An investigation of the mechanical properties of the molten globule state of apomyoglobin
Single molecule force spectroscopy has provided important insights into the properties and mechanisms of protein folding. However, there are still many unanswered questions about how force affects the folding and unfolding of proteins and, in particular, the relationship between force and the rate-limiting transition state. In this thesis, I developed two protein systems to address two specific questions. The first question arose form previous work on E. coli RNAse H, in which a molten globule-like intermediate was observed to have a large distance (5 ± 1 nm) to the transition state. This large distance was in sharp contrast to the smaller distances (< 2 nm) typically observed for natively folded proteins. This raised the question of whether this distance was a general property of the E. coli RNAse H intermediate or a more general property of a molten globule state. To this end, I investigated the equilibrium molten globule state of sperm whale apomyoglobin at pH 5 under force and demonstrated that this state had a large distance to the transition state of 6.1 ± 0.5 nm. Further, this state was shown to have a large distance to the transition state regardless of the axis of the applied force. This work suggests that a large distance to the transition state is a general property of the molten globule state. The second system was developed using the SH3 domain from chicken c-Src in order to investigate if and how the structure of the transition state changes under force. I investigated the behavior of the protein under two different force axes observing significant differences in the mechanical unfolding of the protein. These experiments are ongoing but indicate that the change in behavior is because of a change in the structure of the transition state under force. Finally, investigating the properties of the molten globule state revealed an error in previous methodology using constant force feedback experiments. In this thesis, I identify and explain the origin of this error. Further, work on the molten globule state required higher fidelity data and a more sophisticated approach for the analysis of the data. Working with John Chodera and colleagues, we implemented novel methods for the analysis of the data.