmilton

Company, Reno, NV). The mutant tau protein solution (15 µg/ml) was prepared by diluting the stock protein solution (7.15 mg/ml) with PBS (pH 6.9). After that, the gold- coated probe was soaked in a diluted solution of the mutant tau protein (15 µg/ml) for 18 h at room temperature (Figure 2.4). Then, the coated probe was thoroughly rinsed by 64

sequential following the following solvents through the syringe that holds modified probe: 100 ml of chloroform, 100 ml of absolute ethanol, and 100 ml of DI water. The flow rate of solvents was limited to 0.8ml/min to prevent probe damage by hydrodynamic flow. Finally, the modified coated probe was dried with a stream of nitrogen for 30 min and put in a desiccator under vacuum overnight. The probe modified with the mutant tau protein was tested using a mica substrate.

Figure 2.4 Modification of gold-coated tip directly with tau-mutant protein. (A) Cleaning gold- coated probe (B) Fixing the cleaning probe into the home-built stainless steel probe holder (C)

Placing the probe holder with probe into a clean 250 µl Hamilton syringe (D) Soaking the gold- coated probe in a diluted solution of the mutant tau protein (15 µg/ml)for 18 h at room temperature.

2.1.8.2Modification of the gold coated probe with tau protein after cleaning the probe with solvents.

In this method, the gold coated probe was cleaned with three types of solvents before use.

First, the gold-coated probe was cleaned with UV radiation by placing it one inch from a

UV source (mercury lamp) for 1 h in the laminar flow hood. Then, the gold-coated probe was fixed in a specific type of metal clips, suspended in a 100 ml beaker, and washed three times with 100 ml of hexane, 100 ml of isopropyl alcohol and 100 ml of DI water, in that order, on an orbital shaker (New Brunswick Scientific, INNOVA 2000, Edison,

New Jersey), for 2 hrs each at 90 rpm. The probe was fixed into the probe holder, placed into a clean 250 ml Hamilton syringe, soaked in a diluted solution of (15 µg/ml) of the mutant tau protein for 4 hrs at room temperature. Then, the modified gold-coated probe was thoroughly rinsed with 100 ml of chloroform, 100 ml of absolute ethanol, and 100 ml of water. Finally, the modified coated probe was dried with a stream of nitrogen for 30 min and put it in a desiccator under vacuum overnight. The probe modified with the mutant tau protein was tested using a mica substrate.

Gold – coated tip Gold – coated tip M-tau 1. Cleaning

2. Soaking with M- tau for 4 hrs.

3. Washing

Figure 2.5 Modification steps of gold-coated tip with tau-mutant protein.

2.1.9 Modified gold-coated substrate with heparin

2.1.9.1 Cleaning the gold-coated substrate

First, the gold-coated substrate was irradiated with UV radiation from a mercury pen lamp for 1 hr. The distance between the probe and the radiation source was set at one inch. Then, the gold-coated substrate was fixed in a metal clip, suspended in a 100 ml beaker, and washed with isopropyl alcohol and DI water on a shaker for 2 hrs each at 90 rpm (Figure 2.6). Finally, it was dried with a stream of nitrogen for 5 min.

Figure 2.6 Cleaning of gold-coated substrate using solvents by suspending it in beaker and using shaker.

2.1.9.2 Attaching of MUA and MU to the gold-coated substrate

Attaching of the heparin molecules to the cleaned gold-coated substrate was performed using a mixture of two different functionalized polymers, ((1:3) MUA (11- mercaptoundcanoic acid) and MU (11-mercapto-1-undecanol)), dissolved in methyl alcohol. The immobilization was done by incubating the gold substrate in the mixture solution overnight at room temperature using custom-manufactured Teflon substrate holder to minimize solution volume and prevent sample contamination as shown in

Figure 2.7. After that, the modified substrate was washed three times with DI water overnight on a shaker with 90 rpm.

Figure 2.7 (A) Gold-coated substrate (B) Modification of the gold-coated substrate with sample using home-built polytetrafluoroethylene reaction vessel.

OH COOH OH OH OH COOH

MUA + MU (1:3) S S S S S S Gold-Coated substrate Incubation overnight Gold-Coated substrate

Figure 2.8 Modification of gold-coated substrate with mixture of MUA and MU molecules.

2.1.9.3 Activation of the carboxyl group in the MUA molecules

To facilitate attaching heparin to the gold-coated substrate, the carboxyl groups (-COOH) of the MUA molecules were activated using EDC ((N-3-dimethyl-aminopropyl) -N- ethylcarbodiimidehydrochoride)) as a cross-linker. The EDC was used to covalently connect the heparin molecules to the gold-coated surface. The modified substrate was incubated into the EDC solution (1mg/ml) in DMF at 4 °C for 4 hrs. After that, the

modified gold-substrate was washed with DI water for 2 hrs on a shaker with 90 rpm.

Figure 2.9 illustrates this step.

H H H H EDC + + 3HC N N N CH3 3HC N N N CH3 C C CH 3 CH3 O O

| | || || OH COOH OH OH OH COOH OH C O OH OH OH C || O

MU MUA MU MUA EDC S S S S S S Incubation for 4 hrs S S S S S S Gold-Coated substrate Gold-Coated substrate

Figure 2.9 Activation of the carboxyl groups (-COOH) of MUA molecules using EDC.

2.1.9.4 Conjugation of heparin molecules to EDC cross-linker

The heparin molecules were chemically attached to the carboxyl groups (-COOH) of the

MUA molecules using EDC as a cross-linker. The heparin solution (20 µM) was prepared by diluting the stock solution (100 µM) with a PBS (pH 6.9). The modified substrate was incubated into 20 µM of heparin solution for 24 hrs at room temperature.

Then, the sample was washed with DI water three times for 6 hrs. Finally, the sample was dried by a stream of nitrogen and put it in desiccators under vacuum overnight. This step is illustrated in Figure 2.10. Note that in this figure size of heparin molecule is not to scale.

H H H H + + HC N N N CH 3HC N N N CH 3 EDC 3 3 Heparin C C CH CH 3 3 | | O O N N

| | | |

|| ||

|| || || || OH C O OH OH OH C O OH C O OH OH OH C O

MU MUA MU MUA Heparin 11kDa Incubation overnight S S S S S S S S S S S S Gold-Coated substrate Gold-Coated substrate

Figure 2.10 Conjugation of heparin molecules to EDC cross-linker.

2.2 Force spectroscopy Force measurements were conducted with an Asylum Research (Santa Barbara, CA)

MFP-3D AFM. The initial tuning will be performed before collecting the data [5]. The spring constant of each tip was calibrated by the thermal noise analysis method in air

[6,7]. To limit the force applied to the modified gold-coated tip, a relative trigger threshold of 10 nm was applied on all cycles. All measurements were carried out at room temperature and with force map scan size of 20 μm. This scan size was selected so that when collecting the force maps AFM probe contacts samples fresh area of the surface.

2.3 Data collection and analysis Data collection started with loading a functionalized probe on the AFM instrument. The laser beam was adjusted at the end of the cantilever until we got the highest sum (Figure

2.10) [8]. To convert the cantilever deflection measurements to force, the curve of deflection of the cantilever (d) versus the scanner displacement (z) was measured in air on a clean glass substrate. First, the slope for the force-distance curve was measured which represents the sensitivity of the used cantilever. Then, the spring constant of the cantilever was obtained using a thermal method. All measurements were carried out in a liquid environment using PBS (pH 6.9). The pH value of PBS was adjusted using potassium hydroxide or hydrochloric acid solutions and monitoring the pH value with a pH meter (Denver Instrument UltraBasic, Bohemia, New York). The substrates (cleaved mica and modified gold substrate) were mounted on the disk piezo of AFM. The large number of force curves (~600 curves for each experiment) was collected to ensure adequate statistics. At the maximum compressive load the AFM probe remained in contact with the surface for the pre-defined contact time. This contact time (below called

the dwell time) between modified gold-coated tip and substrates was varied during measurements in order to detect time-dependent changes in tip-sample interaction during the tip-sample contact. The force-distance curves of the interaction were collected at four values (0.0 s, 0.1 s, 0.3 s and 1.5 s) of dwell time.

To get more information about the conformation of tau protein, the modified tip was also moved towards and away from the surface at different rate values. The probe was moved up and down from the substrate with four values (0.5, 1, 1.5 and 2Hz) of scan rate.

Systematically changing the contact times is important in this work because when the protein molecules spend a relatively long time in contact with the surface, it will give them time to reorganize. Changing the scan rate allows to detect dynamic characteristics of unbinding process.

Photodiode

I1 I2 I3 I4

Cantilever

t h L

Figure 2.10 Tuning the laser beam of AFM [4].

The curves of the deflection of the cantilever (d) versus the scanner displacement (z) were collected and analyzed by custom-written code implemented in Matlab program.

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3. Analysis of rupture forces

In our work, we are attempting to carefully analyze the collected force curves and to look at various aspects of the interaction that we can extract. The ideal curve consists of four basic regions: An adhesion peak due to the short range of nonspecific interaction between the tau and the surface, a gradual increase in force region due the stretching of the attached tau molecule, a complete rupture event point, and finally, the region where the tip comes free from the surface.

150

100

0 N p

, e

c -50 r

o Rupture force ( F ) F -100

-150 Stretching Rupture point -200 slope Separation distance ( d ) -250

-300 0 10 20 30 40 50 60 70 80 90 100 Separation, nm

Figure 3.1 Typical force curve with multiple rupture events obtained from the interaction of tau protein molecules with mica shows the variables that extracted from the collected force-distance curves. The red curve represents the approach process of tip from surface, and the blue curve represents the retraction process. The retraction curve appears with multiple rupture events. The red arrow indicates to the value of rupture force (F), the green arrow indicates to the values of separation distance (d), and the black arrow indicates to the magnitude of the spring constant.

From the collected force curves, three different variables were obtained. First, we characterized the force (F) which shows how strongly molecules interact. Then, we determined the separation distance (d), because it corresponds to the length of the tau protein molecule. Also, we characterized the stretching slope that reflects on the spring constant of the molecules before the rupture. The higher is the stretching slope the stiffer is the molecule. Zero of negative values of the stretching slope indicate “sliding” contact.

We considered the force curve with multiple rupture events as shown in Figure 3.1.

3.1 The selecting sample preparation procedure

To characterize the conformational dynamics of tau protein molecules, a series of experiments were carried out in this study. Typically, most of the methods that have been done by others to study the dynamic properties of proteins have been based on using a single molecule approach. In the single molecule approach, molecules of interest are tethered via specific types of linkers. In this way, the extracting force-distance curves are related to the interaction for the two molecules (one on the tip and the other attached to the surface). Typically, the interaction between the members of that pair will show only one rupture event on the force-distance curve [1] (Figure 3.2, A). On the other hand, the idea in our work was not to use a single molecule approach. Instead of that, we used a multiple molecule approach. Because of that, the extracting force-distance curves consist of multiple rupture events [2] (Figure 3.2, B).

