UC Berkeley

The dynamic nature of proteins is often an underappreciated aspect of biology necessary for understanding protein function and turnover in the cell. This is in part because within the multitude of states that a protein occupies throughout its lifetime, at most a few are significantly populated and have long enough lifetimes to study using traditional structural biology methods. In this thesis, I develop and explore experimental methods (amide hydrogen-deuterium exchange and cysteine labeling) to selectively label and characterize these rare and transient conformations methods. Specifically, I used cysteine labeling as a means to validate computational predictions of rare conformational fluctuations that expose potential drug-binding sites, I used pulse-labeling amide hydrogen exchange coupled with mass spectrometry detection to identify intermediates formed during protein folding and developed new data analysis procedures for analyzing hydrogen exchange rates measured determined by mass spectrometry on proteolytic fragments.

The first project used thiol labeling rates to validate computational predictions of cryptic binding sites. Analysis of millisecond-long molecular dynamics (MD) simulations of β-lactamase uncovered several potential conformational fluctuations that expose hidden, or cryptic binding pockets. Targeting these rare conformations presents a potential avenue for drug development. I validated the existence of these pockets by introducing cysteine residues in select positions and characterizing their accessibility to chemical modifications. In addition to validating the existence of these fluctuations, modification of cysteines at these sites modulates activity allosterically.

In the second project, I used pulsed labeling hydrogen exchange to follow the folding pathway of a protein family over evolutionary time (>3 billion years). I determined the conformations populated during folding (folding intermediates) for a family of RNase H proteins, including two extant and seven ancestral proteins. Each of these proteins was shown to populate a similar folding intermediate prior to the rate-limiting step in folding; however, the details of the steps leading up to this intermediate varied. We further showed that we can alter these early folding steps for a given protein using rationally designed mutations.

The third project compared and characterized the refolding and co-translational folding pathway of the protein HaloTag. We found that HaloTag aggregates during refolding but not during cotranslational folding, and it adopts at least one intermediate during refolding that is suppressed during cotranslational folding.

Finally, in the fourth project I developed a modification of a recently published approach for analyzing data from mass spectrometry-based detection of hydrogen exchange. This modification allows me to obtain multiple protection factors per peptide monitored and obtain a quantitative measurement of the free energy of hydrogen exchange in different regions of the protein.