BOTTOM-UP DESIGN OF SYNTHETIC PHOTOACTIVE METALLOPROTEINS
Jiufeng Fan
A Dissertation
Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
December 2009
Comm ittee:
Michael Y. Ogawa, Advisor
Ray A. Larsen Graduate Faculty Representative
Felix N. Castellano
Tho mas H. Kinstle
ii
ABSTRACT
Michael Ogawa, Advisor
This dissertation describes the design of synthetic photoactive metalloproteins and their
metal binding properties. Four peptides are prepared to study the interplay between peptide
backbone and metal ion clusters. The four peptides synthesized are:
KIEALEGKCEACEGKIEALEGK-GGY mini-C9C12-GGY
Q IAALEQK IAALEQK CAACEQK IAALEQK GGY AQC16C19-GGY
Q IAALEQK IAAVEQK CAACEQK IAALEQK GGY AQVC16C19-GGY
Q IAAVEQK CAACEQK IAALEQK GGY miniAQVC16C19-GGY
Starting from a previously designed peptide C16C19-GGY, a smaller peptide, mini-
C9C12-GGY was designed. Mini-C9C12-GGY has the same peptide sequence with C16C19-
GGY except that it has less amino acids than C16C19-GGY at N-terminus. It was shown that mini-C9C12-GGY also can bind to Cu(I) to form a tetrameric metalloprotein Cu(I)/mini-C9C12-
GGY which displays an intense room-temperature luminescence around 600 nm. It was also shown that the Cu(I) coordination in Cu(I)/mini-C9C12-GGY might change at high loading of the Cu(I). The emission of the Cu(I)/mini-C9C12-GGY follows biexpotnetial decay and can be quenched by a series of [Ru(NH3)5X] (X= chloro, amine, lutidine, pyridine, nicotinamide, and
3,5-dimethyl pyridine dicarboxylate). The quenching mechanism is assigned to a photoinduced
electron-transfer event by transient spectroscopy. The bimolecular electron-transfer of
Cu(I)/mini-C9C12-GGY occurs in the diffusion limit region. Such results support our hypothesis iii
that the hydrophobic condition provided by the coiled coil structure in Cu(I)/C16C19-GGY
results in the prohibition of close approach between the donor and acceptor.
The effect of the stability of the apopeptide folding on the oligomerization states of the metalloprotein was demonstrated by a series of peptides derived from a dimeric peptide motif:
Q(IAALEQK)nGGY (n>3). The peptides are AQC16C19-GGY, AQVC16C19GGY, and miniAQVC9C12-GGY. AQC16C19-GGY and AQVC16C19GGY exists as coiled coil at their
apo-states. MiniAQVC9C12-GGY exists as a random coil due to the short peptide sequence
compared to AQC16C19-GGY and AQVC16C19GGY. Although the peptides have different
free energies of folding at their apo-states, all the resulting metalloproteins after the addition of
Cu(I) exist as trimers as determined by ultracentrifugation analysis and/or high performance size
exclusion chromatography. These results indicate that metal ions are still the dominating factor
in determining the oligomerization structures of metalloproteins in the researched system. iv
To my parents,
Henjun Fan, Yuyun Sun
Thanks my wife Dongxia Liu for her love and support
Thanks my brother Jiuyue Fan for his support v
ACKNOWLEDGMENTS
One of the pleasures of finishing this dissertation is the opportunity to thank so many people who have helped me through the past years. Foremost, I would like to thank my advisor,
Dr. Michael Ogawa, for his guidance, support, and unselfish transfer of his knowledge. He gave me a lot of freedom to do the research, encouraged me to think independently to grow as a scientist, and lighted me to new research fields with his creative advices.
I would like to thank my committee members, Dr. Felix N. Castellano, Dr. Thomas H.
Kinstle, and Dr. Ray A. Larsen, for being on my committee over my PhD study. Their kind support and encouragement are always there for me to pursue my degree till this last hurdle.
The Ogawa’s group has been the second family of mine and a good source of support and entertainment for me to enjoy. Jing Hong welcomed me to the lab and taught me to be a meticulous and skeptical scientist. Xianchu Zhu, Fei Xie and I always have some informative controversy about our projects. Mikhail V Tsurkan, Daniil Viatcheslavovic Zaytsev, Liu Liu, and
Madhumita Mukherjee are always ready for proving me help with research and life.
My special thanks go to Dr. Felix N. Castellano for providing me the laser facilities and the Ohio Laboratory for Kinetic Spectrometry for permitting me using the instruments. I also would like to thank Evgeny Danilov for the nano-transient experiments assistance.
Finally, I would like to express my gratitude to my friends for all the great memories that I can take with me, and my family for their encouragement and support. Special thanks to my wife
Dongxia Liu for her love and understanding.
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BOTTOM-UP DESIGN OF SYNTHETIC PHOTOACTIVE METALLOPROTEINS
TABLE OF CONTENTS Page
CHAPTER I. INTRODUCTION …………………………….……………………….…………..1
I.1 Introduction to metalloproteins ………………………………………………………....1
I.2 Metalloprotein types and functions ..…………………………………………………....1
I.3 Interaction of metal ions with proteins in metalloproteins …………...……...…….…...3
I.4 Metalloprotein design ………………….…………………..…………………...……....4
I.4.1. Goals for metalloprotein design …. .………………………….…………....4
I.4.2 Design strategies…………………………………...………….….……….....5
I.5 Electron transfer study in metalloproteins…………………...……………....…...... 16
I.6 Research objectives and thesis outline….……………………………………..…..…...19
References ……………………..……………………………..……………………...……22
CHAPTER II. EXPERIMANTAL …………………………………………...………...….…... 28
II.1 Materials ……………………………………………………………………..…...….. 28
II.2 Preparation of peptide and Ru(NH3)5X(BF4)3 ……………………………….…..….. 28
II.3 Electrochemical measurements of metalloproteins ……………………………....…..30
II.4 Molecular weight determination of peptides and metalloproteins …………...……....32
II.5 Spectroscopic investigations ……………………………………………….…………34
References ……………………………………...………………….……………….…..…35
CHAPTER III. PHOTOPHYSICS AND PHOTOINDUCED ELECTRON TRANSFER
PROPERTIES OF CU(I)/MINI-C9C12-GGY………..…………….....…….40
III.1 Introduction ………………………………………………………....…………….…40
III.2 Synthesis of mini-C9C12-GGY and Cu(I)/mini-C9C12-GGY.………...... ……47 vii
III.2.1 Mini-C9C12-GGY peptide synthesis ………………………...... 47
III. 2.2 Synthesis of Cu(I)/mini-C9C12-GGY ……………………………….….48
III.2.3 Determination of oligomeric state of Cu(I)/mini-C9C12-GGY ……….....48
III.3 Spectroscopic property investigation ………………...…..……….………...…..…...51
III.3.1 Circular dichroism (CD) spectroscopy …………….….………...….….…51
III.3.2 UV-Vis spectroscopy ………………………………………...... …….…58
III.3.3 Emission spectroscopy at room temperature …………………….……….60
III.3.4 Emission at 77 K and excitation spectra ………………………...…….…65
III.3.5 Summary on photophysics of Cu(I)/mini-C9C12-GGY ……..……...…...70
III.4 Electron transfer study ……………………………………...….…………….………71
III.4.1 Lifetime at room temperature ……………………………………..….…..71
III.4.2 Redox potential of Cu(I)/mini-C9C12-GGY and ruthenium quenchers….73
3+ III.4.3 Effect of [Ru(NH3)5Lut] on the decay kinetics and quenching
mechanism………………………………………………………………..77
III.4.4. Stern-Volmer behavior of Cu(I)/mini-C9C12-GGY quenching………....80
III.4.5 Electron transfer of Cu(I)/mini-C9C12-GGY with Ru(III) complexes
…………………………………………………….………...…..….82
III.4.6 Bimolecular electron transfer events ………………………..………....…83
III.4.7 Summary on electron transfer reactions ……………………….….……...86
References …………………………………………………………………………...……87
Chapter IV. THE EFFECT OF THE FREE ENRGY OF PEPTIDE FOLDING ON
OLIGOMERIZATION STATE OF METALLOPROTEIN ………...………....…91
IV.1 Introduction ………………………………………………...………………….….…91
IV.2 Peptide synthesis and characterization …………………………....………………....93 viii
IV.2.1 Peptide synthesis …………………………………...... ……...93
IV.2.2 Conformational analysis ...... 96
IV.2.3 Oligomeric state analysis …………………………………...…..………..97
IV.3 Synthesis and characterization of Cu-peptide ………………………………...……100
IV.3.1 Circular dichroism (CD) titration ………………...... ……...... ….…..….100
IV.3.2 UV-vis titration ...... 105
IV.3.3 Emission titration ...... 109
IV.3.4 Oligomeric states determination of metalloproteins ……..………...…...116
IV.4 Conclusions ………………..……………………………………..………………...121
References ………………………………………………………………………...……..125
Appendix. CRYSTAL STRUCUGTRE AND OLIGOMERIZTION STATE DETERMINATION
OF Cd(II)/AQC16C19-GGY……………………………..………………...……...129
References………………...... …………………………………………………..……...137
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LIST OF FIGURES
Figure Page
Figure I.1 Schematic illustration of the interaction between proteins and metal cofactors. ….…..4
Figure I.2 Schematic views of synthetic oiled-coils ……………………………………….….….9
Figure I.3 Helical wheel diagram of the coiled coil ………………………………………….…13
Figure I.4 Computer model of the ET heterodimer ruthenium derivatized H21(30mer) peptide..15
Figure I.5 A schematic illustration of bi-molecular electron transfer mechanism……...…...... 18
Figure I.6 Plot of lnk vs. -ΔG0……………………………………………………………..….….19
Figure III.1 Computer generated model of the Cu(I) adduct of C16C19-GGY …...……….…...41
Figure III.2 Plot of the electron transfer rates of the long lifetime component in Cu(I)/C16C19- GGY adduct with Ru(III) complexes ……………….……………………….…….44
Figure III.3 Plot of the electron transfer rates of the short lifetime component in Cu(I)/C16C19- GGY adduct with Ru(III) complexes……………………………………….….…..45
Figure III.4 MALDI mass spectrum of synthesized peptide mini-C9C12-GGY……………..….48
Figure III.5 dn/dc measurement of Cu(I)/mini-C9C12-GGY by MALLS…………………..…..49
Figure III.6 Zimm plot showing the angle dependence of the scattered light intensity from a representative time slice of the HPSEC elution peak……………...………………49
Figure III.7 Determination of molecular weight of Cu(I)/mini-C9C12-GGY velocity ultracentrifugation, (10 mM acetate buffer, pH 5.4)………..………………..…….50
Figure III.8 Circular dichroism spectra of mini-C9C12-GGY (■) and mini-C9C12-GGY upon the addition of 1 equivalent of Cu(I) (●)……………………………..…………..……52
Figure III.9 Mini-C9C12-GGY upon the addition of Cu(I) (0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, and 1.1 equivalents)……………………………………………………….56
Figure III.10 Mini-C9C12-GGY upon the addition of Cu(I) (1.3, 1.5, 1.7, 2.1, 2.5 equivalents) ………………………………………………………...... …………57
Figure III.11 The near-UV CD titration of mini-C9C12-GGY by Cu(I)………….……….…….57
x
Figure III.12 Different spectra of a 100 M solution of mini-C9C12-GGY (0.2 M acetate buffer, pH 5.4) upon the addition of 0.1-1.1 equivalents of Cu(CH3CN)4PF6……………………………………………………………...……59
Figure III.13 Different spectra of a 100 M solution of mini-C9C12-GGY (0.2 M acetate buffer, pH 5.4) upon the addition of 1.1-2.0 equivalents of Cu(CH3CN)4PF6 (10 mM in acetonitrile)…………………………………………………………...... ……….60
Figure III.14 Absorption change of mini-C9C12-GGY at 262 nm (■), 298 nm (●), and 236 nm (▲) vs. the Cu(I) equivalent. Spectra were measured in 0.2 M acetate buffer, pH 5.4………………………………………………………………………...………60
Figure III. 15 Emission spectra of a 100 M solution of mini-C9C12-GGY (0.2 M acetate buffer, pH 5.4) upon the addition of of Cu(CH3CN)4PF6 (10 mM in acetonitrile)……...62
Figure III.16 The change of maximum emission intensity of a 100 M solution of mini-C9C12- GGY (0.2 M acetate buffer, pH 5.4) upon the addition of Cu(CH3CN)4PF6 (10 mM in acetonitrile)…………………………………………………..…….…….62
Figure III.17 The emission maximum wavelength of mini-C9C12-GGY after the addition of Cu(I)……………………………………………………………………...………65
Figure III.18 Emission spectrum at 77 K of a 100 μM solution of mini-C9C12-GGY (0.2 M acetate buffer, pH 5.4) upon the addition of 1.0 equivalent Cu(CH3CN)4PF6 (10 mM in acetonitrile)………………………………………………………………66
Figure III.19 600 nm excitation spectrum of Cu(I)/mini-C9C12 at room temperature (A) and 77K(B) in pH 5.4 acetate buffer…………………………………………………67
Figure III.20. 425 nm excitation spectrum of Cu(I)/mini-C9C12 at 77K in pH 5.4 acetate buffer with 50% (v/v) glycerol…………………………………...……………………..67
Figure III.21 Emission decay of Cu(I)/mini-C9C12-GGY recorded at 77 K………….……...…68
Figure III.22 Proposed qualitative scheme describing the luminescence properties of Cu(I)/mini- C9C12-GGY……………………………..…………...…………...... ……..71
Figure III.23 Triplet decay trace of ca. 25 µM Cu(I)/mini-C9C12-GGY in argon-saturated solution……………………………………………..……………………….……73
Figure III.24 Emission spectra of Cu(I)/C16C19-GGY and Cu(I)/mini-C9C12-GGY recorded at 77 K…………………………………………….………………………….……..74
Figure III.25 Redox titration curve of Cu(I)/mini-C9C12-GGY monitored by optical spectroscopy………………………………………………………………..…..….75
Figure III.26 The emission of 100uM Cu(I)/mini-C9C12-GGY without oxidation (black) and the xi
reduced Cu(I)/mini-C9C12-GGY by 40 μl TCEP (25 mg/ml concentration) after oxidation by excessive addition of K2IrCl6 (red)…………………….…………..76
Figure III.27 Cyclic voltammograms of two modified Cu peptides ………………………….…76
Figure III.28 Triplet decay traces of 100 µM mini-C9C12-GGY and 100 µM Cu(I) in 0.2 M pH 3+ 5.4 acetate buffer in the (a) absence and (b) presence of 60 µM [Ru(NH3)5Lut] ……………………………………………………..……………………………..78
Figure III.29 Transient absorption spectrum of a solution of Cu(I)/mini-C9C12-GGY (330 μM) and [RuA5(lut)]3+ (330 μM) measured 20μs after the laser flash……………..…79
Figure III.30 Pseudo first-order kinetic plots of the observed rate constants (kobs) for quenching of the emission for the fast (●) and slow (■) decay components of Cu(I)/mini- C9C12-GGY as a function of quencher concentration……………………....…..80
Figure III.31 Stern-Volmer plots of the quenching of 100 µM Cu(I), 100 µM mini-C9C12-GGY 3+ in 0.2 M pH 5.4 acetate buffer by [Ru(NH3)6] , showing the linear fit through the data points…………………………………….………………………………….81
Figure III.32 Driving force dependence of bimolecular quenching rate constants for the short lifetime component of Cu(I)/C16C19-GGY (▲) and Cu(I)/mini-C9C12-GGY (●)………………………………………………………………….…………….85
Figure III. 33 Driving force dependence of bimolecular quenching rate constants for the long lifetime components of Cu(I)/C16C19-GGY (▲) and Cu(I)/mini-C9C12-GGY (●)…………………………………………………..……………………………86
Figure IV.1 MALDI-TOF mass spectrum of the purified AQC16C19-GGY…………..…....….94
Figure IV.2 MALDI-TOF mass spectrum of the purified AQVC16C19-GGY……………..…..95
Figure IV.3 MALDI-TOF mass spectrum of the purified mini-AQVC9C12-GGY…………..…95
Figure IV.4 The plot of the denaturation of AQC16C19-GGY (▲) and AQVC16C19-GGY (■) with increasing concentration of guanidinium chloride……………...... ….…..…..97
Figure IV.5 Determination of molecular weight of AQC16C19-GGY velocity ultracentrifugation, (10 mM acetate buffer, pH 5.4)……………………..…….…..99
Figure IV.6 High performance size exclusion chromatography (HPSEC) chromatogram of the AQC16C19-GGY and AQVC16C19-GGY monitored at 275 nm…………….....100
Figure IV.7 Circular dichroism spectra of aqueous solutions of AQC16C19-GGY (111 μM) (■) and Cu(I)/AQC16C19-GGY (●) (with 111uM Cu(I) in pH 5.4 acetate buffer, 298 K)…………………………………………………………………………..……..101 xii
Figure IV.8 CD spectra obtained by successive addition of Cu(I) to 100 μM AQVC16C19-GGY peptide in 10 mM, pH 5.4 acetate buffer. (Cu(I) equivalents: 0-2.75)...... …103
Figure IV.9 Plots of signal at 222 nm (θ222 nm) (A) and signal ratio at 222 nm to that at 208 nm (θ222 nm/θ208 nm) (B) as a function of Cu(I) equivalents…………………….……...103
Figure IV.10 Circular dichroism spectra of mini-AQVC9C12-GGY (100 μM) (■) and Cu(I)/ mini-AQVC9C12-GGY (●) (100 μM) in pH5.4, 10mM acetate buffer 298 K…...... 104
Figure IV.11 The difference UV–vis absorption spectra obtained upon successive adding Cu(I) (0.0-2.0 equivalents) to 98.9 μM AQC16C19-GGY in pH 5.4, 0.2 M acetate buffer (A), and plot of delta absorbance at 262 nm and 298 nm vs. Cu(I) equivalents (B)…………………………………………………………..………………….…106
Figure IV.12 The difference UV–vis absorption spectra obtained upon successive adding Cu(I) to 100 μM AQVC16C19-GGY in pH 5.4, 0.2 M acetate buffer (A), and plot of delta absorbance at 268 and 298 nm vs. the Cu(I) equivalents (B)………………….....107
Figure IV.13 The difference UV–vis absorption spectra obtained upon successive adding Cu(I) to 90.4 μM mini-AQVC9C12-GGY in pH 5.4, 0.2 M acetate buffer……………………………………………………………………..……….109
Figure IV.14 Emission titration of AQC16C19-GGY by [Cu(CH3CN)4]PF6: Emission spectra obtained upon addition of Cu(I) to the 131.8 μM peptide solution 0.2 M acetate buffer (pH5.4)…………………………………………………………………….110
Figure IV.15 Emission decay of Cu(I)/AQC16C19-GGY excited at 355 nm monitored at 590 nm ……………………………………………………………………………...……..111
Figure IV.16 Emission titration of AQVC16C19-GGY by [Cu(CH3CN)4]PF6……...... …...…112
Figure IV.17 Emission decay of Cu(I)/AQVC16C19-GGY excited at 355 nm monitored at 590 nm…………………………………………………………………………..…….112
Figure IV.18 Emission titration of Cu(I)/mini-AQVC9C12-GGY by [Cu(CH3CN)4]PF6….....114
Figure IV.19 Emission decay of Cu(I)/mini-AQVC9C12-GGY excited at 355 nm monitored at 600 nm……………………………………………………………………………115
Figure IV.20 Genetic algorithm analysis 3D distribution plot of AUC data of Cu(I)/AQC16C19- GGY in 0.2 M pH 5.4 acetate buffer……………………………………..…….108
Figure IV.21 High performance size exclusion chromatography (HPSEC) chromatogram of Cu(I)/AQC16C19-GGY monitored at 275 nm……………………………….…118
xiii
Figure IV.22 The calibration curve obtained by using peptide standards on a Superdex 75 column………………………………………………………………………..…119
Figure IV.23 High performance size exclusion chromatography (HPSEC) chromatogram of the apo-AQVC16C19-GGY (—) and Cu(I)/AQVC16C19-GGY (—) monitored at 275 nm……………………………………………………………………...…..120
Figure IV.24 High performance size exclusion chromatography (HPSEC) chromatogram of the apo-mini-AQVC9C12-GGY (—) and Cu(I)/mini-AQVC9C12-GGY (—) monitored at 275 nm……………………………………………………………121
Figure A1 Crystal structure of Cd(II)/AQC16C19-GGY (A) and Cd4 cluster structure in Cd(II)/AQC16C19-GGY (B)…………………………………………...... ……129
Figure A2 Helical wheel diagrams for parallel three stranded coiled coil peptides…..…….….130
Figure A3 Genetic algorithm analysis of velocity ultracentrifugation results of Cd(II)/AQC16C19-GGY………………………………………………………...…..136
Figure A4 Genetic algorithm analysis of velocity ultracentrifugation results of Cd(II)/AQC16C19-GGY…………………………………………………………….136
xiv
LIST OF TABLES
Table Page
Table I.1 The peptide sequences of the TRI family………………………………….…………..11
3+ Table III.1 Redox potentials of [Ru(NH3)5X] and observed bi-molecular electron transfer rate 3+ constants between [Ru(NH3)5X] (X = chloro, ammine, 3,5-lutidine, pyridine, nicotinamide, and dimethyl 3,5-pyridine dicarboxylate) and Cu(I)/C16C19-GGY. …………………………………………………………………………………….….44
Table III.2 Schematic illustration of sequence difference between C16C19-GGY and mini- C9C12-GGY……………………………………………….…..……..……………..47
Table III.3 Near-UV CD spectra maxima of Cu(I) metallothioneins……...………….……...….54
Table III.4 Luminescence Properties of Cu8-MT, Cu12-MT,Cu(I)4, Cu(I)6 polyhedra and Cu(I)/mini-C9C12-GGY at 77 K ……………..……………………………………..70
3+ Table IV.5 Redox potentials of [Ru(NH3)5X] and observed bimolecular electron transfer rate constants……..……………………………………………………………………..82
Table IV.1 Sequence of synthesized peptides……………………………………...……....……94
Table IV.2 The molecular weight of AQC16C19-GGY and AQVC16C19-GGY in solution measured by SEC and AUC…………………………………………………..…….99
Table IV.3 The molecular weight distribution and percentage of AUC data of Cu(I)/AQC16C19- GGY in 0.2M pH5.4 acetate buffer………………..……………………………....117
Table IV.4 The molecular weight of the Cu(I)/peptide and their oligomerization states…...….125
Table IV.6 Properties of Cu(I)/peptides……………………………………………….……….125
Table A1 Cadmium coordination bond lengths (Å)…………………………………...... ….133
Table A2 Cadmium coordination bond angles (degrees)…………………………....…...….....133
Table A3 Geminal S···S distances……………………………………………..………….…….134
Table A4 Cd···Cd···Cd angles degree)………………………………………………..…...……134
1
CHAPTER I. INTRODUCTION AND RESEARCH MOTIVATION
I.1 Introduction to metalloproteins
Metalloproteins are a special class of proteins that utilize the unique chemical properties of metal atoms in conjunction with the macromolecular assembly to perform life-sustaining processes.1 In metalloproteins, the metal ion, such as iron, calcium, copper, and zinc, is usually coordinated by nitrogen, oxygen or sulfur atoms belonging to amino acids in the polypeptide chain and/or a macrocyclic ligand incorporated into the protein. The presence of the metal ion allows metalloproteins to perform functions that cannot easily be performed by the limited set of functional groups found in amino acids. Metalloproteins comprise approximately one-third of all structurally characterized proteins and play vital roles in many biological processes in cells, such as energy conversion occurring in photosynthesis, enzymes, oxygen carriers in respiration, and governing gene regulation and expression in a growing number of signaling processes.2
I.2 Metalloprotein types and functions
Metalloproteins are normally classified into the following categories: storage and transport metalloproteins, metalloenzymes, signal-transduction metalloproteins, and other functional metalloenzymes.2 These are defined below.
(a) Storage and transport metalloproteins This type of metalloproteins mainly functions to
carry and store essential elements such as oxygen and metal ions in cells. For example, the
principle oxygen carrier metalloproteins include hemoglobin, myoglobin, and hemerythrin
metalloproteins, which are iron-containing proteins.3 The iron ion (Fe(III)) is also stored in 2
ferritin and can be delivered by transferrin, a blood plasma protein. 4 Ceruloplasmin is a major
copper-carrying protein in the blood, which is associated with possible oxidation of Fe(II) into
Fe(III) to assist its transport by transferrin.5
(b) Metalloenzymes The metalloenzyme catalyzes reactions which are difficult to achieve
in organic chemistry with high yield. One example is that superoxide dismutase reduces the
superoxide ions near the diffusion limited rate, which is difficult to achieve in the organic
chemistry field.6 One common feature of metalloenzymes is that the metal ion is bound to the
protein with one labile coordination site. Such a coordination site is important to fit the substrate
and catalyze the reaction. Well-known examples of metalloenzymes include carbonic anhydrase,
vitamin B12-dependent enzymes, nitrogenase (nitrogen fixation) superoxide dismutase, and
chlorophyll.4
(c) Signal-transduction metalloproteins Calmodulin is an example of a signal-transduction metalloprotein. Binding of calcium causes a conformational change to occur in the protein to transfer signals.7, 8 In Calmodulin, the two alpha helices are positioned roughly perpendicular to
one another and linked by a short loop region (about 12 amino acids) that usually binds calcium
ions.