Figure 3.2 Shows the force-distance curve of the single molecule approach (A). Adapted from

“AFM for Analysis of Structure and Dynamics of DNA and Protein-DNA Complexes,” by

Lyubchenko, Y. L. et al, Methods, 2009, 47, p. 212. And the multiple molecules approach (B).

Adapted from “Quantifying Interactions between DNA Oligomers and Graphite Surface Using

Single Molecule Force Spectroscopy,” by Sara, L. et al, American Chemical Society, 2012, 116, p. 13901.

3.1.1 Examination the cleaning of the AFM gold-coated tip

It is worthwhile to note that obtaining relevant and accurate results are related to using appropriate procedures to prepare the samples. The first step in the preparation of samples is to test the cleaning of the AFM probe. Tau protein molecules were covalently attached to an AFM probe as described above in the experimental part (Figure 2.5). First, tau molecules were attached directly to a commercial gold-coated tip after it was cleaned with UV radiation using a mercury lamp but without using any type of solvent. The mica substrate was cleaved and used directly. The interactions between covalently attached tau molecules and cleaved mica substrate were probed in multiple approach-retraction

cycles (~600 force-distance curves) with buffer at pH 6.9 at different dwell times (0 s, 0.1 s, 0.3 s and 1s). Force-distance curves were acquired to characterize both the strength of the adhesive interaction force between the tau and the mica surface and the separation distance between the tip and the surface, which reflects directly the length of the tau protein molecules.

A B Mean 98.2 | STD 24.2 1000 160 Position 94±1.1 nm Width 22.2±1.2 nm 140 500 Amplitude 134±5.9 120 N

p 0

, 100 e c r o # F -500 80

60 -1000 40 -1500 20

-2000 0 0 20 40 60 80 100 120 40 60 80 100 120 140 160 Separation distance, nm nm

Figure 3.3 (A) Illustrates the force distance curve where the red line represents the approach process of the tip from mica substrate, and the blue curve represents the retraction process of tip from surface (B) The Gaussian distribution of the interaction of the tau protein and the mica surface before the cleaning.

Figure (3.3) shows the force-distance curve and the Gaussian distribution of the tau protein and the mica substrate, when the retraction velocity was 1000 nm/s (frequency of approach/ retract cycle 1 Hz, scan size is 500 nm). From this experiment, we obtained the force-distance curve with multiple rupture events. The force curve indicates that there are adhesive force events at long distances between the tau molecules and the mica surfacce (more than 100 nm). This distance is longer than the fully extended length of

the fragment of the tau protein (~68 nm). The very long separation distance on the force curve indicates the maximum possibility of accumulation of contamination materials on the AFM tip. The contamination materials may gather and form a bridge leading to exceeding the expected separation-distance length.

To examine the effect and the presence of contamination materials on the gold-coated tip, we collected multiple force-separation distance curves of the interaction between the free gold-coated tip (an unmodified gold-coated tip) vs. the cleaved mica substrate before and after cleaning the gold-coated tip (Figure 3.4). The free gold-coated tip was cleaned with

UV radiation and three types of solvent (toluene, isopropanol and DI water). Astatistical analysis of the adhesive force values were measured for a set of force-distance curves. In the case of the interactions between the non-cleaned free tip and cleaved mica, we noted that there is a small range of adhesive force (~150 pN). On the other hand, cleaning the gold-coated tip resulted in cancellation of any adhesive force between the tip and the cleaved mica (Figure 3.3.B). From this experiment, we realized that, for the samples measured with AFM, cleaning is required during the sample preparation in order to reduce possible artifacts.

A B

100 100 N p

, 0 e N c p r

, o e F c r

-100 o -100 F

Rupture force ( F ) -200 -200

0 50 100 0 50 100 Separation distance, nm Separation distance, nm

Figure 3.4 The force - distance curve of the interaction of the free gold-coated tip with mica before (A) and after cleaning (B). The red curve represents the approach process, and the blue curve represents the retraction process. The retraction curve in (A) appears with multiple rupture events (brown lines).

3.1.2 Examining the effect of the soaking time of the AFM gold-coated tip with tau protein solution on extracting the separation distance.

Determining proper soaking time required to attach the mutant tau protein sequence to the gold-coated tip was required to prepare samples that exhibit noticeable number of force curves with tip-sample interactions in the length range corresponding to the size of single protein molecule. To determine soaking time, the cleaned probe was soaked with a diluted solution (15 µg/ml) of the mutant tau protein in PBS overnight at room temperature to immobilize the protein chemically on the gold-coated tip. By using this modified tip in the measurement, we achieved a short separation distance (~27 nm), that may be due to the formation of intermolecular cross-linking between tau protein sequence units during the soaking time.

Next, we used the same procedure, but the soaking time was reduced to 4 hrs. The results of the separation distance in the collected force curves using this procedure were consistence with the fully extended length of sequence of the tau protein fragment (~68 nm).

3.2 Studying the interaction of tau protein with a freshly cleaved mica surface

3.2.1 Using different concentrations of heparin solution (1 μM, 10 μM and 20 μM)

Tau protein fragments were chemically attached to a gold coated-tip as mentioned previously in the experimental section. The experiments were performed using freshly cleaved mica substrate. The interactions between tau protein molecules and a negatively charged mica surface were probed in multiple approach-retraction cycles in two environments. First, the interaction took place between the tau protein and mica with a

PBS (pH 6.9) (the control experiment). Second, the interaction was between tau protein molecules and mica using various concentrations of heparin solution (1 μM, 10 μM and

20 μM). Finally, we repeated the control experiment to examine the reversibility of heparin effects. In these experiments, the force-distance curves of the interaction of tau protein molecules and mica surface are measured as a function of the probe dwell time and scan rate. The experimental setup is shown in Figure 3.5.

Gold-coated tip Gold-coated tip M - tau d 0 Gold-coated tip d M - tau 1 M - tau d 2

Mica Mica Mica

A. The probe far away from the B. With buffer C. With heparin solution surface

Figure 3.5 Scheme of experiment setup to the interaction of the tau protein and mica surface (A)

The modified probe is far away from mica surface (B) Interaction of the tau molecules with mica in PBS solution (pH 6.9) & (B) Interaction of the tau molecules with mica in an 11 kDa heparin solution.

Starting with a low concentration of heparin, an 11 kDa fragment solution (1 μM) with 1s of dwell time, the histograms of separation distance show shifting in the separation distance from (~49.7 nm) to (~41 nm) by using heparin (1 μM) (Figure 3.6). Also, the histograms illustrate decreasing in the population of rupture events in the heparin environment. Substituting the heparin solution with buffer for the second time gave us a histogram with a mean value separation (~49.4nm) similar to the original value of the separation distance in the Figure 3.6.A. However, number of detected rupture events further decreased, indicating that decreased binding probability in Figure 3.6.B might be unrelated to heparin incubation.

A 250

200 With buffer (pH 6.9) 150 # 100 50 0 0 20 40 60 80 100 120 B 250

200 1 µM heparin 11 kDa 150 # 100

50 0 0 20 40 60 80 100 120

C 250

200 With buffer (pH 6.9) 150 # 100

50 0 0 20 40 60 80 100 120

Separation distance, nm

Figure 3.6 Histograms of separation distances of the interaction between the tau protein and freshly cleaved mica with a PBS (pH 6.9) (A, C) and an 11 kDa heparin solution (1 μM) (B) with

1 s dwell time. Data were collected in sequence of steps (A→B→C).

On the other hand, the mean values in the histograms of the interaction force between tau protein molecules and mica using both a buffer solution and a heparin solutions didn't change (the mean value with a buffer is ~209 pN and ~206 pN with a heparin solution), but the population of high-force events (>100 pN) decreased slightly when returned back to the buffer solution (Figure 3.7).

300 With buffer (pH 6.9) 200 #

100

0 0 100 200 300 400 500 600 700

B 300 With 11 kDa heparin solution (1 µM)

200 #

100

0 100 200 300 400 500 600 700

C 300 With buffer (pH 6.9) 200 #

100

0 0 100 200 300 400 500 600 700

Force, pN

Figure 3.7 Histograms of interaction forces of the interaction between the tau protein and freshly cleaved mica with a PBS (pH 6.9) (A, C) and an 11 kDa heparin solution (1 μM) (B) with 1 s dwell time. Data were collected in sequence of steps (A→B→C).

The measurements of the spring constant of stretched tau protein before the rupture show that when using a heparin solution there is a slight shifting in the histogram to the negative region (B) leading to an increase in the mean value from ~7 pN/nm using a buffer solution to ~9 pN/nm using the heparin solution. This change is not very

significant, but it occurs with visible shift of the entire peak towards higher values. This indicates that in presence of heparin tau molecules became slightly stiffer. The recovery of the stretching spring constant to the almost original value in panel C indicates that the tau protein molecules didn't change irreversibly after exposure to 1 M solution of heparin solution.

500

With buffer (pH 6.9)

# 250

-40 -30 -20 -10 0 10 20 30 40

500 With 11 kDa heparin solution (1 µM)

# 250

0 -40 -30 -20 -10 0 10 20 30 40

C 500

With buffer (pH 6.9)

# 250

0 -40 -30 -20 -10 0 10 20 30 40

Spring constant, pN/nm

Figure 3.8 Histograms of spring constant of the interaction between the tau protein and freshly cleaved mica with a PBS (pH 6.9) (A, C) and an 11 kDa heparin solution (1 μM) (B) with 1 s dwell time. Data were collected in sequence of steps (A→B→C).

Graphs of the average separation distance vs. dwell time illustrate that the separation distances became shorter when tau protein is exposed to 1 M heparin solution and returned to almost the same values after the heparin molecules were washed out.

Dependence of rupture forces on presence of heparin is smaller, particularly at 0.0 s and

0.1 s dwell time. The interaction forces before adding heparin solution and after removing heparin solution remain nearly the same. Similar results are observed for the spring constant as can be observed in Figure 3.9. These data indicate that addition of 1

M heparin solution results in small changes in interaction parameters that recover back to original values after heparin solution is removed.

A B

100 500 Tau with buf (1) Tau with buf (1) Tau with 11 kDa hep. Tau with 11 kDa hep 80 400 Tau with buf (2) Tau with buf (2)

60 300

40 Force,pN 200

SeparationDistance, nm 100

0 0 0 0.2 0.4 0.6 0.8 1 1.2 0 0.2 0.4 0.6 0.8 1 1.2 Dwell Time, s Dwell Time, s

30 Tau with buf(1) Tau with 11 kDa hep Tau with buf (2) 20

SpringConstant,pN/nm 0

0 0.2 0.4 0.6 0.8 1 1.2 Dwell Time, s

Figure 3.9 The plotting of dwell time (s) vs. (A) separation distance (nm), (B) force (pN) and (C) spring constant o of the interaction between the tau protein and freshly cleaved mica with a PBS

(pH 6.9) and an 11 kDa heparin solution (1 μM).

Measurements with different concentrations of 11 kDa heparin solution (10 μM and 20

μM) were performed, and the data were collected and analyzed. The histogram of the separation distance of the interaction of tau protein with mica in PBS at pH 6.9 at 1s dwell time shows the mean value of tip-separation distance of rupture events is ~ 41 nm

(Figure 3.10.A). This value of separation reduced to ~36 nm when the buffer solution was replaced with a 10 μM of heparin solution (Figure 3.10.B). On the other hand, the distribution of interaction forces between the tau protein molecules and mica in buffer solution displays higher interaction force, with a maximum value of ~130 pN, and the substitution of the buffer solution with 10 μM of a heparin solution resulted in the disappearance of the higher range of the adhesive forces. The shifting of the interaction force distribution to the low region might be explained by noting that increasing concentration of the negatively charged heparin molecules which are known to bind to the tau protein molecules and thus reducing charge on tau protein molecules (making it more negative) and thus leading to increased electrostatic repulsion between the tip and the mica surface [3]. This suggestion is consistent with disappearance of long-range events in panel B because in this case parts of tau protein molecule responsible for long- range binding in panel A might loop back and bind to heparin molecules. Measuring the interactions between the tau protein molecules on the tip and the mica again with a buffer solution (Figure 3.10.C) shows even further reduction in the mean value of rupture distance to ~26 nm. Here unlike in experiment in Figure 3.6, there is no recovery after removing the heparin. This indicates that number of heparin molecules might remain strongly attached to the tau protein molecules so that tau protein molecules remain collapsed.

A 150

100 With buffer sol. (pH6.9) # 50

0 0 20 40 60 80 100 120

150

With 11 kDa heparin sol. (10 µM) 100 # 50

0 0 20 40 60 80 100 120

150 With buffer sol. (pH6.9) 100 # 50

0 0 20 40 60 80 100 120

Separation distance, nm

Figure 3.10 Histograms of the separation distance of the interaction between the tau protein and freshly cleaved mica with a PBS (pH 6.9) (A, C) and with an 11 kDa heparin solution (10 μM)

(B) with1 s dwell time. Data were collected in sequence of steps (A→B→C).

A 150

With buffer sol. (pH6.9) 100 # 50

0 0 50 100 150 200 250 300 350 400 450 500

150 With 11 kDa heparin sol. (10 µM) 100 # 50

0 0 50 100 150 200 250 300 350 400 450 500

150

With buffer sol. (pH6.9) 100 # 50

0 0 50 100 150 200 250 300 350 400 450 500

Max force, pN

Figure 3.11 Histograms of interaction forces of the interaction between the tau protein and freshly cleaved mica with a PBS (pH 6.9) (A, C) and an 11 kDa heparin solution (10 μM) (B) with 1 s dwell time. Data were collected in sequence of steps (A→B→C).

The irreversibility of the separation at rupture upon adding of 10 μM of heparin is in contrast to the dependence of the rupture force as shown in Figure 3.11. Here it can be noticed that probability to register high rupture forces with magnitude ~200 pN and more decreases in 10 μM heparin but then recovers back upon removing of heparin. This can be reconciled with changes in separation distance by suggestion that heparin that remains 88

bound to tau protein after washing it off with PBS is probably bound not to the external surface of tau protein that coats the tip but “hides” to the lower level of tau protein.

Therefore it still affects the structure of bound tau protein but does not affect significantly interaction forces of tau protein with mica. It can be noticed that spring constant of tau protein does not change significantly when adding heparin at 10 μM concentration as shown in Figure 3.12. Similar to the behavior at 1 μM concentration, the spring constant shifts slightly to higher values upon adding of heparin. But unlike behavior at lower concentration, it remains at higher value even after heparin is removed. This is consistent with picture of irreversible change in conformation of tau protein that can be deduced from analysis of separation distance and rupture force.

200 With buffer sol. (pH6.9) 150

# 100

0 -40 -30 -20 -10 0 10 20 30 40

200

150 With 11 kDa heparin sol. (10 µM)

# 100

0 -40 -30 -20 -10 0 10 20 30 40

C 200 With buffer sol. (pH6.9) 150

# 100

0 -40 -30 -20 -10 0 10 20 30 40

Spring constant, pN/nm

Figure 3.12 Histograms of spring constant of the interaction between the tau protein and freshly cleaved mica with a PBS (pH 6.9) (A, C) and an 11 kDa heparin solution (10 μM) (B) with 1 s dwell time. Data were collected in sequence of steps (A→B→C).

Graphs of the average separation distance vs. dwell time before, during, and after removing of 10 μM solution of 11 kDa heparin show that the separation distances continue changing upon adding and after removing of heparin, similar to behavior at 1 s

dwell time discussed above (Figure 3.13.A). Changes in the average rupture forces and average spring constant are less significant (Figure 3.13.B-C).

A B

80 300 Tau with buf (1) Tau with buf (1) Tau with 11 kDa hep 250 Tau with 11 kDa hep 60 Tau with buf (2) Tau with buf (2) 200

40 150

Force,pN 100 20

SeparationDistance, nm 50

0 0 0 0.2 0.4 0.6 0.8 1 1.2 0 0.2 0.4 0.6 0.8 1 1.2 Dwell Time, s Dwell Time, s

20 Tau with buf (1) 15 Tau with 11 kDa hep Tau with buf (2) 10

SpringConstant, pN/nm -5

-10 0 0.2 0.4 0.6 0.8 1 1.2 Dwell Time, s

Figure 3.13 The plotting of dwell time (s) vs. (A) separation distance (nm), (B) force (pN) and

PBS (pH 6.9) and an 11 kDa heparin solution (10 μM).

Similar data analysis was performed for experiment with 20 μM heparin solution. The histograms reveal that in this experiment the separation distances before adding heparin are similar to other experiments range from ~20 nm to ~100 nm. But the decrease in the separation distances is more significant in 20 μM solution of heparin than in 10μM solution (compare Figure 3.14.A-B and Figure 3.10.A-B). Also, it can be noted that here probability to detect rupture events is considerably lower than in 10 μ M solution. Upon removal of heparin, the separation distance did not revert back to the original distribution of separation distances as in panel in A. Instead separation distances cluster around 91

significantly shorter value of ~30 nm. This observation implies that increasing the number of the negative charged heparin molecules increases the possibility of binding of the heparin molecules to the tau protein sequence, and because of that the detachment of the heparin became a more difficult process. The attachment of the heparin molecules induces structural and conformational changes (decreasing the separation at which tau protein separates from the surface).

150

100 With buffer sol. (pH6.9) # 50

0 20 40 60 80 100 120 B

150

With 11 kDa heparin sol. (20 µM) 100 # 50

0 0 20 40 60 80 100 120 C

150 With buffer sol. (pH6.9) 100 # 50

0 0 20 40 60 80 100 120

Separation distance, nm

Figure 3.14 Histograms of separation distances of the interaction between the tau protein and freshly cleaved mica with a PBS (pH 6.9) (A, C) and an 11 kDa heparin solution (20 μM) (B) with 1 s dwell time. Data were collected in sequence of steps (A→B→C).

This suggestion of binding of heparin molecules to tau protein is consistent with the results of dependence of the interaction force between tau protein molecules and a mica surface upon adding and removing 20 μM heparin solution (Figure 3.15). Diminishing the high range of the interaction force is due to the increasing of the negative charge on the tip which leads to an increase in the repulsion force between the tip and the mica surface. Also this suggestion is consistent with the results of the spring constant at rupture (Figure 3.16). The abundance of negative charges that can bind to tau protein greatly reduces probability to detect binding (panel B). Upon removal of heparin this probability returns and spring constant of tau protein molecules interacting with mica surface remains similar but appearance of distribution of ruptures with negative slopes in the histogram (panel C) indicates somewhat different type of interaction with mica surface upon rupture: higher probability of “sliding” rather than “stretching” contacts.

A 150

With buffer sol. (pH6.9) 100 # 50

0 0 100 200 300 400 500 600 700

B 150 With 11 kDa heparin sol. (20 µM) 100 # 50

0 0 100 200 300 400 500 600 700

C 150

100 With buffer sol. (pH6.9) # 50

0 0 100 200 300 400 500 600 700

Force, pN

Figure 3.15 Histograms of the interaction forces of the interaction between the tau protein and freshly cleaved mica with a PBS (pH 6.9) (A, C) and an 11 kDa heparin solution (20 μM) (B) with 1 s dwell time. Data were collected in sequence of steps (A→B→C).

A 200 With buffer sol. (pH6.9) 150

# 100

0 -40 -30 -20 -10 0 10 20 30 40

B 200

150 With 11 kDa heparin sol. (20 µM)

# 100

50 0

-40 -30 -20 -10 0 10 20 30 40

C 200 With buffer sol. (pH6.9) 150

# 100

0 -40 -30 -20 -10 0 10 20 30 40

Spring constant, pN/nm

Figure 3.16 Histograms of spring constant of the interaction between the tau protein and freshly cleaved mica with a PBS (pH 6.9) (A, C) and an 11 kDa heparin solution (20 μM) (B) with 1 s dwell time. Data were collected in sequence of steps (A→B→C).

These dependencies similar to those that are discussed above for dwell time of 1 s can be observed for other dwell times as illustrated in graphs of the behavior of average parameters vs. dwell time that are displayed in Figure 3.17.

A B

120 Tau with buf (1) 600 Tau with buf (1) Tau with 11 kDa hep. 100 Tau with 11 kDa hep Tau with buf (2) 500 Tau with buf (2) 80 400

60 300

40 Force,pN 200

Separationdistance, nm 20 100

0 0 0 0.2 0.4 0.6 0.8 1 1.2 0 0.2 0.4 0.6 0.8 1 1.2 Dwell Time, s Dwell Time, s

C 30 Tau with buf (1) Tau with 11 kDa hep 20 Tau with buf (2)

0 Springconstant, pN/nm

-10 0 0.2 0.4 0.6 0.8 1 1.2 Dwell Time, s

Figure 3.17 The plotting of dwell time (s) vs. (A) separation distance (nm), (B) force (pN) and

PBS (pH 6.9) and an 11 kDa heparin solution (20 μM).

The separation distance distributions of using various concentrations of heparin solution

(Figure 3.6, Figure 3.10 and Figure 3.14) reveal that increasing the concentration of heparin solutions induced more reducing in the separation distance at which tau protein ruptures from the surface, and that process of the length reduction becomes irreversible using higher concentrations of heparin solution.

3.2.2 Using 18 kDa heparin solution

In our work we also examined the effects of a high molecular mass of heparin on the distribution of rupture parameters. We collected the data of the interaction between the tau protein attached to the tip of AFM probe and the mica surface in presence of 10 μM

of 18 kDa heparin solution. The experiments are performed with 1 Hz of scan rate corresponding to 0.6 m/s probe velocity.

200 Tau vs. mica with buffer

# 100

0 0 10 20 30 40 50 60 70 80 90 100 B

200 Tau vs. mica with hep. 18 kDa

# 100

0 0 10 20 30 40 50 60 70 80 90 100 C 200 Tau vs. mica with buffer

# 100

0 10 20 30 40 50 60 70 80 90 100

Distance, nm

Figure 3.18 Histograms of the separation distance of the interaction between the tau protein and freshly cleaved mica with a PBS (pH 6.9) (A, C) and an 18 kDa heparin solution (10 μM) (B) with 1 s dwell time. Data were collected in sequence of steps (A→B→C).

Figure 3.18 shows the histograms of separation distances at rupture obtained using a buffer solution and 18 kDa heparin molecules. Performing experiment using a higher molecular weight (18 kDa) of heparin solution caused the disappearance of the long

distance rupture events and decreases the average value from ~46 nm to ~21 nm. Also, the high adhesive force reduced in presence 18 kDa heparin solution and didn't recover with removal heparin. As result, the decrease in length of separation at rupture upon adding heparin is more pronounces when adding heparin with higher molar mass.

200 Tau vs. mica with buffer

# 100

0 0 50 100 150 200 250 300 350 400

200 Tau vs. mica with hep. 18 kDa

# 100

0 0 50 100 150 200 250 300 350 400 C

200 Tau vs. mica with buffer

# 100

0 50 100 150 200 250 300 350 400

. Force, pN

Figure 3.19 Histograms of the interaction forces of the interaction between the tau protein and freshly cleaved mica with a PBS (pH 6.9) (A,C) and an 18 kDa heparin solution (10 μM) (B) with

1 s dwell time. Data were collected in sequence of steps (A→B→C).

3.3 Characterizing the conformational dynamics of tau protein molecules using heparin grafted to a gold-coated surface

In some of the experiments discussed above, placing tau protein in solution with heparin resulted in irreversible changes in tau protein molecules as detected by AFM force measurements. These findings hint that some heparin molecules remain bound to tau protein molecules even after removing heparin molecules from solution. Attachment of heparin to the tip during incubation with heparin solution is supported by significant decrease in binding probability. In order to determine whether accumulation of heparin on the tip caused the irreversible changes discussed above, we designed another approach where the heparin molecules were grafted to the gold- coated surface. The goal of using this approach is to prevent heparin molecules from leaving the surface and thus decrease number of heparin molecules that might transfer to the tip during the measurements. The heparin molecules were chemically attached to the gold-coated substrate using carboxyl groups (-COOH) of MUA molecules and EDC as a cross-linker, as discussed in section 2 above. Our procedure for the dynamic measurements was carried out first between the tau molecules on the tip and the free gold-coated substrate in PBS solution (pH 6.9) as a control experiment. Then, we collected data for the interactions between the tau protein on the tip and heparin covalently attached to the gold-coated surface. Finally, interactions between the same tip with the tau molecules and free gold substrate in PBS solution (pH 6.9) were performed again to examine the reversibility. The setup of experiment is shown in Figure 3.20. The experiments were performed at various dwell times and scan rates.

A. Interaction between tau protein and gold surface with buffer solution

AFM tip

M-tau AFM tip

M-tau

PBS PBS Gold-Coated substrate Gold-Coated substrate

B. Interaction between tau protein and heparin grafted on gold surface

AFM tip

M-tau AFM tip

Heparin Heparin | | | | N N N N

| | | |

|| ||

|| || || || OH C || O OH OH OH C O OH C O OH OH OH C O

MU MUA MU MUA

S S S S S S S S S S S S Gold-Coated substrate Gold-Coated substrate

Figure 3.20 Schemes of experiment setup to the interaction of (A) the tau protein vs. free gold- coated substrate using buffer solution (green) where d1 is the separation distance between the tip and the gold-coated surface (B) The interaction between the tau protein attached to the tip and the heparin grafted to gold-coated substrate where d2 is the separation distance between the tip and the modified surface. The drawing is not to scale.

The analysis of the rupture parameters reveals that in presence of grafted heparin molecules the average separation distance at rupture decreases from ~55 nm to ~18 nm

(histograms are shown in Figure 3.21.A-B). Using bare gold surface in the following experiment shows an increase in the distance to 63 nm (histogram is shown in Figure 100

3.21.C). From the last histogram it can be seen that the mean value of rupture distance have increased because relative proportion of rupture events at short length scale has decreased. This increase might be due to opening of some collapsed substructures in the tip-grafted tau protein molecules.

150 With buffer sol. (pH6.9) 100 # 50

0 20 40 60 80 100 120 B

150 With 11 k Da hep. grafted on 100 gold surface # 50

0 0 20 40 60 80 100 120

C 150 With buffer sol. (pH6.9) 100 # 50

0 0 20 40 60 80 100 120

Separation distance, nm

Figure 3.21 Histograms of the separation distances of the interaction between the tau protein and free gold-coated substrate with a PBS (pH 6.9) (A, C) and an 11 kDa heparin molecules grafted to gold-coated substrate (B) with 1 s dwell time. Data were collected in sequence of steps

(A→B→C).

Histograms of rupture corresponding to experiments with grafted heparin are shown in

Figure 3.22. Distribution of rupture forces shifts towards lower values on heparin surface

(panels A and B) and when moving back to gold surface the high value rupture forces

101

appear again in the distribution (panel C). The shape of the distribution does not recover completely, similar to the distribution of distances.

150 With buffer sol. (pH 6.9) 100 # 50

0 0 100 200 300 400 500 600 700

150 With 11 k Da hep. grafted on 100 gold surface # 50

0 0 100 200 300 400 500 600 700

150 With buffer sol. (pH 6.9) 100 # 50

0 0 100 200 300 400 500 600 700

Force, pN

Figure 3.22 Histograms of interaction forces of the interaction between the tau protein and free gold-coated substrate with a PBS (pH 6.9) (A, C) and 11 kDa heparin molecules grafted to gold- coated substrate (B) with 1 s dwell time. Data were collected in sequence of steps (A→B→C).

Histograms of the spring constants before the rupture events reveals a decrease in probability of the rupture events with spring constant around zero and increase in probability of stiffer rupture events (~30-40 pN/nm) (Figure 3.23.A-B). Moving back to the gold substrate significantly decreases proportion of the “stiff” rupture events and greatly increases number of rupture events with spring constant in 0-10 pN/nm range.

102

This supports earlier suggestion that after exposure to the heparin-coated surface, tau molecules on AFM tip change conformation so that they extend more at lower forces.

150

100 With buffer sol. (pH 6.9) # 50

0 -20 -10 0 10 20 30 40 50

150 With 11 k Da grafted on 100 gold surface # 50

0 -20 -10 0 10 20 30 40 50

150 With buffer sol. (pH 6.9) 100 # 50

0 -20 -10 0 10 20 30 40 50

Spring constant, pN/nm

Figure 3.23 Histograms of spring constant of the interaction between the tau protein and free gold-coated substrate with a PBS (pH 6.9) (A, C) and an11 kDa heparin molecules grafted to gold-coated substrate (B) with1 s dwell time. Data were collected in sequence of steps

(A→B→C).

Graphs of the separation distance vs. the dwell time illustrate that the length of the tau protein became shorter with replacing the free gold-coated surface and returned to the

103

extended state by using the free gold-coated surface for the second time (Figure 3.24.A).

This dependence is observed for all dwell times, indicating that change in rupture distance does not require significant time to occur. Furthermore, the rupture forces decrease in presence of heparin molecules and increased back to almost original value upon returning to gold surface (Figure 3.24.B). Values of average spring constants measured at different dwell times behave similarly to the dependence at 1 s dwell time that is discussed above: exposure to heparin surface decreases the spring constant of tau molecules at rupture (Figure 3.24.C). Thus, the irreversibility revealed in this experiment is of different kind than the irreversibility when heparin molecules are in solution. When tau protein is exposed to heparin molecules in solution, the separation distance decreases afterwards, indicating more collapsed structure, while exposure of tau molecules to heparin molecules on the surface increases the separation distance, indicating more extended structure. Recently if was demonstrated that when 11 kDa heparin interacts in solution with the same fragment of tau protein that is studied here, it extends the tau protein length in two regions: region between residues 272 and 285 and region between residues 303 and 316 [4]. The suggestion is made that the tau protein adopts the overall extended conformation when interacting with heparin molecules. This suggestion contradicts with observations that are made here. One possible explanations for such discrepancy is that the suggestion of extending the overall structure is made based on observation of behavior of only two relatively short regions and thus might not represent behavior of the entire molecule. Another explanation is that we learn about stretching tau molecules that are attached to the surface together, not molecules in solution.

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Consequently one heparin molecule might bind simultaneously to different tau molecules, compacting the structure.

A B

120 Tau with buf (1) 500 Tau with buf (1) 100 Tau with 11 kDa hep Tau with 11 k Da hep Tau with buf (2) 400 Tau with buf (2) 80

60 300

40 Force,pN 200

SeparationDistance, nm 20 100

0 0 0 0.2 0.4 0.6 0.8 1 1.2 0 0.2 0.4 0.6 0.8 1 1.2 Dwell Time, s Dwell Time, s C

40 Tau with buf (1) Tau with 11 k Da hep 30 Tau with buf (2)

0 SpringConstant, pN/nm

-10 0 0.2 0.4 0.6 0.8 1 1.2 Dwell Time, s

Figure 3.24 Graphs show dependence of several parameters on the dwell time (s): (A) separation distance (nm), (B) force (pN) and (C) spring constant of the interaction between the tau protein and free gold-coated substrate. Measurements are performed in PBS (pH 6.9) using 11 kDa heparin molecules grafted to the gold-coated substrate.

We can compare the force-distance curves measured in this work with the typical force- distance curves that were obtained in the experiment extending the Aβ1–42 fibrils as studied by Karsai et al [4]. In this work a myloid fibrils were mechanically manipulated by using AFM. In both studies the force-distance curves often appear as a set of hierarchical force steps (Figure 3.25.A). The force plateaus and staircases were observed in the extending of the Aβ1–42 corresponding to the unzipping of β sheets from the fibril

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surface, but in our case, the force plateaus and staircases are due to the rupture of the tau protein sequences from the surface, and the adhesion signatures often exhibit sharp nonlinear force peaks [5]. The step heights were calculated by measuring the distance between the average force values of sequential plateaus. During the retraction process, the force level remains constant before the rupture occurs (Figure 3.25.B). Then, the force curves ends with an abrupt, stepwise decrease in force [5,6,7].

Figure 3.25 (A) Force- distance curves of unzipping of Aβ1-42. Adapted from “Mechanical

Manipulation of Alzheimer’s Amyloid β1–42 Fibrils,” by Kellermayer, M. et al, Journal of

Structural Biology, 2006, 155, p. 321 (B) The force plateaus of gradually unzipping of the β sheets of Aβ1-42. Adapted from “Effect of Lysine-28 Side-Chain Acetylation on the

Nanomechanical, Behavior of Alzheimer Amyloid β25−35 Fibrils,” by Kellermayer, M. et al,

Journal of Chemical Information and Modeling, 2005, 45, p. 1643.

The plateau force histograms for Aβ appeared to have multimodal distributions as shown in the Figure 3.26.A. In contrast, the force histograms of the interaction of the tau protein

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with the surface appeared with one or two modal distributions as shown in the figure

3.26.B.

80 Tau vs. mica

50 # 40

0 0 50 100 150 200 250 300 Force, pN Figure 3.26 (A) The plateau force histograms of unzipping of β1-42 and Aβ1-42. Adapted from

“Mechanical Manipulation of Alzheimer’s Amyloid β1–42 Fibrils,” by Kellermayer, M. et al,

Journal of Structural Biology, 2006, 155, p. 323 (B) The Gaussian distribution of the interaction force of the tau protein with mica substrate.

