Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Biochemistry & Molecular Biophysics

First Advisor

Feng Gai


The topic of how a protein folds has been a major area of research for several decades; however, important details about this process are still undetermined. Experimental limitations in the study of protein folding are a result of no technique possessing both the necessary spatial and temporal resolution. This Thesis presents several studies conducted with the goal of expanding upon the experimentalist's toolbox, involving new methods of interrogating and/or perturbing protein systems of interest.

The early chapters of this Thesis describe our efforts using established synthetic methods to extend the utility of infrared spectroscopy in the study of protein folding. Specifically, we show that, using the strategy of cysteine alkylation, we can incorporate novel vibrational probes into proteins in a site-specific manner. We also show that combined sidechain mutagenesis, probing at multiple frequencies, and isotopic labeling to obtain secondary structural resolution in infrared studies of protein folding, in the process uncovering details about the folding mechanism of the Trp-cage miniprotein. Similarly, we illustrated the use of thioamides as site-specific reporters of backbone-backbone hydrogen bonding, and applied this functionalization to the Trpzip2 beta-hairpin system to validate its proposed folding mechanism. Further work involved using D-amino acids to interrogate turn regions in proteins, specifically examining Trp-cage folding.

The later chapters of this Thesis are focused on the effects of extrinsic molecules on the structural ordering of proteins. Taking advantage of the lack of tertiary structure of intrinsically disordered proteins, we examined the effect that trifluoroethanol has on protein folding, and found evidence that this cosolvent acts as a nano-crowder. We also introduced the idea of using phototriggers to modify the free energy landscape of folding for a given protein, and demonstrated that a peptide that typically folds in an activated (barrier-containing) manner can be made to fold in a downhill fashion upon irradiation.