(d) Other functional metalloenzymes Some other metalloenzymes consisting of
magnesium, vanadium, manganese, nickel et al. metal ions play important roles in biological
systems. For example, DNA polymerases use a magnesium ion to catalyze the polymerization of
deoxyribonucleotides into a DNA strand.9 Hydrogenases use iron and/or nickel at their active
sites catalyses the reversible oxidation of molecular hydrogen.10, 11
3
I.3 Interaction of metal ions with proteins in metalloproteins
Many different kinds of interactions exist between metal ions and proteins in
metalloproteins which are related to properties of both metal ions and proteins. For example, the
metal binding affinity, the redox properties of metal ions, and the ligand geometry of the proteins
all affect interaction in metalloproteins.12 Williams et al. described four models for the
interaction between proteins and the metal ions, as illustrated in Figure I.1 (A)-(D).13 In these models, M and P represent the metal ion and protein, respectively. The model (A) shows one extreme condition where the coordination of metal ions is forced into uncommon coordination geometries by the stable protein fold. One example of this type of interaction is the Cu(II) ion in blue copper proteins, where the rigid protein geometry forces Cu(II) into a distorted ‘tetrahedral’ coordination sphere consisting of two sulphurs from methionine and cysteine, and two nitrogens from histidine.14 The electron transfer rate can be enhanced by this irregular and high energy
arrangement at the metal center because of the transition-state geometry between the tetrahedral
and square planar equilibrium configurations of the two oxidation states involved. The opposite
extreme condition (shown in model (D)) shows that the protein structure is organized by the
coordination of the metal ions. In this condition, the metal ion enforces the protein to fold into
different conformation. The zinc finger protein is one of the best-studied examples in this interaction condition.12 When zinc ion is absent, the domain of zinc finger protein is in a
denatured state since it is too small to have a hydrophobic core. However, upon binding with
zinc ion, the tetrahedral coordination sphere of zinc ion stabilizes the secondary and tertiary
structures of the peptide so that the biological function-DNA binding can be fulfilled. The other
two models, (B) and (C), are more intermediate cases. In model (B), metal binding occurs when
small changes in protein structure are induced by large changes in metal coordination geometry 4
to induce perfect fitting metal ion into the protein. In model (C), both conformations of the
protein and the metal ion undergo considerable changes upon binding in what is called an
“induced fit model”
Protein Metal ion Combination
A + M P
B + M P
C + M P
D + M P
Figure I. 1 Schematic illustration of the differences between the ways in which the properties of a group, which can be an intrinsic amino acid of a protein or an extrinsic factor such as a metal ion or a coenzyme, can be structurally energised by and/or can energise a protein structure. (from reference 9)
I.4 Metalloprotein design
I.4.1 Goals for metalloprotein design
Many essential biological functions require the presence of metal ions, and most of these
metal ions are involved in metalloprotein functions. Thus, gaining a better understanding of
properties and the working mechanism of metalloproteins is of great biological and medical importance. To this objective, much work has been done to mimic the behavior of the natural metalloproteins via designing synthetic analogs.12, 15-22 Two main goals have been considered in
this work on metalloprotein design: (1) designing a perfect preformed coordination environment 5
for the metal, which must therefore generate the highest affinity. (2) attaining a structure for a
specific purpose by designing precursor structure which is not preformed but is created by
induced fit at the expense of binding energy. The first goal concentrates on building structures
with the lowest energy state of protein. This design generates the highest binding affinity, but
probably at the expense of function. The precursor structure can be inspired by studying the
naturally occurring protein scaffolds and engineering them away from their evolved function.
Thus the two strategies have been developed as following.
I.4.2 Design strategies
To achieve the goals of protein design, two complementary approaches called “top-down”
and “bottom-up” approaches have been developed to synthesize new types of chemically
functional metalloproteins.12, 17, 23, 24 The “top-down” approach starts with a native protein and
attempts to reengineer its structure to perform new functions.25-28 Two strategies are generally utilized to achieve this goal: (a) designing metal ion sites into folded non-metalloproteins, and (b) redesigning the existing metal binding sites to bind different metal ions.
The “bottom-up” design starts with the already designed peptides and inorganic building blocks and assembles them in order to endow the metalloprotein chemical functions. Both structural design and functional design are employed to obtain the aimed protein.
(a) “Top-down” design metalloproteins
Two methods have been developed in the “Top-down” design strategy.
(i) Designing metal ion sites into folded non-metalloproteins
In this design strategy, either natural or unnatural amino acids are introduced into the sequence 6
when a native protein which does not normally bind metal ions. For the natural amino acids,
histidine and cysteine are mostly used in the design of metal binding sites. For example,
Hellinga’s group redesigned the Escherichia coli maltose-binding protein (MBP) to bind a
binuclear copper using this method.24, 29, 30 The MPB has two stable conformations which can
switch in response to the substrate binding. Therefore, there are more chances to arrange the
histidine ligands in the right place for metal coordination. As a result, computational designs of
five candidates consisting of 9 to 10 mutations each were constructed by oligonucleotide-
directed mutagenesis. A 10-fold mutant, MBP.Hc.E, can bind two CuII or two CoII metal ions to
II II II form Cu 2 and Co 2 complexes that interact with H2O2 and O2. The formed Co 2 complex is
spectroscopically similar to a synthetic model that structurally mimics the oxy-hemocyanin core,
II whereas, the Cu 2 complex is not. Another designed metalloprotein in this family is I329F. I329F
with a binuclear copper center has shown a similar spectroscopic signature of oxy-hemocyanin
when reacted with low concentrations of H2O2 and favors a closed conformation because of the
introduction of one additional mutation in the hinge region of MBP.. Through the strategy of
designing metal ion sites into folded non-metalloproteins, the authors not only introduced a
copper oxygenase active site into a non-metalloprotein but controlled the reactivity of the
designed metalloprotein.29-31
Unnatural amino acids can also be incorporated into proteins to design functional metal
ion binding sites in the top-down to design metalloproteins. Several methods have been
developed to conduct this type of metalloprotein design. One method is to disable a particular
amino acid expression in cells to replace one type of amino acid (i.e. an auxotroph) in the protein
with an unnatural amino acid and supplementing the growth media with an analog of the natural
amino acid.32 However, this method can only replace limited amino acids and is not position- 7
specific. Ikeda et al. studied the incorporation of several histidine analogues and successfully
incorporated a novel histidine analogue b-(1,2,3-triazol-4-yl)-DL-alanine into the protein in
vivo.32 The second method is chemical modification of an amino acid residue, but this
modification is limited to some amino acids such as cysteine, and located at the desired
position.33 For example, Jones and co-workers modified the cysteine M222C, a buried residue in
the S1 pocket in subtilisin Bacilus lentus (SBL), with a series of methanethiosulfonates. Such
modification resulted in significant decreases in catalytic efficiency levels, as much as 122-fold
lower than wild type.34 Semi-synthesis is another method which can be used as a compromise
method to incorporate unnatural amino acids into proteins to replace one of the metal ion ligands
via site directed mutagenesis with small amino acids.35
The utilization of the translation of tRNA in vitro is can also be used as a method to insert unnatural amino acids.36-38 The recent development of the expressed protein ligation (EPL)
method makes it possible to link the bacterially expressed peptides with synthetic peptides
without the requirement of protection group. This method not only lowers the cost but also
increases the yield due to the low cost of bacterially expressed peptides and the coupling of
synthetic peptide without the protection group. Using the EPL method, Lu and coworkers used
selenocysteine (SeCys) and selenomethionine (SeMet)/norleucine to replace both Cys and Met
ligands site-specifically in type 1 blue copper azurin respectively.39-41 Those isosteric
replacements in the copper center can finely tune the structural and functional properties without
altering the metal-binding environment. As a result, the azurin with SeCys shows a 50 nm red-
shift of the visible charge transfer band and a two-fold increase in EPR hyperfine coupling
constant. However, the limitation of this method is that it is hard to introduce unnatural amino
acids close to the C- or N-terminus because a cysteine has to be placed to connect the peptides. 8
(ii) Redesigning the metal binding sites to bind different metal ions or cofactors modification
Rather than reengineering the binding site of metal ions in the first strategy, reengineering the natural metalloprotein to bind other metal ions or design a metal binding site in a non- metalloprotein is normally the second method used in metalloprotein design. This method basically aims at the functional metalloprotein design. Gray and coworkers developed appending synthetic redox-active groups to proteins by chemical modification.42, 43 The histidine at different position for example His83 can react with ruthenium-labeling reagents for example
2+ Ru(bpy)2(im) . The resulting ruthenium binding protein is useful to study the distance
dependence of electron transfer along β-strands. Benson et al also used ruthenium complex to
modify MPB at a structurally defined location.44 This modification endows the controllable conformation by changing the potential.
The reconstitution of the natural cofactor, such as heme, can introduce functional group to
modify the properties of metalloproteins. Hamachi and coworkers have successfully modified the
heme cofactor and introduced a hydrophobic long alkyl chain, metal complexes, and other
functional groups into myoglobin to enrich its functions. For example, the incorporation of a
long alkyl chain greatly facilitates conversion of a water-soluble protein into a membrane-bound
protein. Attachment of a photosensitizer has enabled us to switch the enzyme activity by means
of visible light.45
(b) “Bottom-up” design of metalloproteins
The “bottom-up” design of metalloprotein starts with the synthesis of polypeptides that are able to bind metal ions. In recent years, the onslaught of modern protein crystallography and the 9
rapidly improving computational methods for protein design makes this approach more attractive
to the biochemists.21, 46, 47 For example, α-helical bundle or small β-sheet with a high affinity for
different metals and metal cofactors can be easily designed based on the current knowledge. The
organization of the ligand can be assessed by the crystallography which is helpful in designing
the polypeptides. In the early development “bottom-up” design of metalloprotein, most of the
work focused on designing the native-like peptide scaffolds that can bind specific metal ions.48
With more structural information obtained from designed helix-bundle, the functional design has attracted more attention.
(i) Native-like peptide scaffolds design
The α-helix and β-sheet are two common motifs in the secondary structure of proteins.
Using current computational methods, the design of an α helical bundle or a small β sheet having a high affinity for binding different metals and metal cofactors can be conducted.21, 46, 49
Compared to the β-sheet which have a good stability to form a rigid preorganized ligand sphere,13 α-helix is inherently more flexible, and might change its conformation upon the binding
of metal cofactors.
Figure I.2 Schematic views of synthetic coiled-coils. Top: primary amino acid sequence of the two helices of the coiled-coil. Left: Helices in parallel orientation. Right: helical wheel diagram 10
of the coiled coil; view down helical axes of the coiled-coil.( adapted from reference)50
Thus, an interesting candidate for a metal binding peptide scaffold the coiled coil which is a structural motif in proteins based on the α-helix. Here, two or more α-helices are coiled together like the strands of a rope. The synthetic coiled-coil sequence consists of three or more repetitions of the seven amino acid residue heptad, denoted by the letters (abcdefg). Positions a
and d of the heptad are occupied by hydrophobic amino acids, positions b, c, and f of the heptad
repeat are occupied by hydrophilic residues, exposed to the solvent, and positions e and g are
occupied by oppositely-charged residues to form salt bridges between complimentary helices.51
A helical wheel diagram (Figure I.2) illustrates the sequence of a parallel two-stranded α-helical coiled coil. Even though the hydrophobic effect is the driving force for the formation of the coiled coil, the final folded structure of coiled coil can also be affected by small changes in the peptide sequence. Harbury et al. performed a landmark study using an archetypal coiled coil,
GCN4, in which rules were established that govern the way that peptide sequences affect the oligomeric state of coiled-coil.52, 53. For example, a dimer is formed when isoleucine and leucine
residues are placed at the a and d positions of the native coiled coil GCN4 respectively. A trimer
is formed when leucine and isoleucine residues are placed at the a and d positions of GCN4, respectively. Furthermore, replacing both a and d positions with L resulted in the formation of a tetrameric coiled coil.
Using the oligomerization diversity and structural simplicity of the coiled coil, several groups have used this motif in their studies of the metalloprotein design. For example, to mimic the natural dinuclear iron enzymes, DeGrado and co-workers designed two self-associated helix- loop-helix units which can bind two separate iron atoms.26-28, 54 Holm and coworkers designed
II the bridged Ni (μ2-SCys)[Fe4S4] protein which resulting from the helix-loop-helix motif which 11
created a bridged metal-binding site.55 The helical bundles containing cysteine or histidine as the
ligands were also studied to bind different metal ions.56-69
(ii) Functional design
The functional design of metalloproteins targets the creation of new metalloproteins with novel structures, controlled functions, and predictable properties. Such functional design and preparation of synthetic metalloproteins provides the opportunity to recruit new functions into
protein structures and will facilitate the creation of novel systems which can find a successful
application in pharmaceuticals, medical diagnostics, catalysis, affinity chromatography, and
protein engineering.19 There are two possible interplays between metal ions and coiled coil
structures. Metal ions can induce the helix bundle or coiled coil having less stability to a
stabilized structure in which the preference coordination of the metal ion is favored. However,
when the stability of the helix bundle or coiled coil is high enough to accommodate the penalty
of inserting metal ions or cofactors, it is possible that the coordination of the metal ions has to be
changed to fit the coiled coil structure. Pecoraro and co-workers56, 58, 61, 63-66, 68, 69 studied the
metal-binding properties of an important family of coiled-coil peptides prepared by subtle
modifications of the parent peptide known as “TRI”, as shown in Table I.1.
Table I.1. The peptide sequences of the TRI family: (Adapted from reference 35)
Name Tri Sequence BabyTRI Ac-G LKALEEK LKALEEK LKALEEK G-NH2 BabyL9C Ac-G LKALEEK CKALEEK LKALEEK G-NH2 BabyL12C Ac-G LKALEEK LKACEEK LKALEEK G-NH2 TRIL9C Ac-G LKALEEK CKALEEK LKALEEK LKALEEK G-NH2 TRL12C Ac-G LKALEEK LKACEEK LKALEEK LKALEEK G-NH2 GrandL9C Ac-G LKALEEK CKALEEK LKALEEK LKALEEK LKALEEKG-NH2
12
In Table I.1, TRI has the sequence Ac-G(LKALEEK)4G-NH2 which places leucine residues
at each of the heptad “a” and “d” positions. This was found to exist as a stable three stranded
coiled-coil at pH > 7 which is not consistent with Harbury’s rules.52, 53 The metal ion biding sites
are created by replacing one leucine residue with a cysteine at either position 9, 12, or 16 of the
sequence (TRI-L9C, TRI-12C, TRI-16C). The affinity of the peptides for soft metal ions such as
Hg(II), Cd(II), and As(II) was investigated. They found that the metal ion in those peptides can
exist as three-stranded coiled coils containing a very unusual three-coordinate metal center. It
was also found that the placement of the cysteine residues along the sequence had a significant
effect on the metal binding constant due to the different pKa of cysteines at different position. A smaller peptide with the same heptad repeat, called baby L9C, was used to investigate the mechanism of encapsulation of Hg(II) within the interior of coiled coils. They found that the two-strand coiled coil with a linear thiolato Hg(II)-center forms after the initial rapid collapse of two peptide chains. The final structure, a three-stranded coiled coil, then forms after the addition of the third peptide to the linear thiolato Hg(II) complex. Different from the binding mechanism of Hg(II), Cd(II) cannot bind either TRI-12C or TRI-16C at low pH, but Cd exists in an unusual trigonal geometry at high pH. Careful thermodynamic studies confirmed the existence of this relationship by showing a linear free-energy relationship between the self-association affinities of the TRI peptides and their ability to bind Hg(II) and Cd(II) ions in trigonal geometries. These studies proved that, within the TRI family of metalloproteins, the conformational preferences of the protein dictate the coordination geometry of the incorporated metal ion.
In related work, Tanaka et al. investigated the metal-induced peptide folding of a parallel
70 three-stranded coiled-coil peptide based on a preformed three-stranded YGG(IEKKIEA)4. One
or two histidine residues were introduced into the hydrophobic core at the third heptad repeat to 13
generate a metal ion binding site. The introduction of histidine decreased the stability of the
coiled coil structure and resulted in a random coil. However, the addition of metal ions such as
Ni(II), Co(II), Zn(II), and Cu(II) induces the random coil structure to a triple-stranded-α-helical bundle with a octahedral metal-binding site in the hydrophobic core comprised in histidine residues.
d a d a d a d a
IZ YGG IEKKIEA IEKKIEA IEKKIEA IEKKIEA
IZ-3adH YGG IEKKIEA IEKKIEA HEKKHEA IEKKIEA
IZ-AC YGG IEKKIEA IEKKIEA AEKKCEA IEKKIEA
Figure I.3 Helical wheel diagram of the coiled coil; view down helical axes of the coile
dcoil. Adapted from reference 67.67
In order to design a “soft” metal-binding site, Tanaka et al replaced the residues at a and d positions of the third heptad repeat with Cys and Ala residues to make a peptide (IZ-AC) with a
“soft” metal-binding site in its hydrophobic core.67 The designed apo-peptide was shown to exist
as a random coil in the absence of a metal ion due to the destabilization of cysteine. However, in 14
the presence of such “soft” metal ions as Cd(II), Cu(I), and Hg(II), the peptide can fold into a
three-stranded α-helical bundle with a trigonal metal-binding site, as indicated by circular
dichroism spectroscopy. In contrast, “harder” metal ions, such as Ni(II), Co(II), and Zn(II)
cannot induce the peptide self-assembly, which is indicative of metal ion selectivity.
Interestingly, Cd(II) and Hg(II) in the resulted three-stranded α-helical bundle adopt a trigonal coordination as shown in Figure I.4 which shows the enforcement of peptide environment.71
Although the Hg(II) or Cd(II) can be inserted into in a preformed peptide with an energized coordination, they are not redox active metal ions. The design of metalloproteins from the
“bottom-up” strategy with redox active metal ions can help to understand the pathway of long- range donor-acceptor interactions in metalloproteins. The synthetic metalloprotein can be used as a model to mimic the long-range electron-transfer reactions found in natural systems. Our group was focused on the subject over the years by designing a series of metalloproteins and conducting their electron transfer investigations.72 In early work, a 30-residue polypeptide called
H21(30-mer) as a model system for natural electron transfer proteins was designed with formed
two-stranded coiled-coils as a rigid scaffold and modified with inorganic redox centers
2+ 2+ 73 ([Ru(NH3)5-] and [Ru(trpy)(bpy)-] ) in a site-directed manner shown in Figure I.5. Electron- pulse radiolysis was used to study intramolecular electron transfer reaction in the systems and showed that the electron-transfer rate constant for the reaction occurring from the
2+ 3+ -1 [Ru(NH3)5H21] donor to the [Ru(trpy)(bpy)H21] acceptor is kET = 380 s . The electron reaction occurred over an estimated metal-to-metal distance of 24 Å across a noncovalent peptide-peptide interface.73 The intramolecular electron transfer occurring over long distances in
the designed metalloproteins is consistent with the distance-rate behavior observed in native
protein systems.74 15
Figure I. 4 Computer model of the Cu(I) adduct of C16C19-GGY. The metalloprotein exists as a four-stranded coiled-coil which encapsulates a tetranuclear Cu(I) thiolate cluster. (adapted from reference73)
Later work in our group designed functionally active synthetic metalloproteins with similar
metal cofactors as found in nature. A peptide called C16C19-GGY base on “IEALEGK” was designed. C16C19-GGY contains cysteine residues located at both the “a” and “d” positions of
its third heptad repeat to form a suitable metal ion (Cu(I)) binding site to construct a native-like
metal-binding domain within its hydrophobic core.57, 60 It was shown that the binding of Cu(I) ions induces the peptide to undergo a conformational change from a disordered random coil to a metal-bridged four-stranded coiled-coil with a Cu4 cluster in the core. The electron transfer
reactions of the designed metalloprotein with a series of Ru(III) complexes were also
investigated which result in the electron transfer in the Marcus inverted region. A
thermodynamic explanation for this observation is the prohibition of a close approach between
the donor and acceptor resulting from the hydrophobicity of the Cu(I) cofactor resulting in the
low electron transfer rate even at high driving force. It is anticipated that further investigation 16
into these systems will contribute to an understating of electron transfer in native metalloproteins
and the design of new kinds of functionally active synthetic metalloproteins.
I.5. Electron transfer study in metalloproteins and electron transfer theory
Electron transfer reactions are involved in a great many important aspects of biology and
biochemistry. Most significant examples for electron transfer reactions are respiration and
photosynthesis. It is the former that we get energy from food and oxygen and it is the later that
plant makes the food and oxygen we consume. During past decades, electron transfer study has
been greatly advanced both in experimental and theoretical studies.
The most important electron transfer theory is the one developed by R. A. Marcus in
1950’s. 75-78 According to this theory, the electron transfer rate is determined by not only the driving force for the reaction (-ΔG0) but also the reorganization energy (λ) and the electronic coupling (HAB) between reactants and products as expressed by Eq. I.1,
2 20 2π 2 G +Δ λ)( kET = H AB exp[− ] (Eq. I.1) πλ BTkh 4λ BTk
Where kET represents the electron transfer rate, T is the absolute temperature, h is Planck
constant, and kB is Boltzmann constant. The reorganization (λ) includes two contributions from the inner-sphere (λi) and outer-sphere (λo) reorganization, where λi and λo reflect the nuclear perturbations of the redox centers and the needed energy to change the surrounding medium due
to the electron transfer reaction, respectively. The electronic coupling (HAB) denotes the strength
of the interaction between reactants and products at the nuclear configuration of the transition
state. The electronic coupling between electron donors (D) and acceptors (A) is function of the 17
distance (r) between donors and acceptors. The kET has been shown to decrease exponentially with the distance between the redox centers according to Eq I.2. in a wide range of biological systems by Dutton and co-workers.79
G +Δ λ)( 20 β rrkk exp[)](exp[ −−−= ] (Eq.I.2) ET 0 0 4λRT
Meanwhile, the electronic coupling between two proteins is also determined by the specific arrangement of the transient protein-protein complex which is determined by local interactions such as hydrogen bonds, van der Waals interactions as discussed by Beratan80-82 and hydrophobic and electrostatic interactions. Therefore, by determining ET reaction rates under different conditions, such as different temperature and reaction driving forces, much structural information about protein-protein interactions, such as the reorganization energy, electronic coupling, and distance between reaction centers can be obtained. Comparing ET reaction rates of protein complexes exhibiting different configurations but similar driving forces can help understand the relationship between complex configuration and ET reaction kinetics.83
The most important contributions of the Marcus equation is the relationship between reorganization (λ) and the driving force (-ΔG0). When the driving force -ΔG0 < λ, the electron transfer rate will increase with an increase in the driving force (point I in Figure I.4), and it recheas the maximum when -ΔG0 = λ (point II in Figure I.4). However, in the “Marcus inverted region” in which -ΔG0 > λ, the electron transfer rate decreases when the driving force increases
(point III in Figure I.6). Thus, the inverted region where -ΔG0 > λ is mostly easily observed for those reactions with small reorganization energies and large driving forces. Unfortunately, it is rare to observe the inverted region for the bimolecular electron transfer reactions because
Marcus’s classical formalism Eq. I.1 will be concealed due to the diffusion of the reactants. Even 18 when small reorganization energies and large driving forces are present, the diffusional rate constant (kd) has to be considered due to the consecutive mechanism of the bimolecular electron transfer reaction. The electron transfer event is classically described through the following scheme, as shown in Figure I.5 in which D and A represent electron donor and acceptor, respectively; kd is the diffusion rate constant, k-d is the dissociation rate constant, ket are the electron transfer rate constants, and kp is the rate constant for pair separation.
kd ket D* + A D* A D+ A- k-d (Precursor complex) (Ion-pair state) -1 τ0 kp
+ - D + A D + A
Figure I.5 A schematic illustration of bi-molecular electron transfer mechanism.
+ - * The electron transfer rate can be obtained by d[D ···A ]/dt = ket[D ···A]. The concentration of
* [D ···A] can be attained under the steady state approximation, thus kobs = kq = kd/(1+k-d/ket), where kobs and kq are the observed the electron transfer rate constant. Therefore, two extreme cases as following relations (1) and (2) can be reached when ket << k-d and ket >> k-d.
(1) kobs = ket × kd/k-d = ket × Keq when ket << k-d
(2) kobs = kd when ket >> k-d, the reaction rate is determined by the diffusion
Thus the driving forces of bimolecular electron transfer reactions must me high enough to permit the electron transfer rates to lie outside the diffusion limit.84Until now, most of electron transfer reactions in Marcus inverted region were in unimolecular processes or in a rigid condition which eliminates the effect of diffusion.84 With the accumulation of more knowledge over the past decades, the factors of electron transfer are more tailorable, such as reorganization energies, 19 electron coupling, and driving force. Thus, designing of metalloprotein and studying the electron transfer reactions have both experimental and theoretical importance.
Figure I.6 Plot of lnk vs. -ΔG0. Points I and III are in the normal and inverted regions, respectively. Point II occurs at -ΔG0 = λ, in which lnk is a maximum. (adapted from reference85)
I.6 Research objectives and thesis outline
(a) Research objectives
Previous studies in our group sought to introduce electron transfer functionality in synthetic metalloproteins. Recently studies on the random coil C16C19-GGY indicates that Cu(I) ions induce it to a tertrameric coiled coil with strong room temperature emission. The electron transfer study with a series of ruthenium complexes shows the tetrameric coiled coil exhibits electron transfer in the Marcus inverted region.86 It was hypothesized that the hydrophobic core of Cu(I)/C16C19-GGY prohibits close approach between the donor and acceptor to lower the value of coupling energy and results in the lower electron transfer rates even under high driving forces. To test the assumption, the first objective in this dissertation focuses on designing a smaller peptide mini-C9C12-GGY which has same peptide sequence except that it has less 20 amino acids at its N-terminus. The shorter sequence will decrease its hyrophobicity and might result in a smaller metalloprotein. Its electron transfer properties might different from what observed for Cu(I)/C16C19-GGY and useful to test our assumption. The second project is aimed to study the interplays between the peptides and metal ions in the formation of metalloprotein.
C16C19-GGY has been shown to be a random coil, however, a tertrameric coiled coil forms upon the addition of Cu(I). Obviously, Cu(I) stabilized the coiled coil structure. However, intensive study about the formation of metal induced coiled coil is needed. For example, what is the role of peptide backbone in the formation of coiled coil? Whether the preformed coiled coil and its stability will influence the metal binding and the oligomerization state of the resulting metalloprotein? The research conducted by Pecoraro et al has shown that a preformed trimeric coiled coil can force metal ions such as Hg(II) or Cd(II) to an abnormal trigonal coordination geometry and determines the resulting metalloprotein.56, 58 Inspired by those studies, a series of peptides with different stability will be studied and the effect of peptide backbones to the structure of the resulting metalloprotein will be investigated and compared in the second project.
(b) Thesis outline
Chapter II will summarize the experimental principles, set-ups, and procedures for metalloproteins design and property investigation. The peptide synthesis and purification, the procedures for preparation of luminescence quenchers (Ru(NH3)5X(BF4)3 (X=3,5-lutidine, nicotinamide, pyridine and dimethyl 3,5-pyridine dicarboxlyate)) will discuss in sequence. High performance size exclusion chromatography, analytical ultracentrifugation, and static light scattering will be employed to measure molecular weights of peptides or metalloproteins. The techniques, emission lifetimes measurement, steady-state luminescence measurements, 21 nanosecond UV-Vis transient absorption spectroscopy, circular dichroism and near-UV circular dichroism spectroscopy, UV-vis titration, and conformational analysis, will be combined to study the properties of metalloproteins or binding processes of peptides to metal ions. Cyclic voltammetry technique will be utilized to measure the redox potential of ruthenium complexes, and redox potentiometry was used to determine the potential of metalloprotein in the solution.
Chapter III will discuss the synthesis of the mini-C9C12-GGY peptide and events involved when binding with metal ions to form metalloprotein. The electron transfer reactions of
Cu(I)/mini-C9C12-GGY with a series of Ru(III) complexes were also investigated. The UV and emission titration will be used to determine the stoichiometry of Cu(I) to peptide monomer.
