Beyond the native state: Exploring the role of partially folded conformations on the protein energy landscape
- Author(s): Connell, Katelyn Blair
- Advisor(s): Marqusee, Susan
- Klinman, Judith P
- et al.
Proteins can sample a variety of partially folded conformations during the transition between the unfolded and native states. The role of such intermediates is a matter of considerable debate, but it is clear that characterization of these partially folded species is crucial for understanding protein folding and function. A single amino acid change can convert E. coli ribonuclease H from a three-state folder that populates a kinetic intermediate to one that folds in an apparent two-state fashion. We have compared the folding trajectories of the three-state and two-state RNases H, proteins with the same native state topology but altered regional stability, using a protein engineering approach. Our data indicate that that both versions of RNase H fold through a similar trajectory with similar high-energy conformations. This suggests that formation of specific partially folded conformations may be a general feature of protein folding that can promote, rather than hinder, efficient folding.
To better understand the robust role this high-energy species plays in folding, we set out to trap the transient intermediate of RNase H at equilibrium by selectively destabilizing the region of the protein known to be unfolded in this species. We find that the intermediate is undetectable in a series of HSQC's, revealing the dynamic nature of this partially folded form on the timescale of NMR detection. This result is in contrast to studies in which the structures of trapped intermediates are solved by NMR, indicating that the they are well-packed and native-like. The dynamic nature of the RNase H intermediate may be important for its role as an on-pathway, productive species that promotes efficient folding. An analogous intermediate is populated on the kinetic trajectory of RNase H from T. thermophilus, an organism that grows optimally at a temperature 30 oC higher than E. coli. To understand how two proteins that share identical structures can function in such different environments, we looked for differences in their energetics by comparing equilibrium mimics of their high-energy intermediates. We find potential differences in the dynamic properties of the intermediates, which may provide insight into how proteins with the same native structure can exhibit vastly different biophysical behavior.
In contrast to globular proteins such as RNase H, repeat proteins are tandem arrays of repeating structural units that have no long-range contacts. In these modular domains, the majority of native contacts could be maintained in the face of partial unfolding. Repeat proteins therefore offer a unique architecture for exploring the extent of cooperativity and roughness on the energy landscape. To understand how a modular system builds cooperativity into its energetics, and to explore the origins and limits of this cooperativity, we studied the behavior of the Notch ankyrin domain in the optical tweezers, a single molecule mechanical tool. The forced unfolding of the Notch ankyrin domain occurs in one or two steps when manipulated in the optical tweezers. Though the unfolding pathway is heterogenous compared to that observed in bulk studies, there is a limit to the degree of uncoupling of individual repeats. We compare these results to the unfolding behavior of the Notch ankyrin domain in the atomic force microscope obtained by our collaborators for this project. This offers some insight into the apparent difference in solution and AFM unfolding of ankyrin repeat proteins.