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Capturing protein dynamics with time-resolved luminescence spectroscopy

Citation

Ford, Nicole Danielle Bouley (2013) Capturing protein dynamics with time-resolved luminescence spectroscopy. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:05302013-155904165

Abstract

The presented doctoral research utilizes time-resolved spectroscopy to characterize protein dynamics and folding mechanisms. We resolve millisecond-timescale folding by coupling time-resolved fluorescence energy transfer (trFRET) to a continuous flow microfluidic mixer to obtain intramolecular distance distributions throughout the folding process. We have elucidated the folding mechanisms of two cytochromes---one that exhibits two-state folding (cytochrome cb562) and one that has both a kinetic refolding intermediate ensemble and a distinct equilibrium unfolding intermediate (cytochrome c552). Our data reveal that the distinct structural features of cytochrome c552 contribute to its thermostability.

We have also investigated intrachain contact dynamics in unfolded cytochrome cb562 by monitoring electron transfer, which occurs as the heme collides with a ruthenium photosensitizer, covalently bound to residues along the polypeptide. Intrachain diffusion for chemically denatured proteins proceeds on the microsecond timescale with an upper limit of 0.1 microseconds. The power-law dependence (slope = -1.5) of the rate constants on the number of peptide bonds between the heme and Ru complex indicate that cytochrome cb562 is minimally frustrated.

In addition, we have explored the pathway dependence of electron tunneling rates between metal sites in proteins. Our research group has converted cytochrome b562 to a c-type cytochrome with the porphyrin covalently bound to cysteine sidechains. We have investigated the effects of the changes to the protein structure (i.e., increased rigidity and potential new equatorial tunneling pathways) on the electron transfer rates, measured by transient absorption, in a series of ruthenium photosensitizer-modified proteins.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:protein folding; intrachain diffusion; FRET; transient absorption; microfluidic mixing; contact quenching; electron transfer; cytochrome cb562; cytochrome c552
Degree Grantor:California Institute of Technology
Division:Chemistry and Chemical Engineering
Major Option:Chemistry
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Gray, Harry B.
Thesis Committee:
  • Barton, Jacqueline K. (chair)
  • Miller, Thomas F.
  • Rees, Douglas C.
  • Winkler, Jay Richmond
  • Gray, Harry B.
Defense Date:1 May 2013
Non-Caltech Author Email:nbouleyford (AT) gmail.com
Funders:
Funding AgencyGrant Number
National Institutes of HealthDK019038
National Institutes of HealthGM068461
Arnold and Mabel Beckman FoundationUNSPECIFIED
Record Number:CaltechTHESIS:05302013-155904165
Persistent URL:http://resolver.caltech.edu/CaltechTHESIS:05302013-155904165
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/jp403234hDOIArticle adapted for ch.4
http:/dx.doi.org/10.1073/pnas.1221832110 DOIArticle adapted for ch.6-7
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7775
Collection:CaltechTHESIS
Deposited By: Nicole Ford
Deposited On:31 May 2013 22:44
Last Modified:01 Apr 2014 16:54

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PDF (Chapters 6-8 (Cytochrome c552, folding summary)) - Final Version
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PDF (Chapter 9 (Electron tunneling)) - Final Version
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