Light scattering will be employed to determine the molecular weigh and get the oligomerization state of the resulting metalloprotein. The emission at 77 k and room temperature will be investigated. The lifetime measurement will be utilized to study the electron transfer. The electron transfer rates will be compared with those for Cu(I)/C16C19-GGY. These results and the assumption will be compared and analyzed to make the conclusion.
Chapter IV will study the effect of the metal ions on the oligomerization state of metalloproteins. Based on the dimer motif ‘IAALEQK’, three peptides (AQC16C19-GGY,
AQVC16C19-GGY, and mini-AQVC9C12-GGY) will be designed and synthesized. The measurement of the free energies of folding for these peptides will be conducted by denaturation experiment. AQC16C19-GGY is expected to have the highest free energy, then followed by
AQVC16C19-GGY, mini-AQVC9C12-GGY. Circular dichroism spectroscopy will be used to study the stability change upon the addition of Cu(I) ions. UV and emission titration experiment will be used to determined the stoichiometry of Cu(I) to peptides. The molecular weights of the resulting metalloprotein will be measured by analytical ultracentrifugation and/or size exclusion 22 experiments. The oligomerization states of the metalloproteins can be obtained according the molecular weights. The correlation analysis of the oligomerization states and the free energies of the peptide folding will be conducted to obtain the effect of free energies and/or the metal ions on the oligomerization states of the metalloprotein.
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28
CHAPTER II. EXPERIMENTAL
II.1 Materials
The Fmoc-protected L-amino acid derivatives, 2-(1H-benzortiazol-1-yl)-1,1,3,3,- tetramethyluronium hexafluorophosphate (HBTU), piperidine, diisopropylcarbodiimide, and anhydrous hydroxybenzotiazole (HOBt) were purchased from Peptides International Inc.
(Louisville, KY). The reagents chloropentaammine ruthenium (III) chloride, 3,5-lutidine, nicotinamide, pyridine, 3,5-pyridine dicarboxylic acid, and tetrakis(acetonitrile) copper(I) hexafluorophosphate were purchased from the Sigma-Aldrich Company (St. Louis, MO). All reagents were used as received.
II.2 Preparation of peptide and Ru(NH3)5X(BF4)3
II.2.1 Peptide synthesis and purification
Four peptides mini-C9C12-GGY, AQC16C19-GGY, AQVC16C19-GGY, and mini-
AQC16C19-GGY were synthesized using the solid-phase methods in an Applied Biosystems
Model 433 A peptide Synthesizer by standard Fmoc chemistry according their peptide sequence.
The 0.25 mmol scale protocol with followed by N-terminal capping protection strategy by acetic anhydride was used as the synthesis program. Activation was achieved by 2-(1H-benzotriazol-1- yl)-1,1,3,3,-tetramethyluronium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole
(HOBT) in DMF. The GGY tag in the peptide sequence was used to determine the peptide
-1 - concentration by measuring the absorption of the tyrosine residue at 275 nm (ε275 = 1450 M cm
1).1 Deprotection of the amino acid side chains and cleavage from the resin was performed by a reaction with a mixture of trifluoroacetic acid (88% v/v), phenol (5% v/v), triisopropylsilane (1% 29 v/v), 2-mercaptoethanol (1% v/v) and water (5% v/v) for 2.5 hours at room temperature. The crude peptide was filtered into cold anhydrous diethyl ether. The white precipitation was collected by vacuum filtration and dried under vacuum.
Waters Model 515 HPLC system equipped with a preparative Vydac C-18 column (10 μM particle size, 22×250 mm) and a Waters Model 994 diode array detector/spectrophotometer were used to purify the peptides. Linear gradient acetonitrile/water mixture containing 0.1 (v/v) % trifluoroacetic acid was used as the mobile phase. The flow rate of mobile phase is 5 or 6 ml/min.
210 nm, 230 nm, and 275 nm were set as the monitoring wavelengths. The collected peptide was verified by MALDI mass-spectroscopy and by re-injecting collected peptide to HPLC to confirm the purity.
II.2.2 Ru(NH3)5X(BF4)3 (X=3,5-lutidine, nicotinamide, pyridine and dimethyl 3,5-pyridine
dicarboxlyate) synthesis
Ru(NH3)5X(BF4)3 (X=3,5-lutidine, nicotinamide, pyridine and dimethyl 3,5-pyridine dicarboxylate) were prepared through a literature method2 with the following modification:
Firstly, an Ag(I) solution was prepared by dissolving 0.0749 g of silver oxide (0.323 mmol) in 2 ml of hot water by dropwise addition of trifluoroacetic acid. Then, 100 mg of [(NH3)5RuCl]Cl2
(0.343 mmol) was added to the prepared Ag(I) solution. The formed white AgCl precipitation was removed by filtration. The light yellow Ru(III) complex solution was bubbled with argon for
30 minutes followed by the addition of several pieces of zinc mercury amalgam. 0.3 g of X (X =
3,5-lutidine, nicotinamide, pyridine and dimethyl 3,5-pyridine dicarboxylate) was added to the ruthenium solution and the solution was stirred for 2 hours under argon condition at room temperature. Finally, the reddish reaction solution was filtered to remove zinc amalgam, and was 30 oxidized by adding hydrogen peroxide (30%) till the solution turned colorless. The formed yellow precipitation by adding absolute ethanol was collected by filtration and washed with ether. If no precipitation was formed, the solution needed to be concentrated and then absolute ethanol and a large amount of anhydrous ether were added in sequence. The product was dried under vacuum and purified by preparative reversed-phase C18 HPLC with a linear gradient of acetonitrile from 0% to 5% in 20 minutes. The purity of the product was checked by HPLC, electrochemistry, and extinction coefficient, respectively.
II.3 Electrochemical measurements
II.3.1 Cyclic voltammetry (CV)
Cyclic voltammetry (CV) technique performed on a BAS 100W Electrochemical Analyzer was used to measure the redox potential of ruthenium complex. The ruthenium aqueous solution was placed in a small cell equipped with a platinum working electrode, a platinum wire auxiliary electrode and a salt bridge. An Ag/AgCl reference electrode and the small cell were then put in a
2 ml cell with 3M NaCl solution to measure the redox potential.
The following equation was used to recalculate the redox potentials:
E (vs NHE) = E (vs Ag/AgCl) + 196 mV (Eq. II.1)
+ where E (vs NHE) and E (vs Ag/AgCl) represent the standard H2/H electrode Ag/AgCl
2+ electrode potential, respectively. The instrument was calibrated using an external Ru(bpy)3
3 3+ standard (E = 1.26 V vs. NHE). All electrochemical measurements of [Ru(NH3)5L] (L=3,5- 31 lutidine, nicotinamide, pyridine and dimethyl 3,5-pyridine dicarboxylate) were performed in 0.2
M pH 5.4 acetate buffer.
II.3.2 Redox potentiometry
The redox potentiometry was used to determine the potential of metalloprotein in the solution. The electrochemical redox potentials of the Cu(I)-metalloproteins were determined by monitoring the decrease of emission intensity at 600 nm upon the excitation at 300 nm. The titration was performed in a quartz cuvette equipped with a miniature reference electrode, a platinum working electrode and syringe needle to degas. Cu(I)/peptide complex solution were prepared in 0.2 M sodium acetate buffer, pH 5.4 in chamber, and transferred the solution to the cuvette. All potential measurements were operated in the cuvette with a small magnetic stir bar under argon flow at room temperature. The initial potential and spectra were recorded after 5 minutes argon bubbling and magnetic stirring. The oxidation of Cu(I)/peptide complex was achieved by addition of small aliquots of the fresh prepared solution of K2IrCl6. After the addition of the oxidant, the solution was allowed to equilibrate and stir for 10-15 minutes before recording the measurement. After completion of the titrations, the emission intensity was plotted against the measured potential, and the data were fitted to the single Nernst equation (n=1) (Eq.
II.2):
⎛ 1 ⎞ Y=Ireduced + ∆I⎜ ⎟ (Eq. II.2) ⎜ − mh /)( RTEEnF ⎟ ⎝ 10( + )1 ⎠
where Ireduced is the emission intensity at 600 nm corresponding to the reduced Cu(I) metalloprotein, ∆I is the change in absorbance or emission from reduced to oxidized forms, Eh is 32
the measured potential of the solution, and Em is the midpoint potential, F/RT=60 mV. The number of electrons (n) was set to 1, but other parameters were allowed to vary in the nonlinear fit.
Protein Film Voltammetry (PFV) experiments were carried out using a three electrode configuration in a glass cell. A platinum wire was used as the counter electrode and a calomel electrode as the reference. The working electrode used was either edge plane graphite
(Cu(I)/C16C19-GGY peptide) or basal plane graphite (Cu(I)/Mini) set in epoxy. The electrodes were cleaned manually using 1.0 μm grit alumina, sonicated, and rinsed with water before protein application. Protein films were developed by directly applying the solution of desired protein to the graphite surface. Experiments were preformed in a 0.200 M acetate (pH 4.0) solution at 25°C with a scan rate of 100 mV/s. Data was collected with the GPES software package.
II.4. Molecular weight determination of peptides and metalloproteins
II.4.1. High performance size exclusion chromatography (HPSEC)
HPSEC experiments were performed on a Waters Model 515 HPLC systems equipped with a Superdex 75 Amersham Pharmacia Biotech column. A Waters Model 996 diode array detector was connected to the column and monitored at a wavelength of 210 nm or 275 nm. The peptide samples were eluted using 0.1 M KCl / 0.05 M KH2PO4, with 0.4 ml/min flaw rate. The estimation of molecular weight was based on K , which can be calculated using Eq. II.3.4, 5 d
K = (V -V )/(V -V ), (Eq. II.3) d act 0 t 0
33
Where V is the elution volume of the solute, V is the void packing volume obtained by using act 0 blue dextran, and V is the total accessible volume of the column obtained by using 2- t mercaptoethanol.
II.4.2 Analytical ultracentrifugation
Analytical ultracentrifugation (AUC) experiments were performed in collaboration with Dr.
Borris Demeler, at Center for Analytical Ultracentrifugation of Macromolecular Assemblies,
University of Texas Health Science Center, San Antonio, Texas. The samples were prepared in
10 or 200 Mm sodium acetate buffer, pH 5.4 - 5.5 with 1-fold of metal ion or in 10 mM Tris buffer, pH 8.5 with 1-fold of metal ion. In the experiment, 280 nm or 230 nm (for the Cd(II)- metalloprotein) scans were taken at equilibrium from 3 concentrations (OD=0.3, 0.5, 0.7) and 4 different speeds (50.0, 53.3, 56.7 and 60.0 krpm). The obtained data were globally analyzed with
UltraScan (Demeler, B. (2005) UltraScan version 7.0).
II.4.3 Static Light Scattering
Static light scattering experiments were performed on a Wyatt Technologies (Santa
Barbara, CA) miniDAWN Tristar HPLC detector set up in-line with a Wyatt Technologies
Optilab rEX refractive index detector. The value of dn/dc for the metal-peptide complexs was determined by plotting the absolute value of the refractive index of samples with known concentration against the peptide concentration, and can be determined by nsample = (dn/dc) csample
+ nH20. Sample concentrations were determined directly from the intensity of the UV signals. The manufacturer’s Astra V software was used to fit the light scattering data obtained during the
HPSEC elution profile to Eq. II.4, 34
2 2 2 2 Rθ = K.c.Mw [1 - (16π /3λ )
where Rθ is the observed angle dependent Rayleigh ratio of the scattered light intensity, Mw is the weight-averaged molar mass of the metal peptide, Rz is the z-average root mean square radius, K is a constant describing the optical properties of the analyte, c is the weight concentration of the analyte, and θ is the scattering angle.
II.5 Spectroscopic investigations
II.5.1 Emission lifetime measurements
Emission lifetimes were measured with a nitrogen-pumped broadband dye laser (2-3nm
6 fwhm) from PTI (GL-3300 N2 laser, GL-301 dye laser) as described previously. BPBD was used as the laser dye (356-390nm) from a laser excitation at 337 nm. The emission of
Cu(I)/peptide complexes was monitored at 600 nm. Pulse energies were typically attenuated to
~100 μJ / pulse measured with a Molectron Joulemeter (J4-05). An optical filter (550 nm) was equipped in front of the Hamamatsu R928 PMT. The PMT signal was enlarged through a 50 Ω resistor to a Tektronix TDS 380 digital oscilloscope (400 MHz). Each data trace
The PMT signal was terminated through a 50 Ω resistor to a Tektronix TDS 380 digital oscilloscope (400 MHz). Typically, 128 laser shots collected at 2-3 Hz were averaged for each data trace. Lifetime measurements were performed on argon-saturated solution of Cu(I)/peptide dissolved in 0.2 M pH 5.4 acetate buffer. For the quench experiment for Cu(I)/mini-C9C12-
3+ GGY, the concentrated stock solution of [Ru(NH3)5X] (X=chloro, ammine, 3,5-lutidine, nicotinamide, pyridine and dimethyl 3,5-pyridine dicarboxylate) was added to the Cu(I)/mini- 35
3+ C9C12-GGY under the assumption that volume of [Ru(NH3)5X] will not affect the concentration of Cu(I)/mini-C9C12-GGY. The concentration of the peptides was determined by
-1 -1 the by UV-Vis spectroscopy using value of ε275 = 1450 M cm . The concentration of the
3+ [Ru(NH3)5X] was determined by measuring absorption of MLCT bands from the reduced
2+ [Ru(NH3)5X] by adding excess ascorbic acid. 0.01 M [Cu(CH3CN)4]PF6 stock solution were prepared by adding 0.049g [Cu(CH3CN)4]PF6 to 4.0 ml argon-saturated acetonitrile in glove box.
All the samples are freshly prepared before the quench experiments.
II.5.2 Steady-state luminescence measurements
Emission spectra were obtained on a Photon Technology International (PTI) fluorometer.
The excitation wavelength was set at 300 nm with a 2 nm slit. The measurements were recorded over 400-800 nm range with a 340 nm or 400 nm long-pass filter. Low temperature (77 K) emission spectra were obtained by using a cylindrical quartz Dewar vessel. Samples containing
50% glycerol were put in quartz NMR tubes and immersed in the Dewar filled with liquid nitrogen to obtain emission spectra.
II.5.3 Nanosecond UV-Vis transient absorption spectroscopy
Nanosecond UV-Vis transient absorption was obtained from a UV-visible laser flash photolysis spectrometer equipped pulses Continuum Surelite Q-switched YAG:Nd laser (355 nm, 5 Hz repetition rate, pulse energy was 5–10 mJ, pulse duration ca. 7 ns). Solution with
Cu(I)/mini-C9C12-GGY was put in a an open top 10 × 10 × 45 mm3 rectangular quartz cuvette with four clear windows. The transient absorption was monitored using a white light probe beam placed at right angles to the laser beam. The light from a 150 W Xe passes a fast shutter 36
(Uniblitz) and focuses on the sample solution. The re-imaged absorption information enters the slit of a SPEX 1681 (0.22m) monochromator. The monochromatic light was then detected with a modified (5-stage dynode amplifier) R928 PMT obtained from Hamamatsu (response time < 2 ns). Based on the output spectral irradiance from the white light source, the detector spectral response, and the optics transmittance, the information from PMT was enlarged with an 50 Ω input impedance and input to a Tektronix TDS-380 digital oscilloscope (400 MHz bandwidth) in a range from ca. 300 and ca. 800 nm and time scale 2.5 ns - 500 us.
II.5.4 UV-vis titration
The UV-vis spectra were obtained on a Hewlett-Packard model 8452 A diode array spectrophotometer. A cuvette with 1 cm path length was used. The spectra were collected over a range of 190 nm - 900 nm with a 2 nm resolution. The peptide solutions were prepared in glove box, and the peptide concentration was determined using the molar extinction coefficient, ε(275 nm) = 1450M-1cm -1.7 100 uM peptide solution was made and transferred to cuvette equipped with an argon filled balloon to do the measurement.
II.5.5 Circular dichroism (CD) and Near-UV CD spectroscopy
Circular dichroism spectra were obtained using an Aviv and Associates model 62 DS circular dichroism spectrometer (Lakewood, NJ). The experiment temperature was set as 25oC controlled by a thermoelectric temperature controller. A rectangular cuvette with 1 mm or 5 mm path length was used. Mean residue molar ellipticities were calculated according to the Eq. II.5,
[θ]=[θ]obs/(10*l c n) (Eq. II.5) 37
where [θ]obs is the observed ellipticity measured in degrees, l is the path length of the cell in centimeters, c is the molar peptide concentration, and n is the number of amino acid residues in the peptide. The wavelength range 190 nm - 260 nm or 230 nm - 400 nm are used for CD and near UV-CD, respectively. The peptide concentration is ca. 100 uM for the titration experiments.
The solutions were allowed to equilibrate for 5 minutes after the addition of metal ions.
The helicity content of apopetides and metallopeptides were calculated by comparison with the calculated ellipticity for a 32-residue peptide with 100% helicity, which can be obtained by using Eq. II.6,
[θ]100% helix = - 40000*[(n - 4)/n] (Eq.II.6)
where n is the number of residues.8, 9
II.5.6 Conformational analysis
The conformational stability of the peptides was determined from GdnHCl denaturation studies which were performed by monitoring the change of ellipticity at 222 nm with increasing
GdnHCl concentrations.10, 11 The experiment was carried out in 50 mM phosphate buffer (100 mM KCl, pH 7.0) with 0-8 M concentration of GdnHCl. Analysis of GdnHCl denaturation
curves was performed using a two-state unfolding model to determine the fraction folding. The fraction folding (F ) can be calculated from Eq. II.7, f
F = (Yobs-Yn)/(Yu-Yn) (Eq. II.7) app 38
where Yobs is the observed molar ellipticity measured in degrees, Yn and Yu are the ellipticities of the native folded and unfolded states, respectively.
The two-state unfolding model can be represented by → 2UD , in which D is the folded peptide and U is the unfolded peptide. K=[U]2/[D] is the equilibrium constant. Using the two- state model, Fapp can be derived by Eq. II.8,
2 1/2 Fapp=[(K +8K[Ptot] -K]/4[Ptot], (Eq. II.8)
where [Ptot] described total protein concentration and equals to 2[D]+[U]. According two additional equations K=exp(-ΔG/RT) and ΔG =ΔGH2O +MG×[GdnHCl], where R is the molar gas constant, T is the temperature in Kelvin, ΔG is the apparent free energy difference between folded and unfolded forms at a specific GdnHCl, and MG is the slope describing the dependence ofΔGH2O on GdnHCl concentration.
0 Yn and Yu can be obtained by the following calculations, Yn=Yn +Mn[GdnHCl] and
0 Yu=Yu +Mu[GdnHCl], where Mn and Mu are the slopes describing the dependence of Yn and Yu on GdnHCl concentration, respectively. All of the equations are combined into one equation
(Eq.II.9) and the unfolding curve is fit by Origin nonlinear fitting program.
0 0 Yobs=(Yu -Yn -(Mu-Mn)*x)*((((exp(-(ΔGH2O+m*x)/RT))^2+8*[Ptot]*exp
(-(ΔGH2O+MG*x)/RT))^0.5-exp(-(ΔGH2O+MG*x)/2477))/(4*[Ptot]))+
0 Yn +Mn*x (Eq. II.9)
39
References:
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Press: 1976.
2. Taube, H.; Gaunder, R. G., Inorg. Chem. 1970, 9, 2627-2639.
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Chem. Rev. 1988, 84, 85-277.
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20862.
40
CHAPTER III. PHOTOPHYSICS AND PHOTOINDUCED ELECTRON TRANSFER
PROPERTIES OF Cu(I)/MINI-C9C12-GGY
III.1 Introduction
The α-helical coiled-coil is a common structural motif in proteins in which more than two
(typically 2-7) α-helices are wound together to form a left-handed supercoil.1-5 The amino acid sequence of synthetic coiled-coil peptides can be based on a representative seven-residue
(heptad) repeat. A detailed description and schematic illustration of the positions of these residues in coiled coils can be referred to Section I.2.3 and Figure I.2 in Chapter I. Because of its relatively simple design and rich structural properties, the coiled-coil motif has been commonly chosen as a model system to the investigate protein properties and functions of natural protein.
Based on the coiled-coil motif, the peptide called C16C19-GGY, was previously designed by our group as a model electron transfer protein.6, 7 The sequence of C16C19-GGY consists of four repetitions of the seven amino acid residue heptad, IEALEGK, which was previously used by our group to prepare a variety of metal-substituted, two-stranded α-helical coiled-coils.8 To construct a potential metal-binding site within the hydrophobic core of the coiled coil, the isoleuciene at a and leucine at d positions of the third heptad in C16C19-GGY were replaced by cysteine residues. However, C16C19-GGY exists as a random coil due to the destabilization of cysteine residues. More interestingly, binding Cu(I) ions produces a dramatic conformational change to a four stranded coiled-coil containing a tetranuclear copper cluster as determined by analytical ultracentrifugation (AUC). Figure III.1. shows the Computer generated model of the
Cu(I) adduct of C16C19-GGY. 41
Figure III. 1. Computer generated model of the Cu(I) adduct of C16C19-GGY. The metalloprotein exists as a four-stranded-helical bundle which contains a cyclic Cu(I)4S4(N/O)4 cofactor.
Kharenko et al.9 reported that the Cu(I) adduct of C16C19-GGY has an intense (Ф =
0.053) luminescence at room temperature centered at 600 nm which persists upon allowing the protein to stand overnight under ambient conditions. It is noteworthy that similar photoluminescent properties have been reported for Cu(I) derivatives of other cysteine rich metal-binding proteins, such as metallothionein,10, 11 the Cox17 copper chaperone,12 as well as the copper responsive transcription factors ACE113 and CopY14. In these systems, polynuclear copper(I) clusters are buried within the protein to shield them from bulk solvent.15 The photoluminescence properties of Cu(I)/C16C19-GGY at room temperature suggests that it also likely contains a polynuclear Cu(I) cluster. The existence of polynuclear Cu(I) cluster in
Cu(I)/C16C19-GGY was verified with metal-ion titrations and X-ray absorption studies.6 The results suggested the presence of a cyclic Cu(I)4S4(N/O)4 cofactor in the protein in which each
Cu(I) atom is coordinated to two bridging cysteine residues and has a terminal nitrogen or oxygen ligand. 42
Besides the ambient temperature luminescence, the photophysical properties of
Cu(I)/C16C19-GGY have been further studied in our group by examining the luminescence quenching by the addition of ferricyanide, oxygen, and urea, respectively. The addition of ferricyanide can cause oxidation of Cu(I) to Cu(II). The luminescence quenching by ferricyanide addition indicates that the emitting species of Cu(I)/C16C19-GGY are associated with the reduced Cu(I) state as is the case for the native copper proteins mentioned above. It was also shown that exposing Cu(I)/C16C19-GGY to the well known triplet quencher molecular oxygen results in the decrease in emission intensity. The luminescence quenching with oxygen suggests that the luminescence of Cu(I)/C16C19-GGY has significant triplet character in the luminescent state. Urea is a well known and widely used chemical denaturant for proteins. The addition of urea in this system causes the unfolding of Cu(I)/C16C19-GGY, which increases the accessibility of the Cu(I)-luminophore to solvent molecules that results in the efficient deactivation of the emissive excited states through a radiationless pathway to the ground state.
This study shows that Cu(I)/C16C19-GGY exists in a hydrophobic, solvent-shielded environment to generate the strong luminescence. The ambient temperature luminescence and quenching studies suggest that Cu(I)/C16C19-GGY has potential to function as a photo-induced electron-transfer agent which can be monitored by emission lifetime experiments.
To conduct the electron transfer study with Cu(I)/C16C19-GGY as an electron donor, the electron reduction potential of Cu(I)/C16C19-GGY was determined by redox potentiometry.
Here UV-Vis absorption and/or steady-state emission spectra were monitored in the measurement as the difference in spectra of oxidized Cu(II)- adduct and Cu(I)/C16C19-GGY.
The obtained results were fit to a single (n = 1) Nernst equation (Y = Ireduced +
⎛ 1 ⎞ ∆I ⎜ ⎟ , already defined in Chapter II), yielding a single midpoint reduction ⎜ − mh /)( RTEEnF ⎟ ⎝ 10( + )1 ⎠ 43 potential (E0 (CuI/CuII)) of 343 mV vs. NHE. The reduction potential of excited state can be obtained by combining the emission measurement of Cu(I)/C16C19-GGY at 77 k (E00) according to equation E0* = E0 - E00. A series of ruthenium ammine with variable reduction potentials (E0(RuIII/RuII)) was synthesized as the electron acceptor as shown in Table III.1. Thus, the driving force of the electron transfer reaction can be calculated by -ΔG0 = E0(RuIII/RuII) - E0*.
The transient absorption of Cu(I)/C16C19-GGY and ruthenium ammines confirmed that the quench process is via the electron transfer reaction, instead of energy transfer. The study also showed that the emission quenching rate constants decrease with increasing driving force, indicating that the system is in the Marcus inverted region. The description for Marcus behavior with Eq. I.7 can be referred to Chapter I.3. This behavior is rarely observed for bimolecular electron transfer due to the diffusional effect of reactants in solution. In the case of
Cu(I)/C16C19-GGY, all the electron transfer rates are lower than the diffusion limit, thus the observation of Marcus inverted region is possible. The fits of the data to Marcus equation yield
-1 reorganization energy λ = 1.7 eV and electronic coupling element HDA ~ 5 cm (Figure III.2 and
Figure III.3, respectively). It was speculated that the small coupling energy may result from the separate distance between the donor and acceptor in the studied system since electronic coupling between electron donors (D) and acceptors (A) decreases exponentially with the increase of the distance (r) between donors and acceptors.16
44
3+ Table III.1. Redox potentials of [Ru(NH3)5X] and observed bi-molecular electron transfer rate 3+ constants between [Ru(NH3)5X] (X = chloro, ammine, 3,5-lutidine, pyridine, nicotinamide, and dimethyl 3,5-pyridine dicarboxylate) and Cu(I)/C16C19-GGY.
0 9 -1 -1 9 -1 -1 Quencher -∆G (eV) kS(10 M s ) kL(10 M s )
Ru(NH3)5Cl 1.687 3.27±0.14 2.53±0.09 Ru(NH3)6 1.787 5.86±0.21 3.47±0.05 Ru(NH3)5(lutidine) 1.982 4.43±0.06 2.44±0.08 Ru(NH3)5(pyridine) 2.027 2.46±0.07 1.36±0.05 Ru(NH3)5(nicotinamide) 2.080 3.46±0.04 1.96±0.06 Ru(NH3)5(dimethyl-3,5- 2.119 1.71±0.21 0.96±0.08 pyridine dicarboxylate)
Figure III. 2. Plot of the electron transfer rates of the long lifetime component in Cu(I)/C16C19- GGY adduct with Ru(III) complexes. The solid line is drawn based on the Marcus theory of electron transfer.
45
Figure III. 3. Plot of the electron transfer rates of the short lifetime component in Cu(I)/C16C19- GGY adduct with Ru(III) complexes. The solid line is drawn based on the Marcus theory of electron transfer.
The reorganization energy of Cu(I)/C16C19-GGY can be calculated by the reorganization energy of the electron transfer reaction λ=(λD+λA)/2, in which λD and λA are the reorganization energies of electron donor and electron acceptor, respectively.17 The reorganization of ruthenium pentammine complexes calculated by self exchange electron transfer reaction is around 1.20 eV.18 Thus the reorganization energy of Cu(I)/C16C19-GGY was calculated to be λ = 2.2 eV,
2+/+ 11 which is larger enough to be comparable with [Cu(phen)2] (λ = 2.4 eV) . The larger reorganization energy means that the redox center undergoes a large solvent reorientation and/or
2+/+ large conformational change before and after the electron transfer. In the case of [Cu(phen)2] ,
+ the reduced state [Cu(phen)2] has a tetrahedral geometry, however, its coordination geometry
2+ undergoes a dramatically changes upon oxidation of the copper ions. The complex [Cu(phen)2]
+ is significantly more planar than the corresponding [Cu(phen)2] complex. The large
2+/+ conformational change results in the large reorganization of [Cu(phen)2] . The similar large 46 reorganization energy observed for Cu(I)/C16C19-GGY suggests that Cu(I)/C16C19-GGY may involve a considerable conformational change due to one-electron oxidation of Cu(I) to Cu(II) under the investigated conditions. Thus the large value of λ and small value of HDA apparently lowers the values of kET below the diffusion limit permitting the inverted Marcus behavior to be observed. It was therefore hypothesized that the hydrophobic core of Cu(I)/C16C19-GGY prohibits close approach between the donor and acceptor to lower the value of HDA.
To test the above hypothesis, an analogous Cu(I) metalloprotein, Cu(I)/mini-C9C12-GGY, was synthesized for present study. The sequence of mini-C9C12-GGY is identical to that of
C16C19-GGY except that it has been shortened by seven residues at its N-terminus (shown in
Table III.2). Although the sequence similarity to that of C16C19-GGY and the expected emission, Cu(I)/mini-C9C12-GGY metalloprotein might exist as a random coil instead of the coiled coil structure found in Cu(I)/C16C19-GGY because shortened sequence decreases the hydrophobic interactions. The random coil structure of Cu(I)/mini-C9C12-GGY offers greater accessibility to surrounding solvent so that the quencher can stay closer to the Cu(I) cluster center compared with that in the coiled coil structure of Cu(I)/C16C19-GGY. Therefore, the distance between the donor and acceptor is expected to be shorter and the electron transfer rate is enhanced due to the larger HDA. This study will assist us to elucidate the mechanism of electron transfer occurring in Cu(I)/C16C19-GGY and related systems, such as the effect of minor change of protein environment on the electron transfer rate. It also furthers our understanding in designing functional metalloproteins. The photophysics of Cu(I)/mini-C9C12-GGY is also investigated in order to understand its structure and properties for assisting electron transfer studies in the present chapter.
47
Table III.2. Schematic illustration of sequence difference between C16C19-GGY and mini- C9C12-GGY
C16C19-GGY KIEALEGKIEALEGKCEACEGKIEALEGKGGY
mini-C9C12-GGY KIEALEGKCEACEGKIEALEGKGGY
III.2 Synthesis of mini-C9C12-GGY and Cu(I)/mini-C9C12-GGY
III.2.1. Mini-C9C12-GGY peptide synthesis
Using solid-phase peptide synthesis, the 25-residue peptide, mini-C9C12-GGY, was prepared with the following sequence:
Ac-K(IEALEGK) (CEACEGK)(IEALEGK)-GGY-amide
Where GGY provides the spectroscopic tag introduced for determination of the peptide concentration in aqueous solutions on the basis of the extinction coefficient of tyrosine (ε275
=1450 M-1cm-1).19 Purification of the apo-peptide mini-C9C12-GGY was conducted by preparative reverse-phase C18 chromatography. The identification of mini-C9C12-GGY peptide was carried out by MALDI mass-spectroscopy (m/z ion), (calculated: 2667.06 found:
2667.85.[M]+, 2689 [M+Na]+, 2705 [M+K]+), as shown in Figure III. 4.
48
Figure III. 4. MALDI mass spectrum of synthesized peptide mini-C9C12-GGY.
III. 2.2 Synthesis of Cu(I)/mini-C9C12-GGY
A previous study has shown that C16C19-GGY can bind to Cu(I) in a 1:1 ratio with highest binding affinity. Excess Cu(I) causes new species to form in Cu(I)/C16C19GGY metalloprotein system with decrease of helicity and decrease of emission. The resulting
Cu(I)/C16C19-GGY peptide existed as a coiled coil tetramer having a strong emission at 600 nm.6 Mini-C9C12-GGY has the same sequence as C16C19-GGY except that it has one less heptad repeat. The synthesis of Cu(I)/mini-C9C12-GGY was carried out using the same procedure as Cu(I)/C16C19-GGY as described in Chapter II.
III.2.3 Determination of oligomeric state of Cu(I)/mini-C9C12-GGY
High performance size exclusion chromatograph (HPSEC) and multi-angle laser light scattering (MALLS) were used to determine the molecular weight of Cu(I)/mini-C9C12-GGY 49
(the adduct of 1 equivalent Cu(I) adding to mini-C9C12-GGY). High performance size exclusion chromatography followed by static light scattering experiment (HPSEC-LS) was carried out to acquire more accurate information on the molecular weight of Cu(I)/mini-C9C12-GGY metalloprotein in aqueous solutions.20-23 The principle behind this techniques and the experimental details on these techniques are described in Sections II.10 and II.13 in Chapter II, respectively.
Figure III. 5. dn/dc measurement of Cu(I)/mini-C9C12-GGY by MALLS.
8.65 5
)] * 10 * )] 8.60 θ
[Kc/R( 8.55
8.50 0.0 0.2 0.4 0.6 0.8 1.0 2 Sin (θ/2) 50
Figure III. 6. Zimm plot showing the angle dependence of the scattered light intensity from a representative time slice of the HPSEC elution peak. The straight line represents the best fit to eq -1 II.1 to yield Mw= (11,400 ± 800) g mol .
Figure III.5 shows the differential refractive index change with the concentration of Cu(I)/mini-
-1 -1 C9C12-GGY which is determined by UV absorption (ε298 = 3300 M cm ). The dn/dc, also called the “specific refractive index increment”，can be calculated by nsample = (dn/dc)csample +
nH2O , where n is the refractive index, c is the concentration of protein. As shown in Figure III.5.
The fitting result shows the dn/dc is 0.1665±0.0045 mL/g, close to the reported number for
Bovine Serum Albumin (BSA), with a dn/dc value of 0.185 mL/g (at 690 nm).20-23 Figure III.6 shows the static light scattering experiment for Cu(I)/mini-C9C12-GGY. The straight line represents the best fit to Eq II.2 in Chapter II to yield Mw= 11.44 ± 0.8 kD. This value is very close to 10.92 kD, which is the calculated molecular weight of four mini-C9C12-GGY peptide chains with four Cu ions. Thus we conclude that the Cu(I)/mini-C9C12-GGY metalloprotein exists as a tetramer.
Figure III. 7 Determination of molecular weight of Cu(I)/mini-C9C12-GGY velocity ultracentrifugation, (10 mM acetate buffer, pH 5.4)
51
Analytical ultracentrifuge (AUC) was also performed to determine the oligomeric states of the Cu(I)/mini-C9C12-GGY. Figure III.7 shows the results obtained at 60.0 krpm speed (peptide concentration was checked by the absorption at 280 nm, A= 0.3, pH=5.4, 10mM acetate buffer) for Cu(I)/mini-C9C12-GGY system. Genetic algorithm analysis was employed to fit the data, yielding three major components with molecular weight of 9.98 kDa at percentage of 32.1 %,
17.7 kDa at percentage of 34.1 %, and 2.68 kDa at percentage of 18.0 %. Considering the molecular weight of mini-C9C12-GGY is 2.7 kDa, the component with molecular weight of 2.68 kDa is apo-mini-C9C12-GGY, and the component with molecular weight of 9.98 kDa is the tetrameric Cu(I)/mini-C9C12-GGY. However, no component with high molecular weight is observed in HPSEC experiment. Therefore, further analysis is necessary to fit the AUC results considering the component with high molecular weight and the high percentage of apo-peptide.
III.3 Spectroscopic property investigation
III.3.1. Circular dichroism (CD) spectroscopy
CD spectroscopy has long been used as a simple technique to detect the secondary structure of proteins. In general, the α-helical structure displays two negative CD signals in far-
UV region at 222 and 208 nm. The signal at 222 nm and signal ratio of 222 nm to 208 nm are usually used to estimate the content of the α-helix and the formation of coiled coil, respectively.
Generally, values of [θ]222/[θ]208 = 1.03 for coiled coil conformations in aqueous solutions and a
[θ]222/[θ]208 ≈ 0.86 for single-stranded α-helix polypeptides as the magnitude of the CD band at
208 nm is sensitive to the presence of interacting α-helices, as this band at 208 nm is polarized parallel to the helix axis. 24 Comparatively, random coil conformation is characterized by a negative absorption between 195 and 200 nm.25 In present study, CD spectroscopy was 52 employed to examine changes in the secondary structure of mini-C9C12-GGY peptide upon binding Cu(I) and the nature of the copper cluster so formed. Spectral changes were monitored sequentially when Cu(I) was added to mini-C9C12-GGY peptide solution fowling the titrating method as described in Chapter II. Figure III.7 shows the CD spectra of mini-C9C12-GGY before and after Cu(I) addition. Comparing these two negative peaks at 208 nm and 222 nm for
Cu(I)/C16C19-GGY, only one negative peak at 199 nm was observed for apo-mini-C9C12-GGY, which indicates that apo-mini-C9C12-GGY exists as a disordered random coil in aqueous solutions. The intensity of the negative peak at 199 nm increases slightly after the addition of
Cu(I). However, no characteristic signals of a coiled-coil were observed. Thus, different from
C16C19-GGY which forms the coiled coil structure upon Cu(I) addition, mini-C9C12-GGY exists as a random coil in both apo-state and after binding Cu(I) ions. The short sequence of mini-C9C12-GGY may be responsible for the random coil structure as discussed in Section III.1.
mini-C9C12-GGY 0 mini-C9C12-GGY with Cu(I) ) -1 -4 dmol 2 -8 deg cm
3 -12 ] (10 θ
[ -16
200 210 220 230 240 Wavelength/nm
Figure III. 8 Circular dichroism spectra of mini-C9C12-GGY (■) and mini-C9C12-GGY upon the addition of 1 equivalent of Cu(I) (●). Spectra were taken in 0.2 M acetate buffer at pH 5.4.
53
Near-UV CD spectroscopy has been shown to be quite sensitive to the formation of Cu(I) cluster in the metallothionein since the low energy bands are related to the spin forbidden 3d-4s metal cluster centered transition that is favored by the d10-d10 interactions of adjacent Cu(I) ions.
This characteristic transition requires multiple Cu(I) ions binding in close proximity each other, with bridging thiolate ligation necessary for the formation of cluster.26 This technique was also used here to follow the spectral changes when titrating Cu(I) into mini-C9C12-GGY. Figures
III.9-III.10 show the near-UV CD spectra of mini-C9C12-GGY upon introducing different amounts of Cu(I) into the peptide solution. As shown in Figure III.8, the addition of 0.1 to 1.1 molar equivalents of metal ion per peptide monomer produce three negative peaks at 262, 298, and 328 nm. These results are compared to the near-UV CD features for Cu(I) clusters found in many Cu(I)-metallothioneins in literature (Table III.3). By ananlogy to Cu(I)-metallothioneins, the high energy peak at 262 nm is from CysS-Cu(I) ligand to metal charge transfer (LMCT) transitions27. The low energy peaks at 298 nm and 328 nm can be assigned to spin-forbidden 3d-
4s transitions in copper clusters. 54
Table III.3. Near-UV CD spectra maxima of Cu(I) metallothioneins
Metalloprotein CD spectra maxima Reference Cu(I)/mini-C9C12-GGY (-)262 (-)298 (-)328 Cu(I)/C16C19-GGY (+)270 (-)295 (+)328 28 Native Cu,Zn-GIF (+)260 (-)284 (+)320 (-)355 29 Cu12-MT from Zn7MT (+)255 (-)280 (+)300 30 Cu8-MT (+)260 (-)283 (+)308 (-)325 (+)357 27 Cu12-MT from apo- MT (+)261 (-)283 (-)317 (+)356 31 Cu6 α-domain (+)245 (-)285 32 Cu6 β-domain (+)262 (-)285 32 Bovine fetal Cu, Zn- (+)250 (-)285 33 MT-1 Cu4-GIF(1-32) (+)262 (-)290 (+)310 (-)325 (+)350 34 Cu6-GIF(1-32) (+)262 (+)292 (-)313 (+)335 34 Cu4-GIF(32-68) (+)260 (-)285 (+)305 (-)323 (+)358 35 Cu6-GIF(32-68) (+)260 (-)285 (+)305 (-)323 (+)358 35
The low energy bands are from the Cu(I)-Cu(I) interactions within a polynuclear Cu(I)- cluster，27 which suggests that Cu(I)/mini-C9C12 also contains a multinuclear site. A close examination of the peak intensity in Figure III.10 shows that the signals at 262 and 298 nm are more negative and the peak at 328nm loses intensity in a linear manner upon addition of Cu(I) up to 1.1 equivalents of Cu(I). It is also seen from Figure III.8-10 that the shape of the CD spectra are similar for samples with Cu(I)/ mini-C9C12-GGY ≦ 1.1. However, upon the addition of >
1.1 equivalents of Cu(I), the spectra shows two obvious changes as shown in Figure III.9. Firstly, a new isodichroic point appears at 298 nm when 1.3 to 2.1 equivalents of Cu(I) are added compared to 280 nm when < 1.1 equivalents of Cu(I) were added. This indicates that another cluster might form in Cu(I)/mini-C9C12-GGY at high metal loadings. Secondly，the trend of peak intensities change with the addition of Cu(I) (Figure III.9). The peak at 262 nm undergoes a rapid increase and gets more positive with increasing Cu(I) addition, reaching a maximum at 2.1 equivalents of Cu(I). 55
Near-UV CD spectroscopy also has shown to be useful to examine the changes of coordination geometry of Cu(I) ions in the Cu(I) clusters. The coordination changes of metal ions in metallothionein have been reported by titrating out the metal ion such as Cd(II) or Zn(II) with metal ions with higher binding affinity such as Cu(I) or Hg(II). Those studies indicate that the band maxima in near-UV CD spectrum are only dependent on the MLCT energies, and the
MLCT energies are dependent on the metal ions and the coordination of metal ions. Thus the band maxima will change when (i) the binding metal ions change; (ii) the coordination of the metal ions changes. For example, the band maximum of Zn7-rabbit- liver-metallothionein (Zn7-
MT2) is around 244 nm which results from the cysteine-Zn LMCT. However the band maximum moves to 255 nm when up to 12 equivalents of Cu(I) were added. Continued addition up to 15 equivalents of Cu(I) results in the decrease at 255 nm and the appearance of a low energy peak at
335 nm. The former process can be explained by the change of binding metal ions. The later process is due to the change of Cu(I) coordination from trigonal to diagonal.30 By analogy to the results in metallothioneins, the maxima band at 262 nm in near-CD spectra of Cu(I)/mini-
C9C12-GGY originates from cysteine-Cu(I) LMCT. And the change in LMCT band at 262 nm and the appearance of the low energy peak at 328 nm (Figure III.9-10) can be assigned to the coordination change of Cu(I). Before 1.1 equivalents of addition of Cu(I), Cu(I) ion most likely bind the peptide with a trigonal geometry which is the most common coordination geometry when copper ions bind to cysteine thiolate ligands of proteins and peptides15,27. However, immediately after the addition of 1.1 equivalents of Cu(I), the spectrum exhibits an rapid change at the LMCT band at 262 nm (Figure III.10) and a new low energy band at 330 nm. These changes can be interpreted by the coordination change of Cu(I) from trigonal to diagonal geometry at high loading of Cu(I).30 Similar coordination change was observed in Cu(I)- 56 metallothioneins and Ag(I)-metallothioneins. Further discussion about coordination changes of
Cu(I) in Cu(I)/mini-C9C12-GGY is also presented in III.3.2. Summing up, the CD data are consistent with the existence of two discrete species before and after 1.1 equivalents of added
Cu(I) in which the Cu(I) ion might have different coordination at different loading stage of Cu(I).
0
-300 -1
-600 dmol 2 -900
0.1 -1200 deg cm 1.1
-1500 Δ[Θ]
-1800 250 300 350 400 450 Wavelength/nm
Figure III.9. Mini-C9C12-GGY upon the addition of Cu(I) (0.1,0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9,1.0, and 1.1 equivalents). Spectra were taken in 0.2 M acetate buffer at pH 5.4.
57
600 -1 0 dmol 2 -600
deg cm 1.3 -1200 2.5 Δ[Θ] -1800
250 300 350 400 450 500 Wavelength/nm
Figure III. 10. Mini-C9C12-GGY upon the addition of Cu(I) (1.3, 1.5, 1.7, 2.1, 2.5 equivalents). Spectra were taken in 0.2 M acetate buffer at pH 5.4.
-1 400 dmol
2 0
-400 deg cm
262nm -800 Θ] [ -1200 0.0 0.5 1.0 1.5 2.0 2.5 Equivalents of Cu(I)
Figure III. 11. The near-UV CD titration of mini-C9C12-GGY by Cu(I). Spectra were taken in 0.2 M acetate buffer at pH 5.4.
58
III.3.2 UV-Vis spectroscopy
UV-Vis absorption titration experiments were also employed to determine the
Cu(I):peptide binding stoichiometries, and to gain some insight into the binding process of Cu(I) to the peptide. The experiments were performed using the titration method as described in the
Chapter II. Figures III.12-III.13 show the difference UV spectra of mini-C9C12-GGY obtained upon successive addition of Cu(I). In Figure III.11, two absorption bands centered at 236 nm and
262 nm with a shoulder of 298 nm are observed when variable Cu (I) equivalents from 0.1 to 0.9 equivalents of Cu(I) were introduced. Further addition of Cu(I) to 1.1 equivalents, however, causes a sudden decrease in the intensity of the 262 nm band with a slight blue-shift to 256 nm.
Interestingly, the intensities of the other two bands (236 nm and 298 nm) keep increasing intensity until the addition of Cu(I) to 1.1 equivalents, as shown in Figure III.11. Such features indicate the formation of a copper cluster in the Cu(I)/mini-C9C12-GGY complex, since that the bands around 236 nm and 262 nm are attributed to ligand to metal charge transfer (LMCT) transitions27, and the band at 298 nm can be assigned to spin-forbidden 3d-4s transitions in copper clusters as report for Cu(I) metallothioneins,27 and several copper chaperones.36
Figure III.13 plots the absorption intensity at 262 and 298 nm upon the addition of Cu(I) to mini-C9C12-GGY. The absorbance at 262 nm absorbance increase increases linearly with the incremental addition of Cu(I) until reaches the maximum when approximately 0.9 fold of is
Cu(I) added. With further addition of Cu(I) (1.0 ~ 1.1 fold), the absorbance at 262 nm decreases a little bit then increases back to approximately the maximum value (as when ~1.5 fold of Cu(I) was added). As for the absorbance at 298 nm, it does not reach the maximum until approximately
1.1 equivalent of Cu(I) was added to the mini-C9C12-GGY solution. With further addition of
Cu(I), the absorbance at 298 nm decreases slowly and then reaches plateau. two break points can 59 be observed during the loading of Cu(I) to mini-C9C12-GGY. The first break point is around 0.9 equivalent of Cu(I), the second one is around 1.1 equivalents of Cu(I).
Breaking point and isosbestic or isodichroic points can be used to identify the transition of new states or complexes formed during the Cu(I) titration30. The break points at 0.9 and 1.1 equivalents together with two isosbestic points centered at 288 nm and 316 nm indicate Cu(I) and mini-C9C12-GGY form a complex with a 0.9:1 metal to peptide stoichiometry first. With more Cu(I), they eventually form complex with 1.1:1 metal to peptide stoichiometry. This result is consistent with results obtained from CD spectra analysis, as discussed in Section III.3.1. This transition (from 0.9:1 to 1.1:1) brings relatively little change to the absorbance at 262 nm but a noticeable increase to the absorbance at 298 nm. It is likely the Cu(I) clusters changed/grew a lot in this transition while Cu-S bonds in Cu(I)/mini-C9C12-GGY metalloprotein did not change much. This transition also is observed in emission titration, as discussed in Section III.3.3.
0.8
0.6 0.1-1.1
0.4 A Δ
0.2
0.0
200 250 300 350 400 450 Wavelength/nm
Figure III. 12. Different spectra of a 100 M solution of mini-C9C12-GGY (0.2 M acetate buffer, pH 5.4) upon the addition of 0.1-1.1 equivalents of Cu(CH3CN)4PF6.
60
1.2
0.8 A Δ 0.4
0.0
200 250 300 350 400 450 Wavelength/nm
Figure III. 13. Different spectra of a 100 M solution of mini-C9C12-GGY (0.2 M acetate buffer, pH 5.4) upon the addition of 1.1-2.0 equivalents of Cu(CH3CN)4PF6 (10 mM in acetonitrile).
0.9 262nm 298nm
0.6 A Δ 0.3
0.0
0.0 0.5 1.0 1.5 2.0 Cu(I) Equivalent
Figure III. 14. Absorption change of mini-C9C12-GGY at 262 nm (■), 298 nm (●), and 236 nm (▲) vs. the Cu(I) equivalent. Spectra were measured in 0.2 M acetate buffer, pH 5.4.
III.3.3 Emission spectroscopy at room temperature
Room temperature luminescence measurement can be used to probe the Cu(I)-Cysteine binding process in metalloproteins since the formed Cu(I) complexes are emissive. Emission titrations have thus proven useful in studying the metal binding of Cu(I)-metallothionein and
Cu(I)/C16C19-GGY.6 In the case of mini-C9C12-GGY, the titration Cu(I) to mini-C9C12-GGY was performed as described in Chapter II. Figure III.15 shows the emission spectra of
Cu(I)/mini-C9C12-GGY as 0.1-2.0 equivalents of Cu(I) was added. Only one strong emission 61 band centered at 600 nm is observed after the addition of Cu(I). Similar emission bands at 550-
650 nm have been reported for quite a few metalloproteins, such as Cu(I)/metallothionein,15
Cu(I)/C16C19-GGY, 6 ACE1 transcription factor,13 and Cox17 copper chaperone.37 The long lifetimes of room temperature emission (τL = 14.8 μs, τS = 2.9 μs) indicate that the emission is spin forbidden. By analogy to Cu(I)/metallothionein and Cu(I)/C16C19-GGY, the emission band of Cu(I)/mini-C9C12-GGY at 600nm can be assigned to a mixed state of 3d-4s/LMCT of copper cluster in the Cu(I)/mini-C9C12-GGY metalloprotein. A detailed discussion for the presence of this emission band can be found in the following section which discusses the 77 K emission spectra.
Figure III.15(A) show that the emission intensity increases steadily with the addition of increasing Cu(I) amounts to 0.1 to 0.8 molar equivalent. A sudden accelerated increase in emission intensity is observed when 0.8 equivalent of Cu(I) is added till the maxima emission reaches at 1.0 equivalent of Cu(I). With further addition of Cu(I), the emission intensity changes in an opposite trend, decreasing with increasing Cu(I) addition. As shown in Figure III.15 (B), the emission intensity around 600 nm decreases rapidly when 1.0 to 1.3 equivalent of Cu(I) is added, and then more slowly when 1.4 to 1.6 equivalent of Cu(I) is added , and finally shows a slightly decrease in band intensity at 2.0 equivalent of Cu(I). To show the band intensity change with equivalents more clearly, the maximum emission intensity was plotted vs Cu(I) addition, as illustrated in Figure III.16. Similar to the tendency shown in Figure III.15, the maximum emission intensity increases near linearly when 0.1 to 0.8 equivalent of Cu(I) was added. A quick increase in emission intensity when 0.8-1.0 equivalent of Cu(I) was added, then a steep decrease when 1.0-1.3 equivalent was added. With further addition of Cu(I), the decrease in the maximum 62 emission intensity continues until reaching plateau value in emission intensity at the addition of
2.0 equivalent of Cu(I).
A B 1.1 8 1.0 6
-1 2.0 s
0.1
-1 6 −4 s
−4 4 ×10 4 ×10
2
2 Counts Counts
0 0
500 600 700 500 600 700 Wavelength/nm Wavelenth/nm
Figure III. 15. Emission spectra of a 100 M solution of mini-C9C12-GGY (0.2 M acetate buffer, pH 5.4) upon the addition of of Cu(CH3CN)4PF6 (10 mM in acetonitrile). (A) Cu(I) equivalent = 0.1-1.0 (B) Cu(I) equivalent = 1.1-2.0.
8 maximum emission wavelength
6 /s -3 4 ×10
2 Counts
0 012 Cu(I) Equivalents
Figure III. 16. The change of maximum emission intensity of a 100 M solution of mini-C9C12- GGY (0.2 M acetate buffer, pH 5.4) upon the addition of Cu(CH3CN)4PF6 (10 mM in acetonitrile). 63
The break points at 0.8 and 1.0 equivalent of Cu(I) is consistent with the results obtained from UV-Vis titration. Such results also suggest that Cu(I) and mini-C9C12-GGY form a complex with a 0.8:1 metal to peptide stoichiometry first. With more Cu(I) added, a complex with 1:1 metal to peptide stoichiometry eventually forms. The emission titration results are very close to the UV and CD titration results. The small deviation of the break points might due to the different sensitivities of titration methods. Besides the band intensity changes with the addition of Cu(I), another obvious characteristic in emission spectra of Cu(I)/mini-C9C12-GGY in Figure
III.15 is the change of emission band maximum. Comparing these two figures in Figure III.15, the band maximum shows an obvious red-shift when the Cu(I) equivalents are within (Figure
III.15(A)) and beyond 1.0 (Figure III.15 (B)). Figure III.17 shows the band maxima data taken from Figure III.15 vs the Cu(I) addition. As shown in this figure, the band emission maximum stays around 600 nm when 0.1 to 1.0 equivalent of Cu(I) was added. The emission maximum gradually changes from 600 nm to 625 nm, when 1.1 to 1.5 equivalent of Cu(I) is added and then followed by a stable emission maximum around 625 nm with more addition of Cu(I). The decreased emission intensity and obvious red-shift of emission maximum suggest that the Cu(I) cluster formed after 1.0 equivalent is very different from the one before 1.0 equivalent. As described in III.3.1 and III.3.2, the change in band emission maximum might be caused by the
Cu(I) coordination of the copper cluster in the Cu(I)/mini-C9C12-GGY complex. The coordination changes of Ag ions in metallothionein (MT) at different loading of Ag(I) have been studied by Stillman et al38 39. Ag(I) has been used extensively in metalloprotein experiments to act as an analogy of Cu(I) complex due to their comparable properties such as oxidation state, binding of types of ligand, and equivalent binding geometries. In Stillman’s study, the emission 64 band maximum of β-domain in Ag-MT shows ~20 nm red shift from 4 equivalents of Ag(I) to 7 equivalents Ag(I). The band maximum shows no change when 1-4 equivalent of Ag(I) was added (~565 nm) or over 7 equivalent of Ag(I) was added (~585 nm). The authors argued that the coordination change of Ag ion in the MT is in charge of the band maximum change: at low
Ag(I) loading (from 1-4 equivalents), Ag ions bind to MT with trigonal coordination geometry; with more Ag ion added, Ag(I) bind to MT with both trigonal and digonal coordination geometry
(from 4-7 equivalents of Ag(I)) until Ag(I) adopt a digonal coordination geometry after 7 equivalents. The trigonal or diagonal coordination geometry was also reported for the copper binding to Zn-rabbit-liver-metallothionein (Zn-MT2) system by using emission spectroscopy and extended X-ray absorption fine structure (EXAFS) spectroscopy. Before Cu ion saturated each domain of MT2, Cu(I) ions adopt trigonal geometry in each domain. More addition of Cu(I) equivalent after the saturation when 12 equivalents of Cu(I) was added causes the Cu(I) coordination shifting to digonal coordination geometry with the rapid decrease in emission intensity and red-shift of emission band maximum.26, 30 In some cases, the trigonally and digonally coordinated Cu(I) can coexist in some systems such as in Cu(I)-thiolate cluster, as reported by the crystal structure of yeast copper thionein.40 By analogy the present study with above systems, it is very likely that the Cu(I) bind to mini-C9C12-GGY with trigonal coordination before the addition of 1.0 equivalent of Cu(I). However, with more addition of
Cu(I), a mixture of trigonal and digonal coordination geometries may coexist since Cu(I) can adopt both trigonal or diagonal coordination geometry with thiolate ligands as reported for Cu(I)- thiolate cluster.40 Continued addition of Cu(I) to Cu(I)/mini-C9C12-GGY system may cause the digonal coordination is the dominating geometry in the Cu(I)/mini-C9C12-GGY complex, and as 65 a result, the emission maximum shows less change when 0.1 to 1.0 or 1.6 to 2.0 equivalent of
Cu(I) was added as shown in Figure III.17.
maximum wavelength 630
620
610
600 maximum wavelength
0.0 0.5 1.0 1.5 2.0 Cu(I) Equivalents
Figure III. 17. The emission maximum wavelength of mini-C9C12-GGY after the addition of Cu(I).
III.3.4 Emission at 77 K and excitation spectra
Low temperature (77 K) luminescence spectroscopy has been useful in studying the photophysical behavior and structural properties of Cu(I)-metallothionein27, 41. This technique was also used here to obtain emission properties of Cu(I)/mini-C9C12-GGY low temperature condition (77 K). Different from the spectroscopic features of emission at room temperature
(shown in Section III.3.3) where only one band at 600 nm (low energy band) is observed, two emission bands at 600 nm and 425 nm are detected for the emission at 77 K as shown in Figure
III.18. To understand the appearance of the new high energy band, the excitation spectra of
Cu(I)/mini-C9C12-GGY at room temperature and 77 K were further investigated. 66
70 100uM mini-C9C12-GGY in pH 5.4 acetate buffer 60 and 1:1 Glycerol emission in 77K excitation wavelength 300 nm 50
3 40
30 Counts /10 20
10
0
400 500 600 700 Wavelength/nm
Figure III. 18. Emission spectrum at 77 K of a 100 μM solution of mini-C9C12-GGY (0.2 M acetate buffer, pH 5.4) upon the addition of 1.0 equivalent Cu(CH3CN)4PF6 (10 mM in acetonitrile).
Room temperature excitation spectrum was obtained by monitoring the emission at 600 nm, while the excitation spectra at 77 K were examined for both emissions at 600 nm and 425 nm, respectively, as shown in Figure III.18-19. Figure III. 19 shows the 600 nm excitation spectra of
Cu(I)/mini-C9C12-GGY at room temperature (A) and 77 K (B) respectively. As shown in Figure
III.19 (A), two poorly resolved peaks at 280 nm and 335 nm are observed for the emission at 600 nm at room temperature. The excitation spectrum for the emission at 600 nm at 77 K shows an obvious peak at 283 nm and a shoulder at 330 nm. Compared with the red-shift of the emission
42 of small molecular Cu(I) cluster Cu4I4(pyridine)4 with decreasing temperature, the similar excitation maximum and emission band at 600 nm at room temperature and 77 K indicate that the low energy band at 600 nm of Cu(I)/C9C12-GGY is independent of the rigidity of the medium. As shown Figure III.20, the excitation spectrum for 425 nm at 77 K shows two well resolved peaks at 272 and 303 nm. The excitation maximum for 425 nm at 77 k show an obvious 67 blue-shift compared with that for 600 nm at 77 K. The nature of the excited states was further probed by the measurement of lifetime at 77 K.
1.0 A 1.2 B
0.8 0.8 0.6
0.4 0.4 Normalization Normalization 0.2
0.0 0.0
250 300 350 400 450 250 300 350 400 450 Wavelength/nm Wavelength/nm
Figure III.19. 600 nm excitation spectrum of Cu(I)/mini-C9C12 at room temperature (A) and 77K(B) in pH 5.4 acetate buffer.
1.0 425 nm excitation at 77k
0.8
0.6
0.4
Normalization 0.2
0.0
250 300 350 400 Wavelength/nm
Figure III.20. 425 nm excitation spectrum of Cu(I)/mini-C9C12 at 77K in pH 5.4 acetate buffer with 50% (v/v) glycerol.
As shown in Figure III.21, the high energy band and low energy band demonstrates different lifetimes. The emission lifetime trace observed at 425 nm can be fit to bi-exponential decay kinetics I(t) = AS exp(-kS t) + ALexp(-kLt), where AS, kS and AL, kL are the amplitudes and rate constants of the shorter and longer lifetime components, respectively. A non-linear least 68
4 -1 squares fit of the data yields values of AS-425nm= 0.025 and kS-425 nm = 7.47 × 10 s (τS-425 nm =
4 -1 13.4 μs), and AL = 0.018 and kL-425 nm = 2.55 × 10 s (τS-425 nm = 39.2 μs). The non-linear least squares fit of the emission lifetime trace at 600 nm yields values of AS-600nm= 0.0113 and kS-600 nm
3 -1 3 -1 = 9.13 × 10 s (τS-600 nm = 109.6 μs), and AL = 0.018 and kL-600 nm = 5.68 × 10 s (τS-600 nm =
175.9 μs). The high energy emission displays shorter lifetimes (long τ=39.2 μs and 13.4μs) than those found for low energy emission (175.9 μs and 109.6μs). The relatively long lifetimes in low-temperature suggest that both emissions are spin forbidden. The sources fo the two emission lifetimes are not clear. It is speculated that the sample measured may contain two different metalloproteins with different emission lifetimes. However, no evidence has been detected so far for the presence of two different metalloproteins in these samples. Summing up, although only the low energy emission is observed at room temperature, the prominent high energy emission and relatively weak low energy emission are observed at 77k. The high energy and low energy emission display different lifetimes as well as distinct excitation maxima.
600 nm decay at 77 k 425 nm decay at 77 k 0.9
0.6
0.3
Normalized Intensity Normalized 0.0
0.0000 0.0003 0.0006 0.0009 Time /s Figure III. 21. Emission decay of Cu(I)/mini-C9C12-GGY recorded at 77 K. Conditions: 100 µM Cu(I) and 100 µM mini-C9C12-GGY in 0.1 M pH 5.4 acetate buffer with 50% (v/v) glycerol.
69
Similar emission spectra on copper clusters at different temperatures have been reported by
Ford et al. 41 and Vasak27 et al with the corresponding data included in Table III.4. For example,
Ford et al. studied the thermochromic behavior of Cu4I4(pyridine)4 and reported that a predominant high energy emission band together with a low energy emission band appeared at
77 K. However, at room temperature, the high energy emission band of Cu4I4(pyridine)4 is hardly discernible, while the low energy band is still present. Ford et al. argued that the two bands are from two different energy states: the high energy state from the triplet cluster-centered state transitions and the low energy state from the mixed LMCT/d-s transitions. A relative large barrier exists between two energy states that might be due to the small Cu···Cu distance (2.69 Å)
(smaller than 2.8 Å, twice the van der Waals radius of Cu). At room temperature, the favorable intersystem crossing from the high to low energy states eliminates the high energy emission. But at 77 K, the intersystem crossing is mostly prohibited so that two emission bands are observed.
The control experiment in their study for the thermochromic behavior of Cu6mtc6 did not show such changes, in which the Cu···Cu distance is larger than 2.8 Å. Based on such analysis, the presence of high energy emission band was attributed to the Cu···Cu short distance, and vice versa, that the high energy emission band can be used to judge if the Cu···Cu distance is smaller than 2.8 Å. Vasak used this rule to explain the emission behaviors of Cu8-MT (Cu···Cu distance
27 < 2.8 Å) and Cu12-MT (Cu···Cu distance > 2.8 Å) in their Cu-MT complex . By analogy with these studies by Ford et al. and Vasak, it is likely that Cu···Cu distance in Cu(I)/mini-C9C12-
GGY is less than 2.8 Å, and a relative large barrier exists between high and low energy emission states of Cu(I)/mini-C9C12-GGY.
70
Table III.4 Luminescence Properties of Cu8-MT, Cu12-MT,Cu(I)4, Cu(I)6 polyhedra and Cu(I)/mini-C9C12-GGY at 77 K
High energy band Low energy band λ(nm) τ (μs) λ(nm) τ (μs) 27 Cu8-MT 425 49 610 128 27 Cu12-MT -- -- 600 129 a 43 Cu4I4py 438 23 619 26 b 43 Cu6mtc6 -- -- 767 14 Cu(I)/mini-C9C12- 425 39.2； 600 175.9；109.6 GGY 13.4 apy=pyridine. bmtc= di-n-propylmonothiocarbamate
III.3.5 Summary on photophysics of Cu(I)/mini-C9C12-GGY
Combining the above CD, near-UV CD, UV-Vis, emission, and excitation spectroscopic studies, a clearer image of the structure of Cu(I)/mini-C9C12-GGY can be obtained. The synthesized mini-C9C12-GGY is a random coil peptide with shorter peptide sequence compared to C16C19-GGY. The formed Cu(I)/mini-C9C12-GGY metalloprotein exists in a random coil structure. In the Cu(I)/mini-C9C12-GGY complex, the copper cluster forms a Cu4 structure in which Cu(I) ions might adopt trigonal coordination geometry when less than 1.0 equivalents of
Cu(I) were added. Successive addition of Cu(I) > 1.0 molar equivalent causes a structure transition and change of copper coordination geometry in Cu(I)/mini-C9C12-GGY complex which is indicated by changes in CD, UV and emission spectra. Cu···Cu distance at equivalent
1.0 is less than 2.8 Å, which results in two observable emission bands at 77 K temperature.
The photoluminescence properties of Cu(I)/mini-C9C12-GGY can be reasonably understood through the following qualitative scheme, as shown in Figure III. 22. Two energy states are present in the excited state of Cu(I)/mini-C9C12-GGY in Figure III. 21. The high energy state is from the triplet cluster-centered state (3Cluster Centered) transitions; while the low energy state is due to the mixed 3LMCT/d-s transitions. At room temperature, the 71 intersystem crossing from 3Cluster Centered state to 3LMCT/d-s state is highly favorable, which results in the observation of a strong low energy emission band, but a diminished emission band from the high energy state. At 77 K, a weak intersystem crossing occurs so that both high and low energy emission bands are detected. All these studies form a good basis for the electron transfer study in following sections.
3LMCT/d-s 3Cluster Centered
Figure III. 22. Proposed qualitative scheme describing the luminescence properties of Cu(I)/mini-C9C12-GGY (Adapted from reference 27).
III.4 Electron transfer study
III.4.1 Lifetime at room temperature
The strong room temperature luminescence of Cu(I)/mini-C9C12GGY suggests that it might function as a photoinduced electron-transfer agent. Due to the structural analogy between
Cu(I)/mini-C9C12-GGY and Cu(I)/C16C19-GGY, the present study was designed to study the unusual electron transfer behavior of Cu(I)/C16C19-GGYto explain the observation of inverted
Marcus behavior as described in section III.1. In that work, the buried Cu4S4 (N/O) cofactor has a very high reorganization energy and experiences a weak electronic coupling to the aqueous acceptors. This latter effect might be due to the positioning of the cofactor within the 72 hydrophobic core of the protein which is well away from the bulk solvent. Cu(I)/miniC9C12-
GGY also exists as a tetramer with a Cu4 cofactor. However, the random coil structure of
Cu(I)/miniC9C12-GGY will be helpful to explain the observation of Marcus inverted behavior in
Cu(I)/C16C19-GGY.
The electron transfer study starts with measuring the lifetime of Cu(I)/mini-C9C12-GGY at room temperature. Cu(I)/mini-C9C12-GGY shows a strong emission at 600 nm (Refer to Figure
III.14), which is similar to that of Cu(I)/C16C19-GGY.6 In the experiment, the emission lifetime trace was measured at 600 nm, as shown in Figure III. 22. Figure III. 22. shows the emission decay trace and corresponding log plot. Figure III.22. (B) indicates that the emission lifetime of
Cu(I)/mini-C9C12-GGY can be best fit to bi-exponential decay kinetics (Eq.II.1), where AS, kS and AL, kL are the amplitudes and rate constants of the shorter and longer lifetime components, respectively.
I(t) = AS exp(-kS t) + ALexp(-kLt) (Eq.III.1)
5 -1 A non-linear least squares fit of the data yields values of AS = 0.00344, kS = 3.4 × 10 s ( S =
4 -1 2.9 s), AL = 0.00647, and kL = 6.8 × 10 s ( L = 14.8 s). L and S are the emission lifetimes of their corresponding components.
73
0.0 B 1.0 A
0.8 -0.4
0.6 -0.8 0.4
0.2 log(intensity) -1.2
Normalized intensity 0.0 -1.6 -20 0 20 40 60 80 100 0 10203040 Time, μs Time/μs
Figure III. 23. Triplet decay trace of ca. 25 µM Cu(I)/mini-C9C12-GGY in argon-saturated solution (A) and corresponding log plot (B), in 0.2 M pH 5.4 acetate buffer at 298 K. The solid line represents fitting to a double exponential kinetics. The intensity is normalized at time zero.
The previous study showed that Cu(I)/C16C19-GGY has two lifetimes and component
6 ratio of long lifetime to short lifetime (AL/AS) is ~ 1. From Figure III.22, Cu(I)/mini-C9C12-
GGY also shows two lifetimes, but the component ratio of long lifetime to short lifetime (AL/AS) is ~ 2. The source of the two emission lifetimes in either Cu(I)/mini-C9C12-GGY or
Cu(I)/C16C19-GGY is not yet known.7 One possible speculation is that the sample measured may contain two different metalloproteins with different emission lifetimes. However, although analytical ultracentrifugation (AUC) experiment indicates that components with high molecular weigh might exists, only one peak can be observed in the HPSEC chromatograph. It means that the two components can not be separated by HPSEC if they really exists. The lifetimes for the
Cu(I)/mini-C9C12-GGY complexes are in the microsecond range. Such long lifetimes have been observed in Cu(I)/C16C19-GGY complex6and Cu(I) metallothioneins.44
III.4.2 Redox potential of Cu(I)/mini-C9C12-GGY and ruthenium quenchers The quantification of driving forces for electron transfer between Cu(I)/mini-C9C12-GGY and Ru(III) complexes was calculated by the Rehm-Weller equation: 74
-ΔG0 = E00 - E0(CuI/CuII) + E0(RuIII/RuII) (Eq. III.3)
where -ΔG0 is the driving force, E00 is the lowest triplet energy, and E0(CuI/CuII) and
E0(RuIII/RuII) are reduction potential of CuI/CuII and RuIII/RuII, respectively. The lowest triplet energy (E00) of Cu(I)/mini-C9C12-GGY can be determined from its emission spectrum measured at 77 K (shown in Figure III.24). Thus, E00 of Cu(I)/mini-C9C12-GGY was calculated from
hc E 00 = = 2.10 eV, where λ = 590 nm is the maximal emission band wavelength of ×106.1 −19 λ
Cu(I)/mini-C9C12-GGY in Figure III.26. E00 of Cu(I)/mini-C9C12-GGY is actually close to E00
= 2.04 eV of Cu(I)/C16C19-GGY, considering they have pretty similar emission spectra when measured at 77 K in aqueous media with 50% glycerol, as shown in Figure III.24.
75 3 50
Counts/10 25
Cu(I)/C16C19-GGY Cu(I)/mini-C9C12-GGY 0 550 600 650 700 Wavelength/nm
Figure III. 24. Emission spectra of Cu(I)/C16C19-GGY and Cu(I)/mini-C9C12-GGY recorded at 77 K. Conditions: 50 µM Cu(I) and 50 µM C16C19-GGY or mini-C9C12-GGY in 0.1 M pH 5.4 acetate buffer with 50% (v/v) glycerol. 75
0.9
0.6
0.3 Emission intensity 0.0
100 200 300 400 mV vs. NHE
Figure III. 25. Redox titration curve of Cu(I)/mini-C9C12-GGY monitored by optical spectroscopy. Emission at 600 nm is plotted against the measured redox potential vs. NHE and the obtained data were fit to the single Nernst equation curve (n=1).
The electrochemical reduction potential, E0(CuI/CuII), of the Cu(I)/mini-C9C12-GGY was measured by the redox titrations through monitoring the change of emission intensity at 600 nm with the potential change controlled by the chemical oxidation by K2IrCl6 as described in
Chapter II.6 Emission at 600 nm is plotted against the measured redox potential vs. NHE as shown in Figure III.25. The obtained data were fit to the single Nernst equation (Eq. II.14) (n=1) and generated with a E0(CuI/CuII) = 276 mV vs NHE. The value of E0(CuI/CuII) for
Cu(I)/C16C19-GGY was measured as 343 mV. It should be noted that the redox titration obtained by monitoring of the decrease in emission intensity of the Cu(I)/mini-C9C12-GGY complex was not reversible, shown in Figure III.26. Even though the values of the measured potentials were changing towards the reduced state of Cu(I) upon the addition of the reducing agent, tris(2-carboxyethyl)phosphine, to the solution, the emission intensity was not fully restored. This result indicates that the oxidation process is accompanied by irreversible changes in the metalloprotein structure. 76
6 without oxidation Reduced by 40uL TCEP(25mg/ml) 5 /s
-5 4
3
2 Counts X 10 Counts 1
0
500 600 700 800 Wavelength/nm
Figure III.26. The emission of 100uM Cu(I)/mini-C9C12-GGY without oxidation (black) and the reduced Cu(I)/mini-C9C12-GGY by 40 μl TCEP (25 mg/ml concentration) after oxidation by excessive addition of K2IrCl6 (red)
Figure III.27. Cyclic voltammograms of two modified Cu peptides. (A) CV of the Cu(I)/mini- C9C12-GGY at a basal plane graphite electrode. (B) Similar CV data collected for the Cu(I)/C16C19-GGY peptide at an edge plane graphite electrode.
The one-electron reduction potential of Cu(I)/mini-C9C12-GGY was also tested by protein film voltammetry and compared with the potential of Cu(I)/C16C19-GGY by the same methods.
Figure III.27 shows the one-electron reduction potential of Cu(I)/mini-C9C12-GGY (A) and
Cu(I)C16C19-GGY (B) observed by protein film voltammetry. In both cases, a quasi-reversible process was observed, at a similar midpoint potential (+230 mV ± 20 mV). However, the 77 potential results obtained by this method are lower than those observed in solution. Such a difference might due to denatured state of the metalloprotein in the film state. In summary, two methods are used to measure the one one-electron reduction potential of Cu(I)/mini-C9C12-
GGY and C16C19-GGY. In both methods, the potentials of Cu(I)/mini-C9C12-GGY and
Cu(I)/C16C19-GGY only shows minor difference.
The electrochemical reduction potential E0(RuIII/RuII) can be measured by cyclic voltammetry (CV) method at room temperature in aqueous solution with 0.2 M pH 5.4 acetate buffer as the support electrolyte. Thus, the driving forces of the electron transfer reaction can be calculated by Eq. III.3.
3+ III.4.3 Effect of [Ru(NH3)5Lut] on the decay kinetics and quenching mechanism
3+ 3+ The addition of [Ru(NH3)5lutidine] ([Ru(NH3)5Lut] ) to a solution of an emissive
Cu(I)/mini-C9C12-GGY complex leads to quenching of the emission intensity and lifetimes. The emission decay measured from a solution containing both Cu(I)/mini-C9C12-GGY and
3+ [Ru(NH3)5Lut] can be accurately fit to a double exponential decay kinetics. Importantly, both emission lifetimes of Cu(I)/mini-C9C12-GGY shorten with the addition of
3+ [Ru(NH3)5Lut] .Figure III.28 shows the emission decay measured from a Cu(I)/mini-C9C12-
3+ GGY solution at the presence of [Ru(NH3)5Lut] . For a comparison, the emission decay of
3+ Cu(I)/mini-C9C12-GGY at the absence of [Ru(NH3)5Lut] was re-plotted in Figure III.22.
3+ Obviously, after adding [Ru(NH3)5Lut] , the emission of Cu(I)/mini-C9C12-GGY decays in a fast speed. The emission decay of Cu(I)/mini-C9C12-GGY in Figure III.23 is also fit to the biexponential decay kinetics (Eq. III.1). The fitting result indicates that Cu(I)/mini-C9C12-GGY 78
3+ has two emission lifetimes (long (τL) and short (τS) lifetimes) after [Ru(NH3)5Lut] addition, but both lifetimes were shortened to τS = 1.46 ± 0.01s and τL = 4.95 ± 0.02s, respectively, with
AS/AL remaining approximately 2:1.
1.0
0.8
0.6
0.4
0.2 (a) (b)
Normalized intensity 0.0
-0.2 -20 0 20 40 60 80 100 Time/μs
Figure III. 28. Triplet decay traces of 100 µM mini-C9C12-GGY and 100 µM Cu(I) in 0.2 M pH 3+ 5.4 acetate buffer in the (a) absence and (b) presence of 60 µM [Ru(NH3)5Lut] .
The lifetime quenching of metalloproteins may involve a variety of mechanisms such as energy transfer or electron transfer process.7 Transient absorption studies were carried out to ascertain the lifetime quenching mechanism of Cu(I)/mini-C9C12-GGY upon adding
3+ [Ru(NH3)5Lut] in present study. The excited state absorption difference spectra of Cu(I)/mini-
3+ C9C12-GGY with [Ru(NH3)5Lut] was obtained 20 µs after a 355 nm laser pulse. As demonstrated in Figure III.29, a broad positive absorption band ranging from 350 to 475 nm is observed after the emission decay of Cu(I)/mini-C9C12-GGY has completely decreases, so the broad peak is not due to the unquenched Cu(I)/mini-C9C12-GGY excited states, but it is
2+ 45 consistent with the appearance of the MLCT band of the reduced [Ru(NH3)5Lut] quencher. 79
These results indicate the lifetime quenching involves a significant contribution from a photoinduced electron-transfer process.7
8
6
3 4 X10
ΔΑ 2
0 350 400 450 500 Wavelength/nm
Figure III. 29. Transient absorption spectrum of a solution of Cu(I)/mini-C9C12-GGY (330 μM) and [RuA5(lut)]3+ (330 μM) measured 20μs after the laser flash.
The luminescence lifetime quenching method has been used to determine the bimolecular electron transfer rates for Cu(I)/C16C19-GGY with ruthenium quenchers.7 The same method is employed here to measure the rate of bimolecular electron transfer between Cu(I)/mini-C9C12-
GGY and ruthenium quenchers. For example, the bimolecular electron transfer rate of
3+ Cu(I)/mini-C9C12-GGY with [Ru(NH3)5Lut] at a range of Ru(III) quencher concentrations
obs obs were analyzed to derive the decay rate constants (kS and kL ) for each quencher concentration.
The relative amplitudes of the emiss3ion decay components (AL and AS) at all studied quencher
obs obs concentrations remain approximately equal (AL : AS = ~2:1). Plots of kS and kL vs the
3+ concentration of [Ru(NH3)5Lut] in Figure III.30. A linear dependence as pseudo-first order quenching kinetics is observed. Analysis of the data yields the bimolecular quenching constants
obs 9 -1 -1 obs 9 -1 -1 of kS = (9.26 ± 1.46) × 10 M s and kL = (2.65 ± 0.24) × 10 M s , respectively. These 80 results indicates that the luminescent copper cluster center in the metalloprotein Cu(I)/mini-
C9C12-GGY can indeed serve as a photoinduced electron-transfer reagent when undergoing a bimolecular reaction with an exogenous acceptor in a free solution.
1.2
) -1 s
-6 0.8
(10 obs k 0.4
0.0 0 20406080100 3+ [Ru(NH ) Lut] (μM) 3 5
Figure III. 30. Pseudo first-order kinetic plots of the observed rate constants (kobs) for quenching of the emission for the fast (●) and slow (■) decay components of Cu(I)/mini-C9C12-GGY as a function of quencher concentration.
III.4.4. Stern-Volmer behavior of Cu(I)/mini-C9C12-GGY quenching
For a bimolecular electron transfer reaction, the luminescence quenching of metalloproteins usually exhibits a Stern-Volmer behavior when no static quenching is involved in the reaction. The Stern-Volmer behavior of Cu(I)/mini-C9C12-GGY quenching by Ru(III) was also examined through the following relation,
τ 0 += τ Ru(III)][k1 (Eq. III.2) τ 0q
where τ0 and τ represent the lifetimes of the Cu(I) complex at the absence and presence of quenchers, kq is the electron transfer rate, and [Ru(III)] denotes the concentration of the Ru(III) 81
quenchers in solution. In the Stern-Volmer relation, the slope of plots of τ0/τ vs quencher concentration is Stern-Volmer constant (KSV = kqτ0) from which the kq can be obtained.