Also, Kellermayer and his group found that the force plateaus of the unzipped Aβ-subunit sheets were repeatable (Figure 3.27.A) [5]. They suggested that the repeatability of the force plateaus is indicative of the successive mechanical cycles and the complete rupture of the Aβ-subunit sheets [5]. In our work we have the same observation as seen in Figure

3.27.B.

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Figure 3.27 (A) Repeatability of the force plateau of plateau force of unzipping of Aβ1-42 and

Aβ1-42. Adapted from “Mechanical Manipulation of Alzheimer’s Amyloid β1–42 Fibrils,” by

Kellermayer, M. et al, Journal of Structural Biology, 2006, 155, p. 321 (B) Repeatability of interaction forces of the tau protein with mica surface.

Stretching the amyloid fibrils with AFM caused the unzipping process of the β sheet.

The peeling off of the fibril while the β strands of the β sheet are mechanically dissociated caused a gradual increase in the apparent contour length. The force step in the force-distance curves occurs in the withdraw trace due to the rupture of the β-sheet during the unzipping process where each bond rupture event causes an increment in the apparent contour length [5,6]. This observation is compatible with our work; the stretching of the tau protein causes a gradual increase in the apparent contour length of the tau protein. The mean values in the distribution histogram that have been extracted from the number of control experiments were ~62 nm, which is close to that of the fully extended length of the tau sequence (~68 nm). However, the findings in work of et al were inconsistent with this expectation, because they expected to get a constant plateau length in the end of each mechanical cycle. The explanation for these short extracted

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lengths compared to the full length of Aβ amyloid fibrils is due to the dynamic effects related to the lifetime and dynamic equilibrium of the interaction that holds the Aβ fibril subunits [5]. In our case, we also obtained different lengths of the stretching tau protein that was immobilized on the gold-coated tip with the same procedures; some of the extracted lengths exceeded the full length of the tau protein sequence and others were less than the full length of the tau protein sequence.

We suggest that the separation distances longer than the extended length of the tau protein are detected because during sample preparation tau protein molecules might join together into longer structure. On the other hand, we explain the short separation distances by three reasons: first, the attaching of tau molecules to the off-apex of the tip which caused reducing in the apparent contour length (Figure 3.28); second, the possibility that the interaction might not be happening between the end of the tau protein sequence and the surface; finally, shortening of the apparent distance might be due to having some secondary structures contained within the tau sequence that lead to reducing the contour length of the tau protein molecules [8].

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Figure 3.28 (A) Off-normal pulling geometry of tether attachment (B) Off-apex geometry of tether attachment. Adapted from “Distributions of Parameters and Features of Multiple Bond

Ruptures in Force Spectroscopy by Atomic Force Microscopy,” by Akhremitchev, B. B. et al,

The Journal of Physical Chemistry, 2010, 114, p. 8758

Moreover, the distribution of the contour length of the interaction of Aβ40 mutant dimers that was obtained by Lyubchenko et al. using a single molecule approach shows a broad distribution as shown in the Figure 3.29.A. The broad range of the contour length distribution obtained contour length of dimers indicates that the monomers in the dimers adopt different conformations [9]. Furthermore, the distribution of the contour length of the tau protein obtained in our research also shows a broad distribution (Figure 3.29.B).

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150 Tau vs. mica with buffer 100 # 50

0 20 40 60 80 100 120 Separation distance, nm

Figure 3.29 (A) The distribution of contour length of Aβ40. Adapted from “Mechanism of

Amyloid β-protein Dimerization Determined Using Single Molecule AFM Force Spectroscopy,” by Lyubchenko, Y. L. et al, Science Reports, 2013, 3, p. 4 (B) The distribution of contour length of tau protein sequence.

The Gaussian distribution of the interaction force of Aβ40 mutant dimers show a rather narrow distribution, and the maxima of Gaussian distribution at rupture was 63.4 ± 3.2 pN. This result indicates that the interaction strengths for Aβ40 mutant dimers with various contour lengths are rather close [9]. On the other hand, the Gaussian distributions of the interaction of tau molecules with the surface were broad compared to those of the

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Aβ40 mutant dimers interaction, which might be due to the several interactions of tau protein and substrate happening at the same time.

We also compared the results of the maximum forces and jump forces of tau-surface interactions with the results of the research that was performed by Berquand et al. on the interaction of the individual antilysozyme antibodies as model for studying protein- protein interaction. The jump force is the force of the rupture transition from minimum to the maximum points. The histograms of unbinding forces of antilysozyme antibody molecules showed several maxima (54 ± 11, 105 ± 9 and 154 ± 19 pN) as shown in the

Figure 3.30.A [10]. Berquand et al. suggested that the low value of the force maxima corresponds to the adhesion strength value between single molecules; this magnitude is similar to the maxima of Gaussian distribution of the jump force of the interaction of the tau protein molecules with substrates (55.4 ± 32.4) (Figure 3.30.B). This means the interaction between the tau protein and surface consists of a sequence of single molecule interaction. Also, the results of the interaction of the tau protein with mica in the buffer solution is consistent with the result of the maximum unbinding force that is obtained from the unbinding avidin-biotin molecules approach that has been used by Sotres et al.

[9]. The mean unbinding force of avidin-biotin molecules was ~48 pN [11]. Moreover, it is also close to the magnitude of the Gaussian distribution of the interaction force of

Aβ42 mutant dimers which was 63.4 ± 3.2pN [8]. In addition, Berquand et al. also suggested that the histograms with strong adhesion forces which are > 150 pN, referred to the multiple interactions of several antilysozyme antibody pairs that take place at the same time [9]. Also, the most probable interaction between mutiple tau protein

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molecules and substrate in our work was found to be >130 pN, which is similar to the strong adhesion forces of multiple interactions of several antilysozyme antibody pairs.

200 Mean 55.4 | STD 32.4 180

160

140

120 # 100

0 50 100 150 200 Jump force, pN

Figure 3.30 (A) The histogram distribution of unbinding forces of antilysozyme antibodies.

Adapted from “Mechanical Manipulation of Alzheimer’s Amyloid β1–42 Fibrils,” by

Kellermayer, M. et al, Journal of Structural Biology, 2006, 155, p. 321 (B) The jump forces of the interactions of tau protein sequence with mica surface.

3.4 Conclusion

In this research, two approaches have been used to characterize the conformational states of tau protein using atomic force microscopy (AFM). First, we studied the interaction of the tau protein with a cleaved mica surface using various concentrations of 11 kDa heparin solution (1 µM, 10 µM and 20 µM), and two different molecular weights of heparin solution (11 kDa and 18 kDa). The comparison of the separation distance distributions using various concentrations of the 11 kDa heparin solution revealed that increasing the concentration of 11 kDa heparin solutions caused reduction in the length of

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separation at which rupture events occur, diminishing the probability in high range of the rupture forces and the irreversibility of the process of exposure of tau proteins to high concentration of heparin molecules. Moreover, using two different molecular weights of heparin solution (11 kDa and 18 kDa) indicated that using molecule with high molar mass further increases the observed irreversibility. We suggest that the observed irreversibility might be caused by heparin molecules that remain bound to tau protein molecules even after removing heparin molecules from solution. The observed effects are concentration-dependent: no significant effects are observed at 1 M concentration of heparin. Second, we characterized the conformational dynamics of tau protein molecules interacting with 11 kDa heparin molecules covalently attached to a gold-coated surface.

The analysis of the rupture parameters of the interaction between tau proteins molecules attached to the tip and 11 kDa heparin grafted to the gold-coated substrate revealed that there are decreases in the mean values of the separation distance and the rupture force after replacing the free gold-coated substrate with a modified gold-coated substrate. Both the separation distances and the interaction force values almost returned back to their original values with the withdrawal of the agent. Further measurements elucidating physico-chemical origin of these results are necessary. Such measurements might include broader range of heparin concentrations and effects of ionic strength that will affect the strength of electrostatic interactions.

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3.5 References

[1] Lyubchenko, Y. L.; Shlyakhtenko L. AFM for Analysis of Structure and Dynamics of

DNA and Protein-DNA Complexes. Methods, 2009, 47, 206–213.

[2] Sara, L.; Wagner, K.; Manohar, S.; Anand, J.; Vezenov, D. Quantifying Interactions

between DNA Oligomers and Graphite Surface Using Single Molecule Force

Spectroscopy. American Chemical Society, 2012, 116, 13896–13903.

[3] Sibille, N.; Sillen, A.; Leroy, A; Wieruszeski, J.; Mulloy, B.; Landrieu, I.; Lippens, G.

Structural Impact of Heparin Binding to Full Length Tau as Studied by NMR

Spectroscopy. Biochemistry, 2006, 45, 12560–12572.

[4] Eschmann, N. A.; Georgieva, E. R.; Ganguly, P.; Borbat, P. P.; Rappaport, M. D.;

Akdogan, Y.; Freed, J. H.; Shea, J. E.; Han, S., Signature of an aggregation-prone

conformation of tau. Sci Rep, 2017, 7, 10.

[5] Karsai, A.; Martonfalvi, Z.; Nagy, A.; Grama, L.; Penke, B.; , Kellermayer, M.

Mechanical Manipulation of Alzheimer’s Amyloid β1–42 Fibrils. Journal of

Structural Biology, 2006, 155, 316–326.

[6] Kellermayer, M.; Grama, L.; Karsai, A.; Nagy, A.; Kahn, A.; Datki, Z., Reversible, B.

Mechanical Unzipping of Amyloid β-Fibrils. The Journal of Biological Chemistry,

2004, 280, 8467–8470.

[7] Karsai, A.; Nagy, A.; Kengyel, A.; Grama, L.; Penke, B.; , Kellermayer, M.; Effect of

Lysine-28 Side-Chain Acetylation on the Nanomechanical Behavior of Alzheimer

Amyloid β25−35 Fibrils. Journal of Chemical Information and Modeling, 2005, 45,

1641–1646.

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[8] Guo, S.; Li, N.; Lad, N.; Desai, S.; Akhremitchev, B. B.; Distributions of Parameters

and Features of Multiple Bond Ruptures in Force Spectroscopy by Atomic Force

Microscopy. The Journal of Physical Chemistry, 2010, 114, 8755–8765.

[9] Lv, Z.; Roychaudhuri, R.; Condron, M. M.; Teplow, D. B.; Lyubchenko, Y. L.

Mechanism of Amyloid β-protein Dimerization Determined Using Single Molecule

AFM Force Spectroscopy. Science Reports, 2013, 3, 1–8.

[10] Berquand, A.; Xia, N.; Castner, D.; Clare, B.; Abbott, N.; Dupres, V.; Adriaensen,

Y.; Dufrene, Y. Antigen Binding Forces of Single Antilysozyme Fv Fragments

Explored by Atomic Force Microscopy, Langmuir, 2005, 21, 5517–5523.

[11] Sotres, J.; Lostao, A.; Wildling, L.; Ebner, A.; Gomez-Moreno, C.; Gruber, H.;

Hinterdorfer, P.; Baro, A. Unbinding Molecular Recognition Force Maps of

Localized Single Receptor Molecules by Atomic Force Microscopy.

