| The energy landscape of a protein is defined as a hyper-dimensional surface constituted by all possible conformations with associated free energies, where the global minimum corresponds to the native structