Heterogeneity in fast-folding beta proteins Open Access

Davis, Caitlin Mearns Marlatt (2015)

Permanent URL: https://etd.library.emory.edu/concern/etds/bk128b64w?locale=en
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Abstract

Most proteins self-assemble, so secondary structure and folding dynamics must be encoded in the protein's sequence. Amyloids, extended β-sheet structures, have been associated with protein misfolding diseases such as Alzheimer's and prion disease. Understanding the secondary structure of these β-sheet structural motifs and the rules that govern their folding will ultimately allow for the design of more effective treatments of these diseases. Of particular interest are the early kinetic events, which structures form first and on what timescale. Laser-induced temperature-jumps coupled with fluorescence or infrared spectroscopy have been used to probe changes in the peptide backbone on the submillisecond timescale. Wavelength dependent infrared measurements allow resolution of the folding mechanism by independently probing the dynamics associated with the β-sheet or β-turn. In this dissertation, a hierarchal approach was taken to study β-sheet structures, starting from the smallest β-sheet structural motif, the β-hairpin, and building up to more complex systems, extended β-sheets and membrane bound β-hairpins. Even for the fastest folding linear β-hairpin, CLN025, folding is heterogeneous and cannot be described by a simple two state model. This work was extended to systems with two β-hairpins, WW domains, which increased the folding complexity. In all WW domains studied, folding was initiated in the turns and the β-sheets form last. Replacing the slower folding β-hairpin with a faster one increased the WW Domain folding speed by an order of magnitude, demonstrating the importance of β-hairpins in the context of the larger domain. β-hairpin formation was also shown to be an early step in the mechanism of membrane-induced folding and insertion of cationic anticancer β-hairpin therapeutics. These studies demonstrate that small fast folding domains are relevant to understanding larger systems, and further, while the folding of these systems is heterogeneous and cannot be described by a simple two-state model there are general rules that govern the folding of all of these systems.

Table of Contents

Chapter 1: Introduction................................................................................................... 1

1.1 The Protein Folding Problem..................................................................................... 1

1.2 Fast Events in Protein Folding................................................................................... 4

1.3 Folding of Beta Proteins............................................................................................ 9

1.4 Dissertation Outline................................................................................................. 12

Chapter 2: Raising the Speed Limit for β-Hairpin Formation.............................. 14

2.1 Abstract.................................................................................................................... 14

2.2 Introduction.............................................................................................................. 15

2.3 Experimental Section................................................................................................ 19

2.4 Equilibrium FTIR Studies........................................................................................ 22

2.5 Temperature-Jump Relaxation Kinetics.................................................................... 28

2.6 Discussion............................................................................................................... 32

2.7 Conclusion............................................................................................................... 36

Chapter 3: WW Domain Folding Complexity Revealed by Infrared Spectroscopy 38

3.1 Abstract.................................................................................................................... 38

3.2 Introduction.............................................................................................................. 39

3.3 Experimental Section................................................................................................ 43

3.4 Far-UV CD Spectroscopy........................................................................................ 46

3.5 FTIR Spectroscopy.................................................................................................. 50

3.6 Temperature-Jump Relaxation Kinetics.................................................................... 54

3.7 Conclusion............................................................................................................... 63

3.8 Appendix.................................................................................................................. 64

Chapter 4: Dynamics of an Ultrafast Folding Subdomain in the Context of a Larger Protein Fold 72

4.1 Abstract.................................................................................................................... 72

4.2 Introduction.............................................................................................................. 73

4.3 Experimental Section................................................................................................ 76

4.4 QUARK Prediction of WW domain Structure........................................................ 81

4.5 Far-UV CD Spectroscopy........................................................................................ 81

4.6 FTIR Spectroscopy.................................................................................................. 85

4.7 Temperature-Jump Relaxation Kinetics.................................................................... 88

4.8 Conclusion............................................................................................................... 97

4.9 Appendix.................................................................................................................. 99

Chapter 5: The Role of Electrostatic Interactions in Turn Stability of β-Proteins 106

5.1 Abstract.................................................................................................................. 106

5.2 Introduction............................................................................................................ 107

5.3 Experimental Section.............................................................................................. 111

5.4 Far-UV CD Spectroscopy...................................................................................... 115

5.5 FTIR Spectroscopy................................................................................................ 118

5.6 Temperature-Jump Relaxation Kinetics.................................................................. 127

5.7 Conclusion............................................................................................................. 133

5.8 Appendix................................................................................................................ 133

Chapter 6: Membrane-Induced Folding of a Cationic Anti-Cancer Peptide......... 141

6.1 Abstract.................................................................................................................. 141

6.2 Introduction............................................................................................................ 141

6.3 Experimental Section.............................................................................................. 144

6.4 Far-UV CD Spectroscopy...................................................................................... 146

6.5 Fluorescence Spectroscopy.................................................................................... 151

6.6 Conclusion............................................................................................................. 156

Chapter 7: Conclusion................................................................................................. 158

Appendix I: Site-Specific Resolution of Protein Folding using IR Labels Incorporated by Recombinant Protein Expression163

A.1 Introduction........................................................................................................... 163

A.2 Experimental Section............................................................................................. 168

A.3 Far-UV CD Spectroscopy..................................................................................... 172

A.4 FTIR Spectroscopy............................................................................................... 175

A.5 Temperature-Jump Relaxation Kinetics................................................................. 181

A.6 Conclusion............................................................................................................ 188

A.7 Appendix............................................................................................................... 189

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