ChemPhysChem, 2008, 9, 590–599.

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4. Analysis of rate of rupture transitions

4. 1 Introduction

Proteins are macromolecules that undergo conformational changes over a wide range of timescales. The biological functions of proteins require conformational changes, and these functions require a large variety of protein rearrangements. Moreover, the mechanical properties of biological macromolecules are related to their function [1, 2].

There have been a few reports of experiments performed to study the mechanical properties of proteins, and most of those reports have focused on studying the mechanical properties of the globular proteins [3]. The majority of studies that have been implemented on the mechanical properties of the folded proteins such as viscoelasticity used AFM technique and were based on the unfolding process, but this process is not suitable for studying the mechanical properties of the folded state, providing information only about the mechanical properties of extended states [4,5]. To date, little has been established about the internal viscosity of proteins. In this work, we used a novel mode to extract time constant parameters that are related to friction in the extended protein molecule.

4.1.1 Previous studies that have been performed to characterize the mechanical properties of proteins.

In 2011, Y. Wang and G. Zocchi designed a nanorheology method to study the mechanical properties of soft nanoparticles (globular proteins). Guanylate Kinase (GK, a

~24 kDa, ~ 24 nm size, ~200 amino acids) was used to couple 20 nm diameter gold nanoparticles (GNPs) to a gold surface (200 μm thick of gold evaporated on a glass slide)

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as shown in the Figure 4.1 [1, 3, 6]. The goal of this gold layer is to serve as a tether for the GK protein through the SH group that is introduced into the protein at position 75 and as a conducting electrode [6].

Figure 4.1 Scheme of the experimental setup including the protein (Guanylate Kinase, GK) attached by the 171 and 75 sites to a gold nanoparticle (GNP). Adapted from “Viscoelastic

Transition and Yield Strain of the Folded Protein,” by Wang, Y.; Zocchi, G., PLOS One, 2011, 6, p. 2.

AC voltage (~1 V in the frequency range10 Hz–10 kHz) was applied to these electrodes to drive the GNPs through the electrophoretic force. In this experiment, evanescent wave scattering technique was used to detect the vertical motion of the GNPs [3]. The relationship between the amplitude (A) and frequency showed that applying low forcing amplitude led to a linear elasticity response of GNP protein where the internal viscosity response disappeared. On the other hand, at higher forcing amplitude, three different

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regimes (linear elasticity, then a regime of viscoelastic but reversible deformations, and finally an irreversible regime) were observed (Figure 4.2) [3].

Figure 4.2 Shows two different curves: one for a low driving force has a characteristic linear elasticity response (a simple spring), and the other, for a higher driving force, is characteristic of a viscoelastic response. Adapted from “The Folded Protein as a Viscoelastic Solid,” by Wang, Y.;

Zocchi, G., Europhysics Letters, 2011, 96, p 3.

By fitting the data for a higher driving force, they got a response curve that is characteristic of viscoelasticity. On the other hand, by fitting the data for a low driving force, they got a response curve that characteristic of a simple spring.

In the previous work, Y. Wang and G. Zocchi noted that applying a higher driving force on the GK folded protein leads the protein to transit reversibly from a linear elasticity regime to a viscoelastic regime [3]. Y. Wang and G. Zocchi continued working on studying the dynamic properties of GK folded proteins and focused on studying the vertical line in the graph of figure 4.2 to investigate more details in this transition [6]. A

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dynamic stretching experiment was carried out with the same setup as in the previous work [1]. The driving frequency was fixed at ν = 10 Hz where the large transition between the elastic and the viscoelastic regimes took place. The driving force was increased gradually with 50 s waiting times between steps [6]. The plotting curve between the amplitude of the driving force and the amplitude response indicated that there is a yield strain at 1 Å (deformation from the elastic regime (z ˂ 1 Å) to the viscoelastic regime (z ˃ 1 Å)) as shown in the Figure 4.3. Also, they found that the transition from elastic to viscoelastic behavior is reversible [6].

Figure 4.3 (a) Experimental force–extension curve vs. deformation measured at 10 Hz indicates that the protein GK exhibiting a yield deformation of 1.1 A°. (b) Direct and indirect measured of the storage modulus vs. applied force at 10 Hz [6]. Adapted from “Viscoelastic Transition and

Yield Strain of the Folded Protein,” by Wang, Y.; Zocchi, G., PLOS One, 2011, 6, p.3.

Y. Wang and G. Zocchi also investigated a method to measure the change in the stiffness of the protein Guanylate Kinase (GK) due to the binding of the substrate guanosine monophosphate (GMP) [1]. The experiment was based on detecting the motion

(mechanical response) of gold nanoparticles due to the application of an AC electric field.

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The driving frequencies ranged from 10 to 10240 Hz. At each frequency the response of amplitude was averaged over 50 s and plotted to get information on the viscoelastic properties of the GK protein [1]. The binding of the GMP substrate to the GK protein caused changes in the conformation of the protein and closed the cleft between the two lobes of the protein (Figure 4.4). By fitting the data in the following equation for the two states (with and without GMP), they found that the protein with a GMP substrate bond became 20% stiffer compared to the free protein (without GMP) [1].

Figure 4.4 Amplitude of the frequency response of the protein Guanylate Kinase (GK) vs. the driving frequencies in the absence ( ) and presence ( ) guanosine monophosphate (GMP) substrate. Adapted from “Elasticity of Globular Proteins Measured from the ac Susceptibility,” by Wang, Y.; Zocchi, G., American Physical Society, 2010, 105, p.3.

A key finding that observed by Zocchi and collaborators an investigation that probed the dynamics of folded protein under the influence of external forces, is that as the driving

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force increases, so does the mechanical response of the folded protein, causing it to exhibit a series of dynamic transitions [1, 3,6]. Furthermore, these researchers also investigated the existence of a relatively abrupt crossover which is known as a viscoelastic transition, as the driving force increases from an elastic response to a viscous response [7]. Based on this investigation, Fogle, C; et al used the Kelvin-Voigt model as a depiction of the driven protein system, as shown in Figure 4.5.

Figure 4.5 Kelvin-Voigt model of viscoelastic system. Adapted from “Protein Viscoelastic

Dynamics: a Model System,” by Fogle, C; Rudnick, J., American Physical Society, 2015, 92, p.1.

This model assumes that the system is over-damping, so the inertial effects are ignored.

The spring constant in this model is not robustly hookean, but the energy of the spring is a function of displacement V(x). According to this model, at low displacement value (x) the dynamic motion of the folded protein behaves like a spring (the potential energy is nearly harmonic and the restoring force is approximately hookean), whereas at sufficient values |x|, it behaves like a viscous slope where the potential energy is linear and associated restoring force approaches a constant value (±F0) as shown in the figure 4.6

[7].

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Figure 4.6 Illustrates the associated restoring force F(x), solid (blue) and the potential energy,

V(x), dashed (red), where the force reaches ±F0 for large displacement x. Adapted from “Protein

Viscoelastic Dynamics: a Model System,” by Fogle, C; Rudnick, J., American Physical Society,

2015, 92, p.3.

Atomic force microscopy has also been used to explore the mechanics of materials in nano scale. In 2011, Radmacheret al. examined the viscoelastic properties of lysozyme, both individual and as an aggregate enzyme, adsorbed on a mica surface using AFM [8].

From their measurements they noted that the slope in the contact region of the enzyme's force curves deviated from a straight line due to elastic deformation compared to the force curves collected on mica (Figure 4.7). Also, they observed the slowing down of the jump of the cantilever on the lysozyme due to its viscosity, in comparison to mica [8].

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Figure 4.7 (a) Force curve on mica (b) Force curve on individual lysozyme enzyme (c) Force curve on aggregate of lysozyme. Adapted from “Imaging Adhesion Forces and Elasticity of

Lysozyme Adsorbed on Mica with the Atomic Force Microscope,” by Radmacher, M. et al,

Langmuir, 1994, 10, p.3811.

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4.2 Using a novel method to characterize the viscous elements of tau protein by analyzing the collected force curves

As I mentioned above in the first part of my research, we don't have a single molecule interactions but we have multiple molecule interactions. During the pulling process, the probe moves up with a certain velocity, and exerts force on the molecules, so the molecules start stretching. In the single molecule approach, when the bond is being ruptured, the cantilever goes from one equilibrium position to another equilibrium position, and snaps back to the baseline. The transition slope should be equal to the spring constant of the cantilever [9,10]. On the other hand, in our work, when the one rupture occurs, it is quite clear from the extracting force-distance curves, it isn't over, and there is still something connecting the tip to the surface. The evidence for that is the several rupture events that appear in the force curves. So, when the first bond is broken, the tip starts to move, but instead of moving freely, it becomes restricted with the second bond, and so on until it becomes free (Figure 4.8. A). Because of that, the collected force curves appear with multiple rupture events (Figure 4.8. B).

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A AFM probe kc AFM k probe c k AFM c probe

M-tau protein M-tau protein M-tau protein a b c

100

N -100 p

e c Transition after c

r -200 a b

o rupture F -300 Rupture points -400

-500 0 10 20 30 40 50 60 70 80 90 100 Separation, nm

Figure 4.8 (A) The cartoons show the rupture process of the tau protein from the surface during retraction process (B) Force –distance curve with multiple rupture events (a, b and c).

Additionally, we observed from analyzing these collected force curves that the extracting transition slope is not equal to the spring constant of the cantilever but it is much lower than the spring constant of the cantilever. This observation implies that there are some molecular processes which have viscous components after the rupture (similar to work of

Radmacher et al. [8]). These viscous elements led to considerably lowering the slope of jump after the rupture. Figure 4.9 illustrates the transition after rupture that extracting from force-distance curves.

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100

0 N

p -100

, e c r o F -200 Transition after rupture Transition Rupture slope -300 point

0 10 20 30 40 50 60 70 80

Separation, nm

Figure 4.9 Shows force-distance curve with the number of stretching slops.

In most of the latest force spectroscopy research, curves with multiple rupture events were not considered interesting, and the researchers threw them away because they did not feature single molecule interaction. In our work, we think that the multiple rupture events curves are interesting and useful because we can use these curves to extract some aspects about molecular viscosity of the molecules under study. To extract the viscous elements, we used the Kelvin-Voigt model (KV). We considered the relaxation of the tip after the rupture.

4.2.1 Kelvin-Voigt model (KV)

Assume that after a bond is broken, there is still connection between the AFM tip and the surface. If ignoring the inertial forces during the jump-off (over damped motion) then the motion of the probe can be modeled using the Kelvin-Voigt and as shown in the figure below.

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A B F, vp Probe’s base

kc AFM kc probe z M-tau protein  k1 x

Kelvin-Voigt model

Figure 4.10 (A) Shows the cartoon of the interaction between the attached molecules and surface

(B) illustrates the Kelvin-Voigt model.

The dashpot in the Kelvin-Voigt model represents viscous elements which contains viscosity that added to force in the viscous term. The motion after bond rupture is described as separation of mass-less AFM tip from the surface. The tip is still connected to the surface with visco-elastic element as shown in the figure above.

The base of the probe moves away from the surface with constant velocity vp:

z() t z0p v t (4.3)

At the same time this position consists of tip-sample separation and cantilever deflection:

z()()()/ t x t F t kc (4.4)

Where kc is the spring constant of the cantilever. In Kevin-Voigt model force is the sum of forces on elastic and viscous elements. In this model force depends on tip-sample

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separation because of the elastic element and also on tip’s velocity due to frictional component:

F()(())() t k10 x t  x   x t (4.5)

where x0 is the position of tip at equilibrium and β is the friction coefficient. Assuming that the motion starts at force F0 then solution of equations 4.3–4.5 above is

1 t/ KV x( t ) x0  F0 kcKv p V  1  e  k c v p t (4.6) kt

where kt = kc + k1 and τKV = β / kt. The tip velocity is then

1 t/ KV x( t ) F0  k c v p KV  F 0  1  e  (4.7)  KVk t

By substituting equations (4.6) and (4.7) into equation (4.5) we can obtain an explicit expression for F(t):

t   kc  KV kc Ft  F0  k c vp KV F 011  e   k c v p t (4.8) k k t   t

4.2.1.1 Characteristic points and features in F (t) and x (t) dependencies

If the rupture occurs when the probe is stationary (vp = 0) then the tip-sample separation and force on the tip eventually become

F0 xxstat()  0  (4.9) kt

and 129

k1 FFstat() 0 (4.10) kt

For the stationary probe the change in force after the rupture is

kc FFFF stat()   0   0 (4.11) kt

After the jump the force reaches minimum at time determined by dF(t)/dt = 0:

F k v t  log 0 c KV p (4.12) min KV  kv1 KV p

This expression indicates the limitation of the Aristotle’s mechanics model: for the time to reach minimum force to be a positive quantity the probe velocity must be in general lower than F0 / β,or for specific values of k1

FF00k1 vp 1   (4.13) kkc c KV

Substituting time of minimum force into the corresponding equations allows calculating values of separation and force at this point. Separation between the minimum force point and the rupture point is

k F k v kc t 0 c p tmin/ KV xmin  x( t min )  x 0  v p t min 2 (1  e ) (4.14) kktt

And decrease in force between these two points is

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Fmin  F 0  k 1  x min   x() t min kv k1 k ctmin/ KV k c cp  vp t min (1  e )  F 0  (4.15) kt k t k t k F k v kc t 0 c p tmin/ KV (1 e )  k1 v p t min kktt

The slope of force plot after the jump is given by

t/ KV dF vp KVk 1 e k c F 0   F  kc (4.16) t/ KV dx vp KVk c e1  F 0

The initial value of the slope is

dF vp   F0 kc 1 (4.17) dx t0  F0 

In the absence of friction the magnitude of the initial slope equals to the spring constant of cantilever kc. At time 푡 ≫ 휏퐾푉 the slope asymptotically approaches k1.

4.2.1.2 Estimation of model parameters from the data

Using eqs. 4.14 and 4.15 we obtain:

kc x min   F min  k c v p t min F k v (4.18)  kv log 0 c p cp  kv1p

Therefore if the time constant τ is known then

F k v Fmin  k c  x min 0 c p kvcp ke1  (4.19) vp 131

Also we can also write another expression with ΔFmin, and Δxmin :

Fmin  k c  x min x k   F 1  ekvcp F  k v (4.20) min 1 min 0 c p  

Substituting eq. (4.19) into eq. (4.20) we obtain transcendental equation:

Fmin  k c  x min  kvcp xmin Fmin  F 0  k c v p 1  e  1   0 (4.21) v p

From force plots the values of F0, ΔFmin, and Δxmin can be estimated and together with experimental parameters kc and vp eq. 4.21 becomes equation of one unknown τ. Solving eq. 4.21 numerically for τ and using the result in eq. 4.19, we obtain unknown parameter k1.

4.2.1.3 Fitting the model to the data

Fitting data with the KV model (as given by equations 4.6 and 4.8) requires adjusting four independent parameters: F0, x0, kt and τKV.

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A B 100 100 0 0 N N -100 p d p

-100 , c , e b e c c r r o Transition after o -200 -200 F rupture F a Fitting the transition -300 Rupture points -300 events with KV model -400 -400 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Separation, nm Separation, nm

C D 100 100

0 0 N N p p

, -100 , -100 e e c c r r c o

o Transition after

a b F F -200 rupture -200 Fitting the transition Rupture points -300 -300 events with KV model 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 Separation, nm Separation, nm

Figure 4.11 Examples of the fitting of two extracting fore-distance curves with KV model. In the

Figure A and C, the brown lines represent the transition event after the rupture. a, b, c and d in the figure A and C represent the rupture points. The red lines in the figure B and D illustrates how

KV model fitting the data. The fitting line of KV model indicates that the KV model has approximately good fitting with data.

Custom software written for Matlab was used to perform the analysis. The software automatically identified position of rupture points labeled with letters (a), (b) and (c) in

Figure 4.8A and fit model described above to the regions between the transitions. The

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initial guess of parameters was obtained as described in section 4.2.1.2 above. Figure

4.11 shows the fitting of two extracting fore-distance curves with KV model.

4.2.1.4 The values of the number of parameters that have been estimated by fitting with the KV model

4.2.1.4.1 Fitting the data extracted from the interaction of tau protein with mica surface using PBS solution (pH 6.9) and 1 μM of 11 kDa heparin solution. The experiment preformed in the order from A to C as shown in the table below. Results reported below were obtained using the same z scan size that was equal to 20 µm.

Table 4.1 Shows the values of the remaining spring constant (k1) that was extracted from fitting data with the KV model for different z scan rates. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 1 μM of 11 kDa heparin solution.

Remaining spring constant (k1, pN/nm)

(A) With buffer solution (1) (B) With 11 kDa (C) With buffer solution (2) heparin solution (1 µM)

mean St. deviation mean St. deviation mean St. deviation

0.5 Hz 13.00 23.50 13.1 22.60 8.00 13.50

1 Hz 5.20 8.50 6.50 12.20 3.60 6.50

1.5 Hz 2.60 6.30 1.70 4.40 2.30 5.00

2 Hz 1.90 4.00 0.90 2.40 4.90 6.60

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15 With buffer (pH 6.9) (1) With 11 kDa hep (10 µM) With buffer (pH 6.9) (2) 10

RemainingSpring Constant, pN/nm 0 0.5 1 1.5 2 Rate, Hz

Figure 4.12 The plots of remaining spring constant (k1) values vs. z scan rate. The magnitudes of the remaining spring constant (k1) were extracted from fitting the data with KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 1 μM of 11 kDa heparin solution. The data were collected in sequence of steps (A→B→C).

Table 4.2 Shows the values of the decay time (τ) that were extracted from fitting the data with the

KV model for different z scan rates. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 1 μM of 11 kDa heparin solution.

Decay time (τ, ms)

(A) With buffer solution (1) (B) With 11 kDa (C) With buffer solution (2) heparin solution (1 µM)

mean St. deviation mean St. deviation mean St. deviation

0.5 Hz 2.40 1.80 2.20 1.50 1.80 1.50

1 Hz 0.92 0.67 1.30 0.90 0.71 0.67

1.5 Hz 0.58 0.49 0.68 0.47 0.54 0.51

2 Hz 0.44 0.35 0.44 0.32 0.64 0.41

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2.5 With buffer (pH 6.9) (1) With 11 kDa hep(1 µM) 2 With buffer (pH 6.9) (2)

1.5

1 Decayms Time, 0.5

0 0.5 1 1.5 2 Rate, Hz

Figure 4.13 The plots of decay time (τ) values vs. z scan rate. The magnitudes of the decay time were extracted from fitting data with KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 1 μM of 11 kDa heparin solution.

The data were collected in sequence of steps (A→B→C).

Table 4.3 Shows the values of the friction coefficient (β) that was extracted from fitting data with the KV model for different z scan rates. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 1 μM of 11 kDa heparin solution.

Friction coefficient (β, g/s)

(A) With buffer solution (1) (B) With 11 kDa (C) With buffer solution (2) heparin solution (1 µM)

mean St. deviation mean St. deviation mean St. deviation

0.5 Hz 0.71 0.57 0.65 0.46 0.52 0.43

1 Hz 0.26 0.19 0.37 0.27 0.20 0.19

1.5 Hz 0.16 0.14 0.19 0.13 0.15 0.14

2 Hz 0.12 0.10 0.12 0.09 0.18 0.12

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0.8 With buffer (pH 6.9) (1) With 11 kDa hep (1 µM) ) s / 0.6 With buffer (pH 6.9) (2) g (

t n e i c i

f 0.4 f e o C

n o i

t 0.2 c i r F 0 0.5 1 1.5 2 Rate, Hz

Figure 4.14 The plots of friction coefficient (β) values vs. z scan rate. The magnitudes of the friction coefficient were extracted from fitting data with KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 1 μM of 11 kDa heparin solution. The data were collected in sequence of steps (A→B→C).

4.2.1.4.2 Fitting the data extracted from the interaction of tau protein with mica surface using PBS buffer solution (pH 6.9) and 10 μM of 11 kDa heparin solution. The experiment preformed in the order from A to C as shown in the below table. Results reported below were obtained using the same z scan size that was equal to 20 µm.

137

Table 4.4 Shows the values of the remaining spring constant (k1) that was extracted from fitting data with the KV model for different z scan rates. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 10 μM of 11 kDa heparin solution.

Remaining spring constant (k1, pN/nm)

(A) With buffer solution (1) (B) With 11 kDa heparin (C) With buffer solution (2) solution (10 µM)

mean St. deviation mean St. deviation mean St. deviation

0.5 Hz 0.30 1.20 0.20 0.80 0.11 0.60

1 Hz 0.30 1.00 0.20 1.10 0.06 0.39

1.5 Hz 0.20 1.00 0.05 0.41 0.01 0.07

2 Hz 0.09 0.44 0.05 0.03 0.02 0.26

m With buffer (pH 6.9) (1) n

/ 0.4 With 11 kDa hep (10 µM) N p

, With buffer (pH 6.9) (2) t

n 0.3 a t s n o C

0.2 g n i r p

S 0.1

g n i n i

a 0 m e

R 0.5 1 1.5 2 Rate, Hz

Figure 4.15 The plots of the remaining spring constant values vs. z scan rate. The magnitudes of the remaining spring constant (k1) were extracted from fitting data with KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 10 μM of 11 kDa heparin solution. The data were collected in sequence of steps (A→B→C).

138

Table 4.5 Shows the values of the decay time (τ) that was extracted from fitting data with the KV model for different z scan rates. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 10 μM of 11 kDa heparin solution.