A variety of Ru(III) quenchers (Ru(NH3)5X, where X=chloro, ammine, 3,5-lutidine, pyridine, nicotinamide, and dimethyl-3,5-pyridine dicarboxylate, respectively) were utilized to study the Stern-Volmer behavior of Cu(I)/mini-C9C12-GGY quenching by Ru(III) in the experiment. The results show that Stern-Volmer plots for all photoredox systems (Cu(I)/mini-
C9C12-GGY with Ru(III) quenchers) are linear. Figure III.30 shows an exemplary Stern-Volmer plot when Ru(NH3)6 was used as the Ru(III) quencher. As expected, the data points in Figure
III.31 follow a linear distribution and exhibit the proper intercept of unity. The linear Stern-
Volmer behavior of Cu(I)/mini-C9C12-GGY quenching by Ru(III) indicates that, under the present experimental conditions, the emission quenching arises from a purely collisional mechanism and not for a ground-state protein–quencher complex.
14 4 -1 12 Long lifetime component Ksv=7.02 X 10 M
10
8 /τ 0 6 τ
4
2 Short liftime component Ksv=3.74 X104 M-1 0 0 20 40 60 80 100 120 140 160 180 Ru(NH ) /μM 3 6
Figure III. 31. Stern-Volmer plots of the quenching of 100 µM Cu(I), 100 µM mini-C9C12-GGY 3+ in 0.2 M pH 5.4 acetate buffer by [Ru(NH3)6] , showing the linear fit through the data points.
82
III.4.5 Electron transfer of Cu(I)/mini-C9C12-GGY with Ru(III) complexes
Hong at al. observed that the bimolecular electron transfer rates between Cu(I)/C16C19-
GGY and ruthenium complexes are below the diffusion limit allowing inverted Marcus behavior to be observed at high driving forces.7 As presented in Section III.1, the observation of inverted
Marcus behavior could be due to the encapsulation the Cu(I) cofactor within the hydrophobic core of the synthetic Cu(I)/C16C19-GGY protein. This may prohibit the close approach of the electron donor and acceptor, and as a result, decreases the electronic coupling between them.
Cu(I)/mini-C9C12-GGY has identical heptad repeat but a random coil structure instead of coiled coil structure in Cu(I)/C16C19-GGY. This structural difference might result in a difference in electron transfer rate with the increasing driving force in Cu(I)/mini-C9C12-GGY.
3+ Table III.5. Redox potentials of [Ru(NH3)5X] and observed bimolecular electron transfer rate 3+ constants between [Ru(NH3)5X] (X = Chloro, Ammine, 3,5-Lutidine, Pyridine, Nicotinamide, and Dimethyl 3,5-pyridine dicarboxylate) and Cu(I)/C16C19-GGY and Cu(I)/mini-C9C12-GGY
Redox Cu(I)/C16C19-GGY Cu(I)/mini-C9C12-GGY potential (mv) ks kL ks kL (109M-1s-1) (109M-1s-1) (109M-1s-1) (109M-1s-1) Ru(NH3)5Cl -40 3.27 ± 0.14 2.53 ± 0.09 5.50±0.58 2.71±0.042
Ru(NH3)6 60 5.86 ± 0.21 3.47 ± 0.05 10.21±1.89 4.60±0.18
Ru(NH3)5 (lutidine) 255 4.43 ± 0.06 2.44 ± 0.08 8.1±0.39 2.175±0.38
Ru(NH3)5(pyridine) 300 2.46 ± 0.07 1.36 ± 0.05 8.61±0.55 3.58±0.38
Ru(NH3)5 353 3.46 ± 0.04 1.96 ± 0.06 11.61±4.06 2.48±0.51 (nicotinamide) Ru(NH3)5(3,5- 392 1.71 ± 0.21 0.96 ± 0.08 10.0±3.16 3.58±0.375 pyridine dicarboxylate)
The values of E0(RuIII/RuII) for a variety of Ru(III) quenchers are listed in Table III.5.
Substituting the measured E00, E0(CuI/CuII), and E0(RuIII/RuII) into Eq. III.3, the driving force (-
ΔG0) for Cu(I)/mini-C9C12-GGY can be finally determined. The same calculation has been used 83 for determining -ΔG0 of Cu(I)/C16C19-GGY by Hong et al, and -ΔG0 values are almost identical to those of Cu(I)/mini-C9C12-GGY at present investigation. This indicates that both Cu(I)/mini-
C9C12-GGY and Cu(I)/C16C19-GGY have the similar driving forces in the electron transfer reactions.
III.4.6 Bimolecular electron transfer events
As described in introduction part, the electron transfer reaction between Cu(I)/C16C19-
GGY and ruthenium complexes with different redox potential lies in the Marcus inverted region.
It was speculated that this observation could be due to the encapsulation of the Cu(I) cofactor within the hydrophobic core of the synthetic Cu(I)/C16C19-GGY. This may prohibit the close approach of the donor and acceptor sites and reduce the electronic coupling between the donor and acceptor. Such as situation might lower the rates of bimolecular electron transfer below the diffusion limit and permit inverted Marcus behavior to be observed at high driving forces. To test this hypothesis, a smaller metalloprotein Cu(I)/mini-C9C12-GGY is designed and synthesized.
Although based on the same heptad repeat with C16C19-GGY, mini-C9C12-GGY has less amino acid than C16C19-GGY. Thus the Cu(I) cofactor in the formed Cu(I)/mini-C9C12-GGY are expected to be closer to quenchers, which can favor the electronic coupling between the donor and acceptor and increase the electron transfer rates even up to diffusion limit. CD experiments have shown that both apo-mini-C9C12-GGY and synthetic metalloprotein
Cu(I)/mini-C9C12-GGY exist as random coil instead of coiled coil observed for Cu(I)/C16C19-
GGY. The strong emission around 600 nm at room temperature and 77 K and the redox potential of Cu(I)/mini-C9C12-GGY are similar to those of Cu(I)/C16C19-GGY, which mean the driving forces for electron transfer reaction between Cu(I)/mini-C9C12-GGY with same ruthenium quenchers are comparable to those for Cu(I)/C16C19-GGY. These results indicates that our aim 84 to make a analogous of Cu(I)/C16C19-GGY is successful. Lifetime measurements are used to obtain the electron transfer rates between Cu(I)/mini-C9C12-GGY and ruthenium quenchers.
With the calculated driving forces and measured corresponding electron transfer rates for both short lifetime (kS) and long lifetime (kL) components (listed in Table III.5) in the experiment, the electron transfer reaction involved in the Cu(I)/mini-C9C12-GGY complex can be understood systematically. Figures III.32 and III.33 shows the close comparison for dependence of the bimolecular electron-transfer rate constants (kS and kL) on driving force between Cu(I)/mini-
C9C12-GGY and Cu(I)/C16C19-GGY based on the data in Table III.4. Two important differences are observed. Firstly, the electron-transfer rate constants for Cu(I)/mini-C9C12-GGY are consistently larger than the corresponding values for Cu(I)/C16C19-GGY. Secondly, the electron transfer rates for Cu(I)/mini-C9C12-GGY are independent of driving force whereas those for Cu(I)/C16C19-GGY appear to follow inverted Marcus behavior.
These differences in electron transfer between Cu(I)/mini-C9C12-GGY and
Cu(I)/C16C19-GGY can be interpreted in terms of a simple model in which diffusional electron- transfer rates can be dictated, in part, by the relative positioning of the redox-active cofactors within the hydrophobic core of the protein. Although, Cu(I)/mini-C9C12-GGY shows similar oligomeric state, luminescence at room temperature and 77K, and redox potential, it exists as a random coiled structure instead of coiled coil structure. The disordered random coil structure offers the ruthenium quenchers greater opportunity to access to the emissive Cu(I) site of
Cu(I)/mini-C9C12-GGY. Therefore, the shorter distance between the emissive part and quencher in Cu(I)/mini-C9C12-GGY increase the value of HDA, and results in faster bimolecular electron transfer rates which is independent on the driving-force. The values of kS for Cu(I)/mini-C9C12-
GGY are shown close to the diffusion limit in the experiment. The small kL values for 85
Cu(I)/mini-C9C12-GGY which is independent of driving force but smaller than the diffusion limit, however, is not yet known from the present result. However, the obtained results support our hypothesis in the beginning of the chapter that the prohibition of close approach between the donor and acceptor resulted in the observation of electron transfer in Marcus region in
Cu(I)/C16C19-GGY.
10.0 s
logk 9.5
k Cu(I)/C16C19-GGY s k Cu(I)/mini-C9C12-GGY s
9.0 -200 0 200 400 600 Reduction Potential (mV vs. NHE)
Figure III. 32. Driving force dependence of bimolecular quenching rate constants for the short lifetime component of Cu(I)/C16C19-GGY (▲) and Cu(I)/mini-C9C12-GGY (●). The black solid line is drawn based on the Marcus theory of electron transfer.
86
10.0
9.5 L logk 9.0 k Cu(I)/C16C19-GGY L k Cu(I)/mini-C9C12-GGY L
8.5 -100 0 100 200 300 400 500 Reduction potential (mV vs. NHE)
Figure III. 33. Driving force dependence of bimolecular quenching rate constants for the long lifetime components of Cu(I)/C16C19-GGY (▲) and Cu(I)/mini-C9C12-GGY (●). The black solid line is drawn based on the Marcus theory of electron transfer.
III.4.7 Summary on electron transfer reactions
In the electron transfer studies, the events involved in Cu(I)/mini-C9C12-GGY are studied and the results are compared to those of Cu(I)/C16C19-GGY. Similar to Cu(I)/C16C19-GGY,
Cu(I)/mini-C9C12-GGY shows two lifetimes at room temperature. Cu(I)/mini-C9C12-GGY undergoes collisional photoinduced electron-transfer to a variety of ruthenium(III) acceptors in solution. However, the electron transfer rates for Cu(I)/mini-C9C12-GGY and Cu(I)/C16C19-
GGY are different. The electron transfer rates for Cu(I)/mini-C9C12-GGY are higher than those for Cu(I)/C16C19-GGY and even close to the diffusion limit. Also, the electron transfer rates for
Cu(I)/mini-C9C12-GGY are independent of driving force whereas those for Cu(I)/C16C19-GGY appear to follow Marcus inverted behavior. Such difference can be understood from the diffusional electron-transfer rates dictated partially by the relative positioning of the redox-active cofactors within the hydrophobic core of the metalloproteins. The disordered random coil 87
Cu(I)/mini-C9C12-GGY offers the ruthenium quenchers greater chance to access to the emissive
Cu(I) site of Cu(I)/mini-C9C12-GGY, and as a result faster bimolecular electron transfer rates close to the diffusion limit and independence on the driving-force are observed. Such results support our hypothesis in the beginning of the chapter that the hydrophobic condition provided by the coiled coil structure result in the prohibition of close approach between the donor and acceptor. This prohibition will cause the small HDA, and decrease the electron transfer rate.
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CHAPTER IV. THE EFFECT OF THE FREE ENRGY OF PEPTIDE FOLDING ON
OLIGOMERIZATION STATE OF METALLOPROTEINS
IV.1 Introduction
Studies on the formation of metalloproteins have shown that metal ions can play important roles in driving the folding of proteins into specific three-dimensional structures.1-3 In such metal-dependent protein folding processes, one or more metal ions are coordinated to ligands in the polypeptides to drive the polypeptide chain in ways that produce a fully functional conformation.4 A good example of this is the metal-dependent folding of zinc-finger proteins in which Zn2+ ions tetrahedrally coordinate to cysteinyl thiolate and histidinyl imidazole nitrogen ligands to stabilize the “finger” conformation which enables the efficient binding DNA, RNA or proteins. The metal-free domain of the zinc finger is too small to fold by itself and the binding of
Zn2+ is needed to trigger the folding of the peptide to its functional form. Although metal- dependent protein folding is intimately connected with protein conformation and the function of the metalloproteins, the folding pathways of metalloproteins are not well understood.5 Therefore, significant efforts are devoted to address the roles of peptide and metal ions in regulating metalloprotein folding.6, 7
Our group has recently studied the folding of the peptide of C16C19-GGY. The addition of
Cd(II) or Cu(I) ion to the same C16C19-GGY, whose apo form is a random coil, induces pronounced conformational changes to form different type of coiled coil structures.3, 8 Thus, addition of Cd(II) ion to C16C19-GGY peptide produces a two-stranded coiled-coil in which a single Cd(II) ion bridges two peptide chains.8 The addition of Cu(I) to C16C19-GGY peptide, however, forms a four-stranded coiled-coil structure which contains four Cu(I) ions within the 92 hydrophobic core of the synthetic Cu(I)/C16C19-GGY metalloprotein.3 These studies indicate that different metal ions can induce different oligomerization states of the resulting metalloproteins even for the same peptide. However, the mechanism of the formation of an ordered C16C19-GGY metalloprotein is still unknown. For example, is the final metalloprotein structure governed by the inherent free energy of the folding of the polypeptide chain? Or is it controlled by the binding of metal ions? To answer these questions and to obtain insight into the mechanism of metalloprotein formation, a series of peptides with different free energies of folding were designed and their metal induced folding behavior was studied.
The peptide with the sequence (IAALEQK)n (n≧ 3) has been shown to be a stable coiled coil dimer.9 In this chapter, three peptides based on this motif have been synthesized. Two core isoleucine and leucine residues are replaced by two cysteine residues to generate a metal-binding position. At the seem time, replacing core leucine residues with smaller and less hydrophobic valine residue, or smaller sequenced peptide consequently leads to varying the free energies of peptide folding. The sequence of the peptides, AQC16C19-GGY, AQVC16C19-GGY, and mini-
AQVC9C12-GGY are shown shown in Table IV.1. The goal of this work was to obtain three peptides having different free energies of folding (AQC16C19-GGY > AQVC16C19-GGY > mini-AQVC9C12-GGY) in their apo-states. By doing this, one can explore the influence of peptide folding on overall structure of the resulting metallopolypeptide. The coiled coil motif used in AQC16C19-GGY and AQVC16C19-GGY are more stable than C16C19-GGY and were exploited to form the coiled coil dimeric structure instead of random coil structure observed for
C16C19-GGY10-12. Thus the oligomerization states the resulting metalloproteins for AQC16C19-
GGY and AQVC16C19-GGY might exist as dimers which reflect the impact of free energies of peptide folding. In contrast, mini-AQVC9C12-GGY is a disordered random coil structure, so the 93 inclusion of this peptide is as a comparison in the exploration of effect of peptide stability on the metalloprotein formation. Therefore, these peptides are expected to form a comparable series of peptides to illustrate the effect of peptide folding on the overall structure of resulting metalloproteins upon adding metal ions. To compare with the metal ion induced tetrameric
Cu(I)/C16C19-GGY, the same metal ion (Cu(I)) was utilized in the present chapter to investigate the influence of peptide folding.
IV.2 Peptide synthesis and characterization
IV.2.1 Peptide synthesis
Three peptides, AQC16C19-GGY, AQVC16C19-GGY, and mini-AQVC9C12-GGY, were prepared by solid-phase peptide synthesis. Their sequences are shown in Table IV.1.
AQC16C19-GGY is based on (IAALEQK)n. Two cysteine residues are replaced at position
“a(16)” and position “d (19)” position. AQVC16C19-GGY shows the same sequence as
AQC16C19-GGY except that a smaller and less hydrophobic valine residue replaces leucine residue at “d(12)” position. Mini-AQVC9C12-GGY shows less amino acid than AQVC9C12-
GGY.
94
Table IV.1 Sequence of synthesized peptides
AQC16C19-GGY Q IAALEQK IAALEQK CAACEQK IAALEQK GGY
AQVC16C19-GGY Q IAALEQK IAAVEQK CAACEQK IAALEQK GGY
Mini-AQVC9C12-GGY Q IAAVEQK CAACEQK IAALEQK GGY
-1 -1 13 The extinction coefficient of tyrosine (ε275 =1450 M cm ) was used to determine the peptide concentration in solution. The purified AQC16C19-GGY, AQVC16C19-GGY, and mini-
AQVC9C12-GGY peptides were identified by MALDI mass-spectroscopy (shown in Figure
IV.1-IV.3.) m/z (ion), AQC16C19-GGY, calculated: 3460.03; found: 3459.76, [M]+, 3483.41
[M+Na]+, 3517.38 [M+K]+. AQVC16C19-GGY, calculated: 3446.01 found: 3445.50 [M]+,
3467.43 [M+Na]+. Mini-AQVC9C12-GGY calculated: 2692.11 found: 2691.83 [M]+, 2730.19
[M+K]+.
Figure IV. 1 MALDI-TOF mass spectrum of the purified AQC16C19-GGY (m/z, observed: 3460.47, calculated: 3459.76, 3483.41 [M+Na]+, 3498.63 [M+K]+, 3517.38 [M+2Na]2+) 95
Figure IV. 2 MALDI-TOF mass spectrum of the purified AQVC16C19-GGY (m/z, observed: 3446.01 found: 3445.50, 3467.43 [M+Na]+).
Figure IV. 3 MALDI-TOF mass spectrum of the purified mini-AQVC9C12-GGY (m/z, observed: 2692.11 found: 2691.83, 2730.19 [M+K]+)
96
IV.2.2 Conformational analysis
The stabilities of the coiled coils formed by the various apo-peptides were studied by monitoring changes in the circular dichroism signal at 222 nm upon the addition of guanidinium chloride (GdnHCl).14, 15 The experiment was carried out in 50 mM phosphate buffer (100 mM
KCl, pH 7.0) with 0-8 M GdnHCl. Analysis of GdnHCl denaturation curves was performed
using a two-state unfolding model to determine the fraction folding. The fraction folding (F ) app can be calculated from Eq. IV.1,
F = (Yobs-Yn)/(Yu-Yn) (Eq. IV.1) app
where Yobs is the observed molar ellipticity measured in degrees, Yn and Yu are the ellipticities of the native folded and unfolded states, respectively. The two-state unfolding model can be represented by → nUF , in which F is the folded peptide and U is the unfolded peptide monomer.
Detailed experimental procedures are described in Chapter II. The denaturation curves of
AQC16C19-GGY and AQVC16C19-GGY are shown in Figure IV.4. the free energy of the coiled-coil folding was determined by fitting of all the folding data to Eq. II.9 and extrapolating the free energy of folding to the point where the concentration of GdnHCl is zero. The free
H2O energy of the dimer folding of AQVC16C19-GGY can be obtained ΔG = 4.47 kcal/mole with a midpoint around 0.35 M GdnHCl, while the free energy of the dimer folding of AQC16C19-
H2O GGY is achieved ΔG = 9.97 kcal/mole with a denaturation midpoint around 1.5 M GdnHCl.
(The data also can be fit to a tetramer model with the free energy 22.4 kcal/mole for AQC16C19-
GGY, 18.4 kcal/mole for AQVC16C19-GGY). For the both models, the structure of
AQC16C19-GGY is more stable than that of AQVC16C19-GGY and both are more stable than 97
C16C19-GGY, which exists as a random coil in its apo-state. Mini-AQVC9C12-GGY is also a random coil structure so that no conformational analysis data is available from this experiment.
1.0
0.8 AQVC16C19-GGY AQC16C19-GGY 0.6 app F 0.4
0.2
0.0 0123456
[GdnHCl]/M
Figure IV. 4. The plot of the denaturation of AQC16C19-GGY (▲) and AQVC16C19-GGY (■) with increasing concentration of guanidinium chloride.
IV.2.3 Oligomerization state analysis
Analytical ultracentrifugation (AUC) and high performance size exclusion chromatography (HPSEC) experiments were performed to determine the oligomerization states of the AQC16C19-GGY and AQVC16C19-GGY, respectively. Figure IV.5 shows the sedimentation velocity ultracenrifugation results obtained at 60.0 krpm speed (peptide concentration was checked by the absorption at 280 nm, A= 0.9, pH=5.4, 10 mM acetate buffer) for AQC16C19-GGY system. The Genetic Algorithm analysis developed by Borris Demeler was employed to fit the data.16 This method can obtain both sedimentation and diffusion coefficients by using a stochastic optimization operation. It is model (for example monomer or dimer model) independent and provides high resolution for these parameters (for example ratio of frictional 98 coefficient) 17,18 For AQC16C19-GGY, the Genetic Algorithm analysis shows that AQC16C19-
GGY exists as a mixture of species in which 75.7% of the peptide exists in a form which has a molecular weight of 12.6kDa, corresponding to that of a peptide tetramer, and 24.3% of the peptide has a diner molecular weight of 6.08 kDa. These results are somewhat surprising when considering that the peptide with “IAALEQK” heptad repeat was shown to forms a dimeric structure.19 However, the two states of the apo-peptide actually offer us more choices to understand the interaction between metal ions and peptide in determinating the structure of metalloprotein. For example, whether is the folding preference of the peptide disturbed by the binding of metal ions? Whether is the oligomerization state of the peptide influenced by the free energies of the peptide folding?
HPSEC experiments were also used to determine the oligomerization state of the apo- peptide. Previous studied peptides/metalloproteins, monomer C16C19-GGY, H21, tetramer
Cu(I)/C16C19-GGY 3, 20, 21, were used as standard. The Cu(I)/AQC16C19-GGY metalloprotein which has been studied by AUC to be a trimer (describes in the following section) was also used as a standard. As shown in Figure IV.6.A, AQC16C19-GGY and AQVC16C19-GGY shows almost the same retention time and peak shape, which indicates they likely have similar oligomerization states considering that they contain the similar sequence. From the calibration curve log(MW) = -(6.6±0.9)×Kd + (27.79±0.3) (Figure IV.6. B) the molecular weight of the
AQVC16C19-GGY and AQC16C19-GGY are ~ 9.2±1.1 kDa which is close to the weighted average molecular weight 11.0 kDa of AUC result of AQC16C19-GGY. The molecular weight measured by the two methods are listed in Table IV.2. These results indicate that AQVC16C19-
GGY might exist as a mixture of dimer and tetramer too. However, the exact ratio of tetramer to dimer cannot be determined by the SEC experiment. Mini-AQVC9C12-GGY is a random coil, 99 and thus exists as a monomer as shown in IV.3.4.
Table IV.2 The molecular weight of AQC16C19-GGY and AQVC16C19-GGY in solution measured by SEC and AUC
Peptide MW (SEC) MW(AUC) AQC16C19-GGY 9.2±1.08 kDa 6.08 kDa (24.3%) 12.6kDa (75.7%) Weighted average 11.0 kda AQVC16C19-GGY 9.2±1.08 kDa --
Figure IV. 5. Determination of molecular weight of AQC16C19-GGY velocity ultracentrifugation, (10 mM acetate buffer, pH 5.4) 100
AQC16C19-GGY 4.2 Cu(I)/C16C19-GGY AQVC16C19-GGY 0.9 Cu(I)/AQC16C19-GGY
4.0 AQVC16C19-GGY 0.6 3.8
3.6 0.3 C16C19-GGY
log(MW) 3.4
0.0 3.2
normalized absorption @275nm HO12 3.0 20 30 40 50 0.36 0.40 0.44 0.48 0.52 Kd Time/min
Figure IV. 6. High performance size exclusion chromatography (HPSEC) chromatogram of the AQC16C19-GGY and AQVC16C19-GGY monitored at 275 nm. The sample was eluted with 0.2 M acetate buffer pH 5.4, 0.4 ml/min. (the peak around 40 min is due to the buffer)
IV.3 Synthesis and characterization of Cu-peptides
IV.3.1 Circular dichroism (CD) titrations
Circular dichroism (CD) titrations were used to characterize the conformational properties of the apo-peptides which occur after the binding of Cu(I) ions. In general, the α-helical structure
22 displays two negative CD signals in far-UV region at 222 and 208 nm with [θ]222/[θ]208 = 0.86 .
Value of [θ]222/[θ]208 = 1.03 are seen for coiled coil conformations as the magnitude of the CD band at 208 nm is sensitive to the presence of interacting α-helices, as this band is polarized parallel to the helix axis.23 Comparatively, random coil conformation is characterized by a negative absorption between 195 and 200 nm.24
The CD spectra of AQC16C19-GGY, AQVC16C19-GGY, and mini-AQVC9C12-GGY were measured and listed in Figures IV.7, IV.8 and IV.10 respectively. Figure III.9 shows the ratio of [θ]222/[θ]208 with the uploading of Cu(I). 101
111uMAQC16C19-GGY 60000 111uM Cu(I)/AQC16C19-GGY
) 0430-07 -1
30000 dmol 2
0 ] (deg cm [Θ -30000
200 220 240 260
Wavelength/nm Figure IV. 7. Circular dichroism spectra of aqueous solutions of AQC16C19-GGY (111 μM) (■) and Cu(I)/AQC16C19-GGY (●) (with 111uM Cu(I) in pH 5.4 acetate buffer, 298 K)
The CD spectra of AQC16C19-GGY taken in the absence and presence of Cu(I) show almost identical characteristics. For example, Figure IV.7 compares the CD spectrum of
AQC16C19-GGY to that of AQC16C19-GGY with 1.0 molar equivalents of added Cu(I). In the
CD spectrum of AQC16C19-GGY, two negative maxima at 208 nm and 222 nm exist, which indicates the presence of the α-helical peptide structure. Examination of the ellipticity ratio of
[θ222nm]/ [θ208nm] shows a ratio of 1.13, indicating the existence of α-helical coiled coil structure in solution. The CD spectrum of AQC16C19-GGY with 1.0 equivalents of added Cu(I) shows identical features compared to that of AQC16C19-GGY. This result shows that Cu(I) addition does not alter the structure of AQC16C19-GGY, although it is known that Cu(I) can bind to the cysteine ligands in AQC16C19-GGY as confirmed by UV-vis and fluorescence spectra (vide infra). The helical content of apo-AQC16C19-GGY and Cu(I)/ AQC16C19-GGY were calculated using Eq. II.2, ([θ]100% helix = - 40000*[(n - 4)/n], where n is the number of residues) defined in Chapter II, and the calculation shows both peptides have a helical content of 92.4%. 102
Figure IV.8 shows the CD spectra of AQVC16C19-GGY taken upon the addition of 0.0-2.8 equivalents of Cu(I) per peptide monomer. Similar to the CD feature of AQC16C19-GGY, two negative maxima at 208 nm and 222 nm, and [θ222nm]/[θ208nm] = 1.0 were observed for
AQVC16C19-GGY, which indicates that apo-AQVC16C19-GGY exists as α-helical coiled coil.
