Capturing Protein Dynamics with Time-Resolved Luminescence Spectroscopy

Author: Ford, Nicole Danielle Bouley

Year: 2013

Degree: Dissertation (Ph.D.)

Advisor: Gray, Harry B.

Committee Members: Barton, Jacqueline K.; Miller, Thomas F.; Rees, Douglas C.; Winkler, Jay Richmond; Gray, Harry B.

Option: Chemistry

DOI: 10.7907/N8B5-4644

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.

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