Decay time (τ, ms)

(A) With buffer solution (1) (B) With 11 kDa heparin (C) With buffer solution (2) solution (10 µM)

mean St. deviation mean St. deviation mean St. deviation

0.5 Hz 0.78 0.80 0.70 2.00 0.70 1.00

1 Hz 0.42 0.36 0.26 0.59 0.37 0.76

1.5 Hz 0.25 0.71 0.16 0.47 0.15 0.39

2 Hz 0.22 0.92 0.048 0.02 0.013 0.018

1 With buffer (pH 6.9) (1) With11 kDa hep (10 µM) 0.8 With buffer (pH 6.9) (2)

0.6

0.4 Decayms Time, 0.2

0 0.5 1 1.5 2 Rate, Hz

Figure 4.16 The plots of the decay time (τ) values vs. rate. The magnitudes of the decay time were extracted from fitting the data with KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 10 μM of 11 kDa heparin solution. The data were collected in sequence of steps (A→B→C).

139

Table 4.6 Shows the values of the friction coefficient (β) that were extracted from fitting the data with the KV model for different z scan rates. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 10 μM of 11 kDa heparin solution.

Friction coefficient (g/s)

(A) With buffer solution (1) (B) With 11 kDa heparin (C) With buffer solution (2) solution (10 µM)

mean St. deviation mean St. deviation mean St. deviation

0.5 Hz 0.26 0.28 0.26 0.70 0.23 0.37

1 Hz 0.15 0.13 0.09 0.21 0.13 0.27

1.5 Hz 0.09 0.25 0.06 0.17 0.05 0.14

2 Hz 0.08 0.32 0.017 0.01 0.04 0.06

0.4 With buffer (pH 6.9) (1) With 11 kDa hep (10 µM) 0.3 With buffer (pH 6.9) (2)

0.2

0.1 FrictionCoefficient, g/s

0 0.5 1 1.5 2 Rate, Hz

Figure 4.17 The plots of the friction coefficient (β) values vs. z scan rate. The magnitudes of friction coefficient were extracted from fitting data with KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 10 μM of 11 kDa heparin solution. The data were collected in sequence of steps (A→B→C).

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4.2.1.4.3 Fitting the data extracted from the interaction of tau protein with mica surface using PBS buffer solution (pH 6.9) and 20 μM of 11 kDa heparin solution. The experiment preformed in the order from A to C as shown in the below table. Results reported below were obtained using the same z scan size that was equal to 20 µm.

Table 4.7 Shows the values of the remaining spring constant (k1) that were extracted from the fitting data with the KV model for different z scan rates. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 20 μM of 11 kDa heparin solution.

Remaining spring constant (k1, pN/nm)

(A) With buffer solution (1) (B) With 11 kDa heparin (C) With buffer solution (2) solution (20 µM)

mean St. deviation mean St. deviation mean St. deviation

0.5 Hz 7.00 9.00 5.90 10.5 0.90 4.50

1 Hz 3.30 5.60 2.20 8.10 0.30 1.50

1.5 Hz 3.00 4.30 1.80 4.60 0.01 0.02

2 Hz 1.80 3.10 0.19 0.57 0.00 0.00

With buffer (pH 6.9) (1) With 11 kDa hep (20 µM) 6 With buffer (pH 6.9) (2)

RemainingSpring Constant, pN/nm 0.5 1 1.5 2

Rate, Hz 141

Figure 4.18 The plots of the Remaining spring constant (k1) values vs. z scan rate. The magnitudes of the remaining spring constant (k1) were extracted from fitting data with KV model.

The data were obtained from the interaction of the tau protein molecules with mica in the buffer

(pH 6.9) and 20 μM of 11 kDa heparin solution. The data were collected in sequence of steps

(A→B→C).

Table 4.8 Shows the values of the decay time (τ) that were extracted from fitting data with the

KV model for different z scan rates. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 20 μM of 11 kDa heparin solution.

Decay time (τ, ms)

(A) With buffer solution (1) (B) With 11 kDa heparin (C) With buffer solution (2) solution (20 µM)

mean St. deviation mean St. deviation mean St. deviation

0.5 Hz 1.70 0.70 1.40 0.90 0.67 0.86

1 Hz 0.88 0.46 0.55 0.74 0.40 0.38

1.5 Hz 0.52 0.49 0.34 0.28 0.14 0.15

2 Hz 0.40 0.18 0.13 0.09 0.13 0.11

2 With buffer (pH 6.9) (1) With 11 kDa hep (20 µM) 1.5 With buffer (pH 6.9) (2)

Decayms Time, 0.5

0 0.5 1 1.5 2 Rate, Hz

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Figure 4.19 The plots of the decay time (τ) values vs. z scan rate. The magnitudes of the decay time were extracted from fitting the data with KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 20 μM of 11 kDa heparin solution. The data were collected in sequence of steps (A→B→C).

Table 4.9 Shows the values of friction coefficient (β) that were extracted from fitting data with the KV model for different z scan rates. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 20 μM of 11 kDa heparin solution.

Friction coefficient (g/s)

(A) With buffer solution (1) (B) With 11 kDa heparin (C) With buffer solution (2) solution (20 µM)

mean St. deviation mean St. deviation mean St. deviation

0.5 Hz 0.56 0.24 0.46 0.32 0.22 0.28

1 Hz 0.29 0.15 0.18 0.24 0.13 0.12

1.5 Hz 0.17 0.17 0.11 0.09 0.05 0.05

2 Hz 0.13 0.13 0.04 0.03 0.04 0.04

With buffer (pH 6.9) (1) 0.6 With 11 kDa hep (20 µM) With buffer (pH 6.9) (2) 0.5

0.4

0.3

0.2

FrictionCoefficient (g/s) 0.1

0 0.5 1 1.5 2 Rate, Hz

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Figure 4.20 The plots of the friction coefficient (β) values vs. z scan rate. The magnitudes of the friction coefficient were extracted from fitting data with KV model. The data were obtained from the interaction of the tau protein molecules with mica in the buffer (pH 6.9) and 20 μM of 11 kDa heparin solution. The data were collected in sequence of steps (A→B→C).

The analysis of the data that were obtained from the interaction of the tau protein with mica using buffer and various concentrations of 11 kDa heparin solutions indicated that there are decreases in the remaining spring constant (k1), the decay time (τ) and the friction coefficient (β) with increases in the rate value. The transition from the buffer to the heparin solution and again to the buffer shows that neither the decay time (τ) nor the friction coefficient (β) changed using 1 µM and 10 µM of the heparin solutions (Figures

4.13, 4.14, 4.16, 4.17). This observation is consistent with changes in the interaction forces and the spring constants that were extracted from the interaction of the tau protein with mica as we could see in the previous chapter in Figure (3.9 and 3.16). On the other hand, using a high concentration of the heparin (20 µM) induced more reduction in the magnitude of the remaining spring constant (k1), the decay time (τ) and the friction coefficient (β) as seen in Figures 4.18, 4.19 and 4.20. The reduction in the extracting values is compatible with the changes in the separation distances and the interaction forces that were measured in the previous chapter (Figures 3.14, 3.15). Moreover, the analysis data with the KV model indicated that after treatment of the tau protein with a high concentration of the heparin (20 µM), the remaining spring constant (k1) considerably decreased as shown in Figure 4.18. The decrease in the remaining spring constant (k1) after incubating the tau protein with a high concentration of heparin is compatible with the decrease in the separation distances and the interaction forces that 144

were measured in the previous chapter as seen in Figure 3.17A and Figure 3.17B. In addition, incubating the tau protein with a high concentration of heparin induced irreversible effect on all the extracting viscous elements (the remaining spring constant

(k1), the decay time (τ) and the friction coefficient (β)). This effect also appeared in the interaction of tau protein with mica using 20 µM of heparin solution, where the separation distances and the interaction forces significantly reduced after incubated the tau protein with heparin solution as can be seen in the previous chapter in Figure 3.17A and Figure 3.17B.

4.3 Conclusion

For the concerning rupture data that have been generated using AFM of the interaction of the tau protein with mica, the analysis of the data revealed that the slope after the rupture appeared significantly lower than the spring constant of the cantilever. That observation gave us a hint that during the transition after the rupture some molecular processes that have viscous components toke place and caused reducing in the slope after the rupture.

Most the existents model that use in analysis data and obtaining viscose elements, even though they are quit useful, they use complicated process and many assumptions. In this chapter, simple model (Kevin-Voigt model) was used to investigate the transition after the rupture, to try to extract some viscous elements and to see how the magnitude of these elements change with change the environment of the interaction of tau protein with mica substrate. From fitting the data with KV model we noted that, systematically changing of the scan rate values caused reduce the extracting viscous elements (the remaining spring constant (k1), the decay time (τ) and the friction coefficient (β)). Also, incubated tau

145

protein with the high concentration of 11 kDa caused significantly decreasing in the remaining spring constant (k1) and irreversible process. In the previous chapter we noted that using high concentration of 11 kDa heparin solution causes accumulation of the heparin on the tip and irreversible changes. With removing the heparin solution, the separation distance, the force and spring constant continue decreasing instead of returning to the original values. On the other hand, the estimating of the remaining spring constant

(k1) values gave similar outcome: adding heparin with concentration of 10 M or of 20

M significantly and irreversibly decreases the mean value of this parameter. Significant irreversible decrease in friction coefficient was observed only at 20 M concentration of heparin. These observations are consistent with suggestion that heparin remains bound to the AFM probe. Further investigations using a larger number of heparin concentrations and in addition controlling electrostatic interactions by changing the ionic strength of solution are needed to elucidate physico-chemical origin of these results.

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4.4 References

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Susceptibility, American Physical Society, 2010, 105, 1–4.

[2] Qu, H.; Landy, J.; Zocchi, G. Cracking Phase Diagram for the Dynamics of an

Enzyme, American Physical Society, 2012, 86, 1–4.

[3] Wang, Y.; Zocchi, G. The Folded Protein as a Viscoelastic Solid, Europhysics

Letters, 2011, 96, 1–6.

[4] Kawakami, M.; Byrne K.; Brockwell, D. J.; Radford, S. E.; Smith, D. A., Viscoelastic

Study of the Mechanical Unfolding of a Protein by AFM, Biophysical Journal, 2006,

91, 16–18.

[5] Oesterhelt, F.; Oesterhelt D.; Pfeiffer, M.; Engel, A.; Gaub, H. E.; M¨uller, D.

Unfolding Pathways of Individual Bacteriorhodopsins, Science, 2000, 288, 143–146.

[6] Wang, Y.; Zocchi, G. Viscoelastic Transition and Yield Strain of the Folded Protein,

PLOS One, 2011, 6, 1–11.

[7] Fogle, C; Rudnick, J. Protein Viscoelastic Dynamics: a Model System, American

Physical Society, 2015, 92, 1–20.

[8] Radmacher, M.; Fritz, M.; Cleveland, J. P.; Walters, D. A.; Hansma, P. K. Imaging

Adhesion Forces and Elasticity of Lysozyme Adsorbed on Mica with the Atomic

Force Microscope, Langmuir, 1994, 10, 3809–3814.

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[9] Staple, D.;Geisler, M.; Hugel, T.; Kreplak, L.; Kreuzer, J. Forced Desorption of

Polymers from Interfaces, New Journal of Physics, 2011, 13,1–21.

[10] Karacsony, O.; Akhremitchev, B. On the Detection of Single Bond Ruptures in

Dynamic Force Spectroscopy by AFM. Langmuir, 2011, 27, 11287–11291.

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