However, in contrast to AQC16C19-GGY, the signal intensity around 208 nm and 222 nm, increases with the addition of 0.0-1.5 equivalents of Cu(I). Continued addition of Cu(I) after 1.5 equivalents causes a slight change in peak intensity around 208 nm and 222 nm. As an example,
Figure IV.9(A) shows changes of signal intensity at 222 nm as a function of Cu(I) equivalents in the peptide solution. The increase in signal intensity of CD spectra at 222 nm with the addition of
Cu(I) demonstrates that Cu(I) can bind to the AQVC16C19-GGY peptide and stabilize the coiled coil structure of the peptide. The maximum helicity (minimum ellipticity at 222 nm) was reached with 1.5 fold of Cu(I) added (Figure IV.9.A), excess addition of Cu(I) shows less effect on the helical change. The observed ellipticity ratios [θ222nm]/[θ208nm] for AQVC16C19-GGY with Cu(I)
(per peptide monomer) as shown in Figure IV.9.B are in the range of 0.97-1.0 which is a typical value for coiled coil peptides.23 As for the change of helical content, the maximum helical content of AQVC16C19-GGY upon adding Cu(I) increased to 46.9%. However, it is still lower than the 92.4% for AQC16C19-GGY. These results indicate that even though the binding of
Cu(I) ions can stabilize the coiled coil structure of AQVC16C19-GGY to some degree, stability of the coiled coil structures also shows the peptide sequence dependence. The AUC results of
AQVC16C19-GGY also indicate the stability of coiled coil since no component of monomer is observed. 103
3 -1 2 dmol 2 1
0 deg cm 4
/10 -1 222nm -2 Θ] [
200 210 220 230 240 250 260 Wavelength/nm
Figure IV. 8. CD spectra obtained by successive addition of Cu(I) to 100 μM AQVC16C19-GGY peptide in 10 mM, pH 5.4 acetate buffer. (Cu(I) equivalents: 0-2.75)
1.10 -1.40 A B -1 1.05 -1.45 dmol 2 1.00 -1.50 208 /θ deg cm deg 4
222 0.95
-1.55 θ /10
222nm 222nm -1.60 0.90 Θ] [
-1.65 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Cu(I) Equivalents Cu(I) Equivalents
Figure IV. 9. Plots of signal at 222 nm (θ222 nm) (A) and signal ratio at 222 nm to that at 208 nm (θ222 nm/θ208 nm) (B) as a function of Cu(I) equivalents. (Data were drawn from CD spectra of AQVC16C19-GGY in Figure IV.8.)
Compared with AQC16C19-GGY and AQVC16C19-GGY, the CD spectrum of mini-
AQVC9C12-GGY consists of only one large negative maximum centered at 199 nm with [θ](199
2 -1 nm) = -12542 deg*cm dmol as shown in Figure IV.10. The CD feature indicates that apo-mini-
AQVC9C12-GGY exists as a disordered random coil in aqueous solution.24 Similar spectral 104 characteristics were observed with the random coil mini-C9C12-GGY peptide (shown in Chapter
III) that has three “IEALEGK” heptad repeats, and two cysteine amino acid residue at the hydrophobic positions “a” and “d” of the second heptad repeat. The shorter sequence of mini-
C9C12-GGY disfavors the formation of coiled coil structure when Cu(I) ions were introduced.
The SEC result of mini-C9C12-GGY also shows that mini-C9C12-GGY exist as a monomer
(vide infra). Even the addition of Cu(I) ion can not induce the formation of coiled coil.
4000
0 -1
dmol -4000 2
-8000
deg cm Cu(I)/mini-AQVC9C12-GGY) mini-AQVC9C12-GGY [Θ] -12000
180 200 220 240 260
Wavelength/nm
Figure IV. 10. Circular dichroism spectra of mini-AQVC9C12-GGY (100 μM) (■) and Cu(I)/ mini-AQVC9C12-GGY (●) (100 μM) in pH5.4, 10mM acetate buffer 298 K.
As shown in Figure IV.10, the Cu(I) addition only causes a minor increase in signal intensity at 222 nm and a decrease at 199 nm. These results indicate that Cu(I) ion might cause a slight conformational change of mini-AQVC9C12-GGY peptide due to Cu(I) binding to cysteine ligand, but no coiled coil structure formed. Although the Cu(I) stabilized the coiled coil structure of AQVC16C19-GGY, the random coil structure of mini-AQVC9C12-GGY indicates that the 105 metal binding is unable to compensate the very low folding tendency of apo-miniC9C12-GGY to form a organized structure due to shorter heptads repeat in mini-AQVC9C12-GGY peptide.
IV.3.2 UV-vis titration
UV-vis absorption titration experiments were carried out to determine the Cu(I) to peptide stoichiometry for the title compounds and gain some insight into the Cu(I) cluster formation in the metalloproteins. The UV-vis titration spectra of AQC16C19-GGY with Cu(I) are shown in
Figure IV.11(A). In the experiments, 0.1 to 2.0 molar equivalent of Cu(I) per peptide monomer were added to 98.94 μM (for peptide monomer) AQC16C19-GGY solution. A new broad absorption band centered at 236 nm with noticeable shoulders at 268 and 298 nm is observed.
The changes of absorbance at 268 nm and 298 nm, which correspond to the thiolate to Cu LMCT transition and Cu(I) cluster-centered transition respectively25, were used to monitor the reaction process as shown in Figure IV.11(B). The absorbance changes saturate after approximately 1.3 equivalents of Cu(I) were added, suggesting a 1.3:1 Cu(I) to peptide monomer stoichiometry.
Such spectroscopic behavior have been reported with the absorption spectra of Cu(I)/C16C19-
GGY and Cu(I)-metallothionein.3, 25 Thus, assuming a peptide trimer conformation as determined by AUC (vide infra), these results suggest four Cu(I) ions are incorporated in the
Cu(I)/AQC16C19-GGY. 106
1.2 A B 98.94uM C16C19GGY-neutral Cu(I) titration 0.6 @268 and 298nm 0502-07
0.8 0.4
0.4 268nm
Absorption 0.2 298nm Absorption
0.0 0.0
200 250 300 350 400 450 0.00.51.01.52.0 Wavelength/nm Wavelength/nm Figure IV. 11. The difference UV–vis absorption spectra obtained upon successive adding Cu(I) (0.0-2.0 equivalents) to 98.9 μM AQC16C19-GGY in pH 5.4, 0.2 M acetate buffer (A), and plot of delta absorbance at 262 nm and 298 nm vs. Cu(I) equivalents (B).
The UV-vis absorption spectra of AQVC16C19-GGY taken upon successive addition of
Cu(I) shows similar characteristics to those of the Cu(I)/AQC16C19-GGY metalloprotein just described. As shown in Figure IV. 12 (A), three absorption bands around 236, 268, and 298 nm are also observed. The changes of absorbance at 268 nm and 298 nm, which correspond to the thiolate to Cu LMCT transition and Cu(I) cluster-centered transition respectively25, were used to monitor the reaction process as shown in Figure IV.12 (B). The absorbance at 268 and 298 nm absorbance reaches the maximum when approximately 1.5 molar equivalents of Cu(I) were added. With further addition of Cu(I) (1.7 ~ 2.5 equivalents), the absorbance at 268 and 298 nm decreases slightly then levels off, suggesting a 1.5:1 Cu(I) to peptide stoichiometry. Thus, assuming a peptide trimer conformation, these results suggest four Cu(I) ions are incorporated in the Cu(I)/AQVC16C19-GGY. 107
1.2 0.8 A 268nm B 298nm 0.6 0.8
0.4 ABS
Δ 0.4
0.2 Abs@268nm
0.0 Δ 100uM AQV12C16C19GGY Cu(I) titration 0.0 in pH5.4 acetate buffer 0730-07 200 250 300 350 400 450 Wavelength/nm -0.5 0.0 0.5 1.0 1.5 2.0 2.5 Cu(I) Equivalents
Figure IV. 12. The difference UV–vis absorption spectra obtained upon successive adding Cu(I) to 100 μM AQVC16C19-GGY in pH 5.4, 0.2 M acetate buffer (A), and plot of delta absorbance at 268 and 298 nm vs. the Cu(I) equivalents (B).
The UV-vis absorption features of mini-AQVC9C12-GGY observed upon Cu(I) addition are different from those of either Cu(I)/AQC16C19-GGY or Cu(I)/AQVC16C19-GGY metalloproteins. As shown in Figure 13(A), two new absorption bands at 236 nm and 264 nm are observed with a noticeable shoulder at 298 nm upon addition of Cu(I). The absorption at around
264 nm is more pronounced compared to the shoulder absorption around 268 nm observed for
Cu(I)/AQC16C19-GGY and Cu(I)/AQVC16C19-GGY. The similar pronounced band around
268 nm has been observed for Cu(I)/C9C12-GGY 26and Cu(I)/mini-C9C12-GGY. This may be a consequence of the properties of the binding ligands in the peptide. It is known that negative charges can be stabilized at the N-terminal of coiled coils due to the helix-dipole effect.27, 28 Thus the cysteinyl thiols in mini-AQVC9C12-GGY are more prone to be deprotonated than those in
AQC16C19-GGY or AQVC16C19-GGY. The changes at 264 and 298 nm, which correspond to the thiolate to Cu LMCT transition and Cu(I) cluster-centered transition respectively, were used to monitor the reaction process as shown in Figure IV.13 (C). The absorbance at 264 nm reaches maximum when 1.0 equivalents of Cu(I) is added. With further addition of Cu(I), the absorbance 108 at 264 nm decrease slightly and then starts to increase when 1.2 equivalent of Cu(I) is added. As for the absorbance at 298 nm, it does not reaches the maximum until approximately 1.2 equivalents of Cu(I) is added. The further addition of Cu(I) results in the decrease in the decrease of the absorbance at 298 nm. These results suggest that Cu(I) and mini-AQVC9C12-GGY form a complex with a 1.0:1.0 metal to peptide stoichiometry first. With more Cu(I), they eventually form complexes with 1.2:1.0 metal to peptide stoichiometry. This transition (from 1.0:1.0 to
1.2:1.0) brings relatively little change to the absorbance at 264 nm but a noticeable increase to the absorbance at 298 nm. It is likely the Cu-S bonds in Cu(I)/mini-AQVC9C12-GGY metalloprotein did not change much while Cu(I) clusters changed in this transition which is also supported by the emission titration results (shows in IV.3.3). Two isosbestic points at 280 and
318 nm, as in Figure IV.13 (B), started to appear after 1.3 molar equivalents of Cu(I) are added.
The appearance of these isosbestic points suggests new species start to form after 1.3 equivalents of Cu(I) were added to mini-AQVC9C12-GGY solution. Similar UV-vis absorption features have been reported for several Cu(I) metalloproteins, such as Cu(I) metalloprotein Ctr1c,29
Cu(I)/C9C12-GGY, Cu(I)/C2C5-GGY,3 Cu(I)/C12C16C19-GGY30 and Cu(I)/mini-C9C12-GGY
(Chapter III). The similarity of those peptides is that they are all exist as a random coil at their apo-states. But for the preformed coiled coil peptide such as AQC16C19-GGY and
AQVC16C19-GGYY, no isosbestic point is observed during the Cu(I) titration. These results probably reflect the effect of the peptide stability on the formation of the metalloprotein. The preformed coiled coil provide a more stable hydrophobic core than the random coil. Thus Cu-S bonds and Cu-cluster might form simultaneously. However, for the random coil peptides, the formation of Cu-cluster formed a loose structure first, and then forms a more tight cluster when 109
Cu(I) equivalents reach some point where the absorption band around 298 nm and the emission intensity will show a faster increase.
0.8 0.11 1.2 1.33 C 0.8 A 0.22 B 1.44 0.33 1.55 0.6 264nm 0.44 1.66 298nm 0.6 0.55 1.77 0.66 0.8 1.88 0.4 0.77 0.4 0.89 1.0 0.2 1.11 0.4 1.22 0.2 Absorrption Absorption Absorption 0.0 0.0 0.0 -0.2 0.0 0.5 1.0 1.5 2.0 200 250 300 350 400 450 200 250 300 350 400 450 Cu(I) Equivalents Wavelength/nm Wavelength/nm Figure IV. 13. The difference UV–vis absorption spectra obtained upon successive adding Cu(I) to 90.4 μM mini-AQVC9C12-GGY in pH 5.4, 0.2 M acetate buffer (A) 0.11 to 1.22 equivalents of Cu(I); (B) 1.33 to 1.88 equivalents of Cu(I); (C) Plot of delta absorbance at 264 nm and 298 nm vs. the equivalents of Cu(I).
IV.3.3 Emission titration
The metal ion to peptide stoichiometry and binding process of Cu(I) to AQC16C19-GGY,
AQVC16C19-GGY, or mini-AQVC9C12-GGY can be further probed by monitoring the changes in the emission spectra obtained upon successive addition of Cu(I) to the peptide solutions.
Details on this experiment are described in Chapter II. Figure IV.14(A) shows the emission spectra of AQC16C19-GGY metalloprotein after 0.1 to 2.0 molar equivalents of Cu(I) per peptide monomer were added to a 131.8 μM solution of AQC16C19-GGY. One broad emission band centered around 590 nm appears after Cu(I) is added, and the emission intensity increases linearly with the sequential addition of Cu(I) until approximately 1.2 equivalents of Cu(I) were added. Further addition of Cu(I) from 1.3 to 2.0 equivalents leads to a sharp decrease in the emission intensity while the emission maximum stays unchanged. This behavior is the same as that observed for C16C19GGY30 , metalloprotein CopA31, and metallothioneins32-34 when excess
Cu(I) was added. Two possible reasons may be responsible for this kind of behavior. One generally accepted reason is that the emission may be quenched by outer solvent as a result of that the Cu(I) cluster is disrupted by the further incoming Cu(I) ions. Figure IV.14. B shows this 110 behavior by replotting the maximum emission intensity in Figure IV.14.A. A break point at 1.2 equivalents of Cu(I) can be seen from Figure IV.14(B). Thus the emission titration experiment supports the 1.2:1.0 Cu(I) to AQC16C19-GGY stoichiometry as the emitting species.
131.8uM C16C19GGY-neutral Cu(I) 25 B emission tiration 0502-07 25 A 20 20 3
3 15 15 10
10 131.8uM C16C19GGY-neutral Cu(I) Counts/10 5 emission tiration @590nm 0502-07 Counts/10 5 0 0 0.0 0.5 1.0 1.5 2.0 400 450 500 550 600 650 700 750 Equivalents of Cu(I) Wavelength/nm
Figure IV. 14. Emission titration of AQC16C19-GGY by [Cu(CH3CN)4]PF6: Emission spectra obtained upon addition of Cu(I) to the 131.8 μM peptide solution 0.2 M acetate buffer (pH5.4) (A) and titration plot of emission intensity (590 nm) vs. Cu(I) equivalents (B).
Emission lifetime measurements of AQC16C19-GGY with 1.2 molar equivalents of Cu(I) was performed at room temperature in argon-saturated solutions. The excitation wavelength was set at 355 nm, and the emission decay was monitored at 590 nm. Figure III.15 shows the emission decay of Cu(I)/AQC16C19-GGY (Figure IV. 17. A) and its corresponding semi log plot (Figure IV. 17. B). The linear decrease of the semi log plot as shown in with time indicates that the emission decay follows mono-exponential decay kinetics: I(t) = Aexp(-kt), in which A, k corresponds to the amplitude and rate constant, respectively. The nonlinear least-square fit of the obtained data yields one lifetime τs = 1/ks = 8.46 μs. The mono-exponential decay kinetics is different from that observed for the decay of Cu(I)/C16C19-GGY metalloprotein which
3 displayed biphasic decay kinetics, for which τs = 1.02 μs, τL = 7.75 μs, and AS/AL =1.0.
However, the lifetime of Cu(I)/AQC16C19-GGY is comparable with the long lifetime of 111
Cu(I)/C16C19-GGY. The different lifetimes of Cu(I)/C16C19-GGY may arise from 2 isomers of
Cu(I)-C16C19GGY metalloprotein which an not be easily separate.
-2 4 100uM Cu(I)/AQC16C19-GGY in A pH5.4 Acetate buffer 0626-07 B -3 3 -3
Data: TEK00003_D Model: ExpDec1 Equation: y = A1*exp(-x/t1) + y0 Weighting: y No weighting
2 Chi^2/DoF = 1.0021E-9 -4 R^2 = 0.99871
y0 0.00002 ±1.8505E-6 A1 0.00364 ±4.8434E-6 t1 8.4613E-6 ±2.2837E-8 1 -5 Amplitude/10 log(intensity)
0 -6
0 1020304050 0 1020304050 Time/us Time/us
Figure IV. 15. Emission decay of Cu(I)/AQC16C19-GGY excited at 355 nm monitored at 590 nm (A) and corresponding log plots (B) at room temperature. The red line in (A) represents the fit of the data to mono-exponential decay kinetics as described in the text.
The Cu(I)/AQVC16C19-GGY metalloprotein shows some similar emission behavior as
Cu(I)/AQC16C19-GGY upon successive addition Cu(I). Only one broad emission band centered around 590 nm is observed and the emission intensity increases until approximately 1.6 molar equivalent of Cu(I) is added to AQVC16C19-GGY solution. This is followed by a decrease in intensity with the further addition of Cu(I). However, the emission maximum stays unchanged throughout the titration processes. Thus the emission titration also support the 1.6:1 Cu(I) to
AQVC16C19-GGY stoichiometry as the emitting species. However, with excess addition of
Cu(I), new species with weak emission forms. This result is consistent with the result analyzed from UV-vis absorption in Section IV.3.2.
The lifetime of Cu(I)/AQVC16C19-GGY was measured under the same conditions as those used for Cu(I)/AQC16C19-GGY (Figure IV.17). The linear decrease of the semi log plot
(Figure IV.17.B) of emission intensity with time indicates that the emission decay follows mono- exponential decay kinetics: I(t) = Aexp(-kt), in which A, k corresponds to the amplitude and rate 112 constant, respectively. The nonlinear least-square fit of the decay data produces one lifetime of τ
= 16 μs which is almost twice the emission lifetime of Cu(I)/AQC16C19-GGY. The longer lifetime might reflect the difference in cluster structure or the hydrophobic condition of the cluster. The structural details of these metalloprotein are still under study.
100uM Cu(I)/AQV12C16C19GGY titration in pH5.4 0.2 M acetate buffer 0801-07 5 B 5 A 4 4
/s 3 -4 3 at 590nm 3 2 2 1
Countsx10 1
Counts/10 0 0 -0.5 0.0 0.5 1.0 1.5 2.0 2.5 450 500 550 600 650 700 750 Cu(I) Equivalents Wavelength/nm
Figure IV. 16. Emission titration of AQVC16C19-GGY by [Cu(CH3CN)4]PF6. Emission spectra obtained upon addition of Cu(I) to the 131.8 μM peptide solution 0.2 M acetate buffer (pH5.4) (A) and titration plot of emission intensity (590 nm) vs. Cu(I) equivalents (B).
0.05 A B -1.4
0.04 Data: TEK00000_B Model: ExpDec1 Equation: y = A1*exp(-x/t1) + y0 Weighting: y No weighting 0.03 -2.1 Chi^2/DoF = 3.1187E-9 R^2 = 0.99996
y0 -0.00018 ±2.241E-6 0.02 A1 0.04479 ±0.00001 t1 1.60016E-5 ±7.0313E-9
Intensity -2.8 0.01 log(intensity)
0.00 -3.5
0 50 100 150 200 0 204060 Time/us Time/us
Figure IV. 17. Emission decay of Cu(I)/AQVC16C19-GGY excited at 355 nm monitored at 590 nm (A) and corresponding log plots at room temperature (B). The red line represents the fit of the data to mono-exponential decay kinetics as described in the text.
113
The emission of Cu(I)/mini-AQVC9C12-GGY was also examined under the same conditions as those used to study the Cu(I)/AQC9C12-GGY and Cu(I)/AQVC9C12-GGY metalloproteins. Figure IV.18 (A)-(B) shows the emission spectra of Cu(I)/mini-AQVC9C12-
GGY and the emission maximum around 618 nm in emission spectra as a function of Cu(I) addition, respectively. The emission intensity of Cu(I)/mini-AQVC9C12-GGY shows a linear increase with the successive addition of Cu(I) until 1.3 equivalents of Cu(I) was added to the mini-AQVC9C12-GGY solution. Further addition of Cu(I) (> 1.3 equivalents) results in a sharp decrease of emission intensity. However, no obvious redshift of the emission maximum is observed as was seen for Cu(I)/mini-C9C12-GGY. Different peptide sequences and oligomerization states might be in charge of the difference in emission properties. Thus the emission titration also supports the stability of the 1.3:1.0 Cu(I) to mini-AQC9C12-GGY as the emitting species. This behavior is also observed for AQC16C19-GGY, Cu-metallothionein, and copper chaperone CopZ.25, 35
Thus, assuming a peptide trimer conformation, these results suggest four Cu(I) ions are incorporated in the Cu(I)/mini-AQVC9C12-GGY.The titration results of AQC16C19-GGY and
AQVC16C19-GGY also support that four Cu(I) ions are incorporated in the resulting metalloprotein. However, the maximum emission wavelength of Cu(I)/AQC9C12-GGY and
Cu(I)/AQVC9C12-GGY complexes is at 590 nm, while the maximum emission wavelength of
Cu(I)/mini-AQVC9C12-GGY presents at 618 nm, and no obvious redshift of the emission maximum is observed during the titration, as shown in Figure IV. 18(A).
114
4.0 90.4uM miniAQV5C9C12GGY Cu(I) titration 618nm 40000 in pH5.4 Acetate buffer 1002-07 A 3.2 B 30000 2.4 /s -4
20000 1.6
0.8 Absorption 10000 Counts10 Δ 0.0
0 0.0 0.5 1.0 1.5 2.0 2.5 500 600 700 800 Cu(I) Equivalents Wavelength/nm Figure IV. 18. Emission titration of Cu(I)/mini-AQVC9C12-GGY by [Cu(CH3CN)4]PF6. Emission spectra obtained upon addition of Cu(I) to the 90.4 μM peptide solution 0.2 M acetate buffer, pH 5.4 (A) and titration plot of emission intensity (618 nm) vs. Cu(I) equivalents (B).
Figure IV.19 shows the lifetimes of Cu(I)/mini-AQVC9C12-GGY measured under the same conditions as those used for the Cu(I)/AQC16C19-GGY and Cu(I)/AQVC16C19-GGY systems. The nonlinear decrease of corresponding semi-log plots of the decay intensity with time indicates that the emission decay of Cu(I)/mini-AQVC9C12-GGY can not be fitted by mono- exponential decay kinetics. Thus bi-exponential decay kinetics are used to fit the decay data: I(t)
= AS exp(-kSt) + ALexp (-kLt) , where AS, kS and AL, kL correspond to the amplitudes and the rate constants for the short lifetime and long lifetime, respectively. The fitting results show AL =
0.00608, τL = 9.87 μs (τS = 1/kL), AS=0.00342, and τs = 1.17 μs (τL = 1/kL). The two emission lifetimes suggest two excited-state manifolds and/or two isomers of the Cu(I)/mini-AQVC9C12-
GGY. The decay kinetics of Cu(I)/mini-C9C12-GGY are different from that of
Cu(I)/AQC16C19-GGY or Cu(I)/AQVC16C19-GGY which shows one lifetime. However, those behaviors are similar to that of Cu(I)/C16C19-GGY, and Cu(I)/mini-C9C12-GGY which also showed two lifetimes. Different from the preformed coiled coil structures of AQC16C19-GGY and AQVC16C19-GGY, C16C19-GGY, mini-C9C12-GGY, and miniAQC16C19-GGY exist as 115 a random coils which might result in the different metal binding mechanisms and affect the conformation of the resulting metalloproteins which displays two lifetimes.
A -2.0 B 0.009
-2.5 0.006 -3.0
0.003 Intesnity -3.5 log(intensity)
0.000 -4.0
0 1020304050 0 1020304050 Time/us Time/us
Figure IV. 19. Emission decay of Cu(I)/mini-AQVC9C12-GGY excited at 355 nm monitored at 600 nm (A) and corresponding log plots (B) at room temperature. The red line represents the fit of the data to mono-exponential decay kinetics as described in the text.
116
IV.3.4 Oligomerization states determination of metalloproteins
The above spectroscopic analyses were used to determine the metal ion to peptide stoichiometry and some secondary structural information of Cu(I)/AQC16C19-GGY,
Cu(I)/AQVC16C19-GGY, and Cu(I)/mini-AQVC9C12-GGY metalloproteins. Further information on the metalloprotein oligomerization state can be further obtained by the analytical ultracentrifugation (AUC) or high performance size exclusion chromatography (HPSEC).Thus, sedimentation velocity AUC experiments were conducted in collaboration with Dr. Borries
Demeler at UTHSC to determine the oligomerization state of the Cu(I)/AQC16C19-GGY adduct. Figure IV.20 shows the fitting results for～ 210 μM Cu(I)/AQC16C19-GGY adduct
(A280nm = 0.9) at 60.0 krpm. The Genetic Algorithm analysis showed that one major species
(86.26% percentage) with a molecular weight of 10.67 kDa presents and other two unknown minor species with low molecular weight and high molecular weight, as shown in Table IV.2.
The molecular weight indicates that Cu(I)/AQC16C19-GGY exists as a trimer considering that the molecule weight of AQC16C19-GGY is 3460. The AUC results of apo-AQC16C19-GGY studied in Section IV.1 have shown that apo-AQC16C19-GGY exists in equilibrium between the dimer and tetramer structures. Interestingly, Cu(I) addition does not form a dimer or tetramer, but a trimer structure with 4 Cu(I) ions in the hydrophobic core as shown by UV, and emission titration results. The mixture of coiled coils with different oligomerization states was also observe for the mutation of Asn16Val36, in which the replacement of asparagine at position 16 of a 33 amino acid peptide GCN4-pl37, 38 results in a mixture of dimers and trimers. However, only the trimer is favored upon the addition of benzene. The metal ion-dependent conformational switches were also studied. For example the Ag(I) ions induced the formation of large fibrous α- helical assemblies39, reversibly switches between a trimeric α-helical coiled coil and a zinc- 117 bound folded monomer.40, 41 The original design of this study is to obtain dimer coiled coils differing in stability, however, the modification of the peptide sequence results in a mixture of dimers and tetramers. More interestingly, a trimer structure is favored upon the addition of Cu(I) ions instead of dimer or tetramer. The metal ions induced switch of oligomerization states is important to de novo designing of metalloprotein design and understand the protein folding.42
Figure IV. 20. Genetic algorithm analysis 3D distribution plot of velocity AUC data of Cu(I)/AQC16C19-GGY in 0.2 M pH 5.4 acetate buffer.
Table IV. 3. The molecular weight distribution and percentage of AUC data of Cu(I)/AQC16C19-GGY in 0.2M pH5.4 acetate buffer.
Molecualr weight Percentage 7.4296e+02 (5.258 %)
1.0676e+04 ( 86.260 %) 2.9880e+04 (8.482 %)
The oligomerization state of Cu(I)/AQC16C19-GGY was then used as a standard in
HPSEC experiment to detect the oligomerization states of the Cu(I)/AQVC16C19-GGY and
Cu(I)/mini-AQVC9C12-GGY metalloproteins. Figure IV.21 shows the HPSEC chromatogram 118
Cu(I)/AQC16C19-GGY where a retention time of 33.9 min can be determined. With the measured retention time, K of Cu(I)/AQC16C19-GGY could be calculated from Eq. II.2 defined d in Chapter II. The calibration curve for molecular weight as a function of K was then d determined based on standards Cu(I)/AQC16C19-GGY and other known peptides or metalloproteins C16C19-GGY, Cu(I)/C16C19-GGY, and HO12, as shown in Figure IV.22. The error of the molecular weight can be evaluated around 12% by the linear fit of the log(MW) vs
Kd.
Cu(I)/AQC16C19-GGY 1.0
0.8
0.6
0.4
0.2
0.0 Normalized Absorption at 275nm -0.2 30 40 50
Time/min Figure IV. 19. High performance size exclusion chromatography (HPSEC) chromatogram of Cu(I)/AQC16C19-GGY monitored at 275 nm. The sample was eluted with 50 mM phosphate buffer at 0.4 ml/min and pH 7.0.
119
4.2 Cu(I)/C16C19-GGY
Cu(I)/AQC16C19-GGY
4.0
Cu(I)/AQVC16C19-GGY
3.8 Cu(I)/Mini-AQVC9C12-GGY
3.6 C16C19-GGY
log(MW) 3.4
mini-AQVC9C12-GGY 3.2 HO12 3.0 0.36 0.40 0.44 0.48 0.52 K d
Figure IV. 20. The calibration curve obtained by using peptide standards on a Superdex 75 column. (All the experiments were done in pH 7.0 phosphate buffer containing 50 mM phosphate and 100 mM potassium chloride. The sample was eluted with 0.2 M sodium acetate buffer at 0.4 ml/min, pH 5.4.)
Figures IV.23 shows the HPSEC chromatograms of apo-AQVC16C19-GGY and
Cu(I)/AQVC16C19-GGY together. The two peaks in Figure IV.23 are similar and overlap with each other. The retention time of HPSEC peak of Cu(I)/AQVC16C19-GGY is 34.5 min, which is slightly longer than 34.2 min of apo-AQVC16C19-GGY. The molecular weights of
Cu(I)/AQVC16C19-GGY and AQVC16C19-GGY are determined from the calibration curve, and a similar value of ～ 9.2±1.1 kDa is obtained. This value is close to the calculated molecular weight of the Cu(I)/AQVC16C19-GGY trimer 10.5 kDa. Thus, Cu(I)/AQVC16C19-GGY metalloprotein likely exists as a trimer structure with 4 Cu(I) ions in the hydrophobic core.
The HPSEC chromatograms of apo-mini-AQVC9C12-GGY and Cu(I)/mini-AQVC9C12-
GGY are shown in Figure IV.24. Different from the overlap of the retention peak of the apo-
AQVC16C19-GGY and Cu(I)/AQVC16C19-GGY, the retention peaks in Figure IV.24 show two almost completely separated peaks. The retention time of apo-mini-AQVC9C12-GGY is 37.8 120 min, while the retention time of Cu(I)/mini-AQVC9C12-GGY is 34.4 min. The calibration curves give the molecular weights of Cu(I)/mini-AQVC9C12-GGY and apo-mini-AQVC9C12-
GGY, 7.0±0.8 kDa and 2.1 kDa, respectively. These values are very close to the calculated MW of the Cu(I)/mini-AQVC9C12-GGY trimer, 8.0 kDa, and MW of mini-AQVC9C12-GGY monomer, 2.7 kDa respectively. Thus, HPSEC results indicate that Cu(I)/mini-AQVC9C12-
GGY likely exists as a trimer.
Cu(I)/AQVC16C19-GGY 1.00 AQVC16C19-GGY
0.75
0.50
0.25
0.00 Normalized absorption at 275 nm Normalized absorption
25 30 35 40 45 50 Time/min
Figure IV. 21. High performance size exclusion chromatography (HPSEC) chromatogram of the apo-AQVC16C19-GGY (—) and Cu(I)/AQVC16C19-GGY (—) monitored at 275 nm. The sample was eluted with 50mM phosphate buffer 0.4 ml/min and pH 7.0.
121
Cu(I)/mini-AQVC9C12 apo-mini-AQVC9C12 0.9
0.6
0.3
0.0 normalized absorption normalized absorption
25 30 35 40 45 50 Time/min
Figure IV. 22. High performance size exclusion chromatography (HPSEC) chromatogram of the apo-mini-AQVC9C12-GGY (—) and Cu(I)/mini-AQVC9C12-GGY (—) monitored at 275 nm. The sample was eluted with 50 mM phosphate buffer at 0.4 ml/min and pH 7.0.
Table IV.4 The molecular weight of the Cu(I)/peptide and their oligomerization states
Peptide MW of Cu(I)/peptide Oligomerization AUC HPSEC State AQC16C19-GGY 10.7 kDa 9.2±1.1 kDa Trimer AQVC16C19-GGY -- 9.2±1.1 kDa Trimer mini-AQVC9C12-GGY -- 7.0±0.8 kDa Trimer
IV.4 Conclusion
In summary, a series of peptides with different free energies of folding at their apo-states were designed and their Cu(I) binding properties were investigated as shown in table III.5 and
III.6. The photoproperites and the oligomerization states of the resulting Cu(I) metalloproteins were also studied. The original design of this series of peptides is studying the free energy effect of the peptide folding on the final oligomerization state of the resulting metalloproteins. Pecoraro et al investigated the enforcement of protein structures in defining the coordination geometry and binding affinity of an active-site metal cofactor by designing peptides with different free energy 122 of peptide folding. Those results indicated that the peptide folding preference is the fundamental driving force for the trigomal geometry attainment. In contrast to their results, the research in our group shows that metal ions can induce a transmission from the random coils to coiled coils to dimer or tetramer which indicate that effect of the metal ions binding in determining the oligomerization preference of the peptide. To investigate the interplay of polypeptide backbone and metal coordination stabilities, the peptides AQC16C19-GGY, AQVC16C19-GGY and miniAQVC16C19-GGY were designed to form a series peptides having different free energies of folding. The peptides are based on a well studied peptide motif (IAALEQK)n which forms a stable coiled coil dimer structure.19 Two cysteine residues are replaced at position “a(16)” and position “d (19)” position to generate a metal ion binding position. AQVC16C19-GGY shows the same sequence as AQC16C19-GGY except that a smaller and less hydrophobic valine residue replaces leucine residue at “d(12)” position. Mini-AQVC9C12-GGY shows less amino acid than AQVC9C12-GGY. However, the disturbing of the peptide hydrophobic cores result in a mixture of dimer and tetramer coiled coil for AQC16C19-GGY and AQVC16C19-GGY and random coil for miniAQVC9C12-GGY. The denaturation experiments show that the free energies of peptides folding in the sequence: AQC16C19-GGY > AQVC16C19-GGY > mini-
AQVC9C12-GGY. The emission titration shows that both Cu(I)/AQC16C19-GGY and
Cu(I)/AQVC16C19-GGY show strong emission at 590 nm while Cu(I)/mini-AQVC9C12-GGY have a strong emission at 618 nm. The 30 nm redshift in emission Cu(I)/mini-AQVC9C12-GGY might reflect the effect of hydrophobic condition of the peptide since Cu(I)/mini-AQVC9C12-
GGY exists a random coil instead of the coiled coil structure observed for Cu(I)/AQC16C19-
GGY and Cu(I)/AQVC16C19-GGY. The emissive Cu(I) cluster in the core of Cu(I)/mini-
AQVC9C12-GGY is more solvent accessible due to the random coiled coil structure. Both 123
Cu(I)/AQC16C19-GGY and Cu(I)/AQVC16C19-GGY show one emission lifetime, however,
Cu(I)/mini-AQVC9C12-GGY shows two lifetime. The similar results also were observed for
Cu(I)/C16C19-GGY and Cu(I)/C9C12-GGY. Two lifetimes may rise from 2 isomers of metalloproteins which can not be separated easily. The two isomer might differs in the orientation of the three peptide chains in the trimer metalloprotein43 or the two emission lifetime may due to two excited-state manifolds.25, 44 Although the lifetime of Cu(I)/AQVC16C19-GY is twice of that of Cu(I)/AQC16C19-GGY, both are in the μs range. No proof so far indicates that the lifetime difference is correlated with the stability of the metalloprotein.
The α-helical content of AQC16C19-GGY shows no increase upon the addition of Cu(I), which indicates that the metal binding can not stabilize the coiled coil structure of AQC16C19-
GGY due to its very stable structure. However the α-helical content of AQVC16C19-GGY increased from 40.9% to 46.9% which shows the stabilization of the metal binding. As for the miniAQVC16C19-GGY, the effect the metal binding is not enough to compensate the peptide folding tendency which is a random coil due to the short peptide sequence.
As for the oligomerization states, the AUC and HPSEC results indicate that all the resulting metalloproteins exist as trimer after the incorporation of Cu(I) although the free energies of the peptide folding are different. This observation is different from the study by
Pecoraro et al,45 where the peptide folding stabilized the unnatural coordination of metal ions and the tertiary structures of metalloproteins are the expected trimer as the original design. In related work, Tanaka et al. investigated the metal-induced peptide conformation transition from random coil to three-stranded coiled-coil coiled coil upon the addition of Ni(II), Co(II), Zn(II), or Cu(II),
46 however the peptide design is base on a preformed three-stranded YGG(IEKKIEA)4. All those study shows the preference of apo-peptide folding instead of metal ions binding. In this study, 124 although AQC16C19-GGY show larger free energy of peptide folding than that of babyL9C45 which folds to timer and forces metal ions to use the unnatural coordination geometries,
Cu(I)/AQC16C19-GGY exists as a metal ions induced trimer structure. These results demonstrate that free energies of apo-peptides folding do not exert great influence on the final oligomerization states of the Cu(I) metalloproteins for our designed peptides. In contrary, it seems that the metal ion plays important role in determining the final oligomerization states of metalloproteins, considering the same metal ion (Cu(I)) causes the same final oligomerization states of the three Cu(I) metalloproteins. The peptide with less free energies of folding,
AQVC16C19-GGY and mini-AQVC16C19-GGY also exist as timers as determined by SEC.
Considering the stoichiometry in terms of peptide monomer, 4-5 Cu(I) ions are incorporated in the trimer metalloproteins. The present result is contrary to our expectation at the beginning of this study. However, it does confirm us another fact that metal ions are still dominating factor in determining the oligomerization structures of metalloproteins, as what we have verified with
Cu(I)/C16C19-GGY and Cd(II)/C16C19-GGY complexes previously.3, 8
125
Table IV.5 Properties of apo-peptides
AQC16C19-GGY AQVC16C19-GGY Mini-AQC16C19- GGY Monomer MW 3460 3446 2692 Free energy of 9.97 (dimer model) 4.47 (dimer model) No data folding 22.4 (tetramer model) 18.4 (tetramer (kcal/mole) model) Oligomerization Mixture of dimer and Mixture of dimer Random coil state tetramer coiled coil and tetramer coiled monomer coil
Table IV.6 Properties of Cu(I)/peptides
Cu(I)/AQC16C19- Cu(I)/AQVC16C19- Cu(I)/miniAQVC9C1 GGY GGY 2-GGY UV 236 nm, 236 nm, 236 nm, 268nm (shoulder) 268 nm (shoulder) 264 nm 298 (shoulder) 298 (shoulder) 298 nm (shoulder) Emission 590 nm 590 nm 618 nm Maximum (nm) Lifetime τ = 7.75 μs τ = 16 μs τL = 9.87, τs = 1.17 μs CD 222 nm (-), 222 nm (-), 208 nm (-) 199 nm (-) 208 nm (-) α-helical content 92.4% 46.9% -- Stoichiometry No data (CD) 1.5 (CD) No data (CD) (Cu(I):peptide 1.3 (UV) 1.6 (UV) 1.3 (UV) monomer) 1.2 (Emission) 1.6 (emission) 1.3 (Emission) MW by SEC 9.2±1.1 kDa 9.2±1.1 kDa 7.0±0.8 kDa Oligomerization Trimer Trimer Trimer State
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APPENDIX
STRUCUTRE AND OLIGOMERIZATION STATE OF Cd(II)/AQC161C19-GGY IN
CRYSTAL AND SOLUTION
To further our investigation on effect of metal ions on the oligomerization state of metalloproteins, Cd(II) ion was introduced to the AQC16C19-GGY apo-peptide, the more “stiff” coiled coil peptide, to study its influences on the peptide chain when metalloprotein forms.
Previous study in our group has revealed that Cd(II) can bind AQC16C19-GGY with a ratio of
1.3 Cd(II) to AQC16C19-GGY.1 The conformation analysis showed that AQC16C19-GGY coiled coil is disturbed by the incorporation of Cd(II) ions to form Cd cluster since the helical content of the metalloprotein is reduced to 79.0% compared to the 92.4% of the apo-peptide. In present study, the trimer structure of Cd(II)/AQVC16C19-GGY metalloprotein and the disturbance of Cd(II) (or Cd4) cluster to the conformation of AQC16C19-GGY coiled coil were further revealed with X-ray crystallography investigation.
A B Cd4 S1a S3a
S2a
Cd1 Cd3 O3 O2 S3b Cd2 S2b S1b O1
Figure A1. Crystal structure of Cd(II)/AQC16C19-GGY (A) and Cd4 cluster structure in Cd(II)/AQC16C19-GGY (B). 130
Figure A2. Helical wheel diagrams for parallel three stranded coiled coil peptides (adapted from reference2).
A general concept on structural information of Cd(II)/AQC16C19-GGY metalloprotein can be understood firstly from illustrations in Figures A1. and A2. shows the crystal structure of
Cd(II)/AQC16C19-GGY metalloprotein obtained from X-ray crystallography, analyzed by Dr.
Ding and Dr. Kennedy in University of Miami. Obviously, the α-helical structure of AQC16C19-
GGY in Cd(II)/AQC16C19-GGY is distorted, deviating away from the original structure. To probe the reasons behind peptide distortion upon metal ion addition, the Cd4 cluster is singled out of the Cd(II)/AQC16C19-GGY metalloprotein, with the structure shown in Figure A1. As shown in Figure A1, gray spheres represent Cd(II), yellow ones represent sulfur from cysteine in positions 16 and 19 of the AQC16C19-GGY peptides, and red spheres are oxygen (COO-) in glutamine acid at position 20 (i.e., “e” position in heptad repeat shown in Figure 2) of peptide chain. Three AQC16C19-GGY peptide chains in Cd(II)/AQC16C19-GGY trimer are labeled as number “1”, “2”, and “3”, respectively; Letters “a” and “b” are used to represent two cysteines in peptide chain. Number “4” in Cd4 (Figure A1.) only means for the 4th Cd atom in the metal cluster, which has no oxygen binding but binds to three Cys-S at position ‘16’ of three individual peptide chains. Cd1, Cd2, and Cd3 each binds to one oxygen from COO- group in glutamine acid of each peptide and three Cys-S, in which two Cys-S originate from one peptide chain (position 131
‘16’ and position‘19’) and another Cys-S from a nearby peptide chain at position ‘19’. Figure
A2. shows the ideal conformation of a trimer coiled coil structure, in which position “e” in heptad repeat is not in the hydrophobic core of the coiled coil structure. Detailed description on
Figure A2. can be referred to Section I.2.3 in Chapter I.
From the binding coordination of atoms in the Cd4 cluster, the disturbance of Cd4 cluster on the coiled coil structure can be easily understood when combining with X-ray crystallographic analysis results. Table A1-A3 lists the cadmium coordination, bond lengths, angles, and geminal S···S distances in Cd4 cluster, respectively, obtained from X-ray crystallographic analysis. For the Cd1 tetrahedron coordination, the three bond lengths of Cd1-S are different, as indicated by Cd1-S1a = 2.618 Å, Cd1-S1b = 2.637 Å, and Cd1-S3b = 2.429 Å in
Table A1. The bond length of Cd1-O3 in Cd1 tetrahedron is 2.189 Å, shorter than any Cd1-S band length due to the smaller radius of O atom than S atom. The bond angles in Cd1 tetrahedron are different, varying from S1b-Cd1-S3b = 84.992° to S1a-Cd1-S3b = 122.105°, as shown in
Table A1. The geminal S···S distances in Cd1 tetrahedron are also different (shown in Table
A3.), ranging from S1b···S3b = 3.426 Å, S1a···S3b = 4.418 Å, to S1a···S1b = 4.544 Å, respectively. The differences in bond lengths, bond angles, and geminal S···S distances of Cd1 tetrahedron indicate that the coordination of Cd1 is not a perfect tetrahedron but a distorted tetrahedron given that all bond lengths, angles, and geminal S···S distances should be equal or very close in an ideal Cd1 tetrahedron coordination. The comparison on bond lengths, angles and geminal S···S distances in Cd2 and Cd3 tetrahedrons (data in Table A1-A3) shows a similar result with Cd1 tetrahedron, which suggests that Cd2 and Cd3 have similarly distorted tetrahedron coordination. The examination on Cd-S distances in all Cd tetrahedron coordination shows that the distances are in the range of 2.429-2.739 Å. The range of such values is triple the 132 range of 2.459-2.544 Å observed for small molecular Cd cluster3. The three times higher deviation in bond lengths suggests that distortion of Cd4 cluster in Cd(II)/AQC16C19-GGY complex is obviously big compared to that of small molecular Cd cluster.
Although distortion of tetrahedron coordination of Cd, the structure of Cd4 cluster as a whole is still pretty stable. Examination on distances (Tablve A1) and angles (Table A2) of Cd atoms at the corner of the Cd4 cluster shows that Cd···Cd distances and Cd···Cd···Cd angles are within the range of 3.93127 Å - 4.03971 Å and 58.6° - 61.0°, respectively. Such close distance and angle values suggest that Cd atoms form almost a ideal tetrahedron structure, which is very likely stable even though distortion happens for the coordination of each Cd tetrahedron. Also, the S-Cd-S angles and geminal S···S distances fall in three groups in the order of ab > aa > bb,
3 which is well in agreement with the results observed in small Cd4 clusters. For example, the largest S-Cd-S angles is S2a-Cd2-S1b = 131.849° > S2a-Cd4-S1a = 109.897° > S2b-Cd2-S1b =
93.001°; the largest geminal S···S distance is S2a···S1b = 4.738 Å >S2a ···S1a = 3.948 Å >
S2b···S1b = 3.878 Å. The analog to small Cd4 clusters suggests that the Cd4 cluster in
Cd(II)/AQC16C19-GGY is not affected obviously by the surrounding peptide chains. On the contrary, the peptide is affect obviously by the incorporated Cd4 cluster. The distorted Cd4 cluster causes the distortion of the AQC16C19-GGY peptide chains and formation of the peptide coiled coil trimer. The binding of Cd(II) to oxygen in glutamine acid of peptide disturbs the position of glutamine acid in position “e” which results in the structure such as α-helix change of peptide chain. The Cd4 cluster also causes formation of the disturbed Cd(II)/AQC16C19-GGY coiled coil trimer structure.
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Table A1. Cadmium coordination bond lengths (Å)
bond bond distance (Å) bond bond distance(Å)
Cd1-S1a 2.618 Cd(3)-O(2) 2.146
Cd1-S1b 2.637 Cd(4)-S(1a) 2.373 Cd(1)-S(3b) 2.429 Cd(4)-S(1a) 2.450 Cd(1)-O(3) 2.189 Cd(4)-S(1a) 2.476 Cd(2)-S(2a) 2.585 Cd(1)···Cd(2) 3.93127 Cd(2)-S(2b) 2.739 Cd(1)···Cd(3) 4.00929 Cd(2)-S(1b) 2.605 Cd(1)···Cd(4) 4.02853 Cd(2)-O(1) 1.910 Cd(2)···Cd(3) 3.98615 Cd(3)-S(3a) 2.476 Cd(2)···Cd(4) 4.00874 Cd(3)-S(3b) 2.463 Cd(3)···Cd(4) 4.03971 Cd(3)-S(2b) 2.739
Table A2. Cadmium coordination bond angles (degrees)
atoms angle atoms angle S(1a)-Cd(1)-S(1b) 119.715 S(1b)-Cd(2)-O(1) 108.862 S(1a)-Cd(1)-S(3b) 122.105 S(3a)-Cd(3)-S(3b) 113.435 S(1a)-Cd(1)-O(3) 114.672 S(3a)-Cd(3)-S(2b) 121.386 S(1b)-Cd(1)-S(3b) 84.992 S(3a)-Cd(3)-O(2) 112.181 S(1b)-Cd(1)-O(3) 89.600 S(3b)-Cd(3)-S(2b) 98.490 S(3b)-Cd(1)-O(3) 117.029 S(3b)-Cd(3)-O(2) 93.341 S(2a)-Cd(2)-S(2b) 106.964 S(2b)-Cd(3)-O(2) 113.502 S(2a)-Cd(2)-S(1b) 131.849 S(1a)-Cd(4)-S(2a) 109.897 S(2a)-Cd(2)-O(1) 112.512 S(1a)-Cd(4)-S(3a) 113.907 S(2b)-Cd(2)-O(1) 94.534 S(2a)-Cd(4)-S(3a) 112.807 S(2b)-Cd(2)-S(1b) 93.001
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Table A3. Geminal S···S distances
atoms distances atoms distances S(1a)···S(1b) 4.544 S(1a)···S(2a) 3.948 S(1a)···S(3b) 4.418 S(1a)···S(3a) 4.065 S(2a)···S(1b) 4.738 S(2a)···S(3a) 4.103 S(2a)···S(2b) 4.280 S(1b)···S(2b) 3.878 S(3a)···S(3b) 4.306 S(1b)···S(3b) 3.426 S(3a)···S(2b) 4.385 S(2b)···S(3b) 3.639
Table A4. Cd···Cd···Cd angles (degree)
atoms angle atoms angle Cd4···Cd1···Cd2 60.5 Cd4···Cd3···Cd1 60.1 Cd4···Cd1···Cd3 60.3 Cd4···Cd3···Cd2 59.9 Cd3···Cd1···Cd2 60.3 Cd2···Cd3···Cd1 58.9 Cd4···Cd2···Cd3 60.7 Cd1···Cd4···Cd2 58.6 Cd4···Cd2···Cd1 61.0 Cd1···Cd4···Cd3 59.6 Cd3···Cd2···Cd1 60.8 Cd2···Cd4···Cd3 59.4
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The X-ray crystallography showed that Cd(II)/AQC16C19-GGY in solid state (crystal) has the distorted trimer structure. Considering the environmental effect on the metalloprotein oligomerization state, sedimentation velocity AUC was employed to determine the oligomerization state of Cd(II)/AQC16C19-GGY in solution. The experimental details on AUC method have been described in Chapter II, and the obtained results are collected in Figures A4-
A5. In Figure A4, the molecular weight of Cd(II)/AQC16C19-GGY was measured at three different concentrations, A280nm = 0.2, 0.3, 0.4 corresponding to Figure A4 (A), (B), and (C), respectively. The molecular weights and percentages of major component determined from these three figures are 8.8 kD, 82.7% (Figure A4 (A)), 9.9 kD, 59.6% (Figure A4 (B)), and 10.5 kD,
75.4% (Figure A4 (C)). The average molecular weight for the major components at different concentration is 9.7±0.86 kDa which indicates that Cd(II)/AQC16C19-GGY in pH 8.5 solution has the trimer oligomerization state, same as that in crystal phase. Lower solution pH might affect the binding constant and then the structure of the metalloprotein, and the pH effect on the metalloprotein structure was then examined at pH = 5.4. Again, Figure A4 (A), (B), and (C) corresponds to Cd(II)/AQC16C19-GGY at three different concentrations, A280nm = 0.2, 0.3, 0.4, respectively. Figure A4. shows that the molecular weight and percentage of the major component ranges from 11.05 kD, 84.6% (Figure A5 (A)), 8.90 kD, 92.8% (Figure A5 (B)), and 8.55 kD,
91.5% (Figure A5 (C)). The average molecular weight of the major components at different concentration is 9.5±1.3 kDa which suggests that Cd(II)/AQC16C19-GGY is still a trimer structure. This result suggests that although lower pH slightly affects the binding constant,
Cd(II)/AQC16C19-GGY still keeps a trimer structure at lower pH pH 5.4, similar to that in
Cd(II)/AQC16C19-GGY metalloprotein crystal.
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Figure A3. Genetic algorithm analysis of velocity ultracentrifugation results of Cd(II)/AQC16C19-GGY in 75 mM tris buffer (pH 8.5) with 1.5 M (NH4)2SO4M at three different concentrations (absorption at 280 nm, A: 0.2, B: 0.3, C, 0.4) at 60 krpm.
Figure A4. Genetic algorithm analysis of velocity ultracentrifugation results of Cd(II)/AQC16C19-GGY in 10 mM acetate buffer (pH 5.4) at three different concentrations (absorption at 280 nm, A: 0.2, B: 0.3, C, 0.4) at 50 krpm.
In summary, these results indicate that Cd(II)/AQC16C19-GGY exists as a trimer structure, with distorted α-helix of peptide chain caused by the formation of Cd4 cluster in metalloprotein. Cd ions in the Cd(II)/AQC16C19-GGY crystal has tetrahedron (Cd1, Cd2, and
Cd3) and trigonal (Cd4) coordination. Although Cd1, Cd2, and Cd3 have distorted tetrahedron coordination, the four Cd atoms keep almost a perfect tetrahedron structure. This result indicates
Cd4 cluster is a very stable and a major reason causing the structure change of the peptide coiled 137 coil. The AUC measurement determined that Cd(II)/AQC16C19-GGY is also a trimer structure in aqueous solutions, even pH was lowered to 5.4.
References
1. Zhu, X., Thesis (Ph.D.)--Bowling Green State University 2009.
2. Woolfson, D. N., The design of coiled-coil structures and assemblies. In Fibrous
Proteins: Coiled-Coils, Collagen And Elastomers, 2005; Vol. 70, pp 79-+.
3. Dean, P. A. W.; Payne, N. C.; Vittal, J. J.; Wu, Y. Y., Inorg. Chem. 1993, 32, 4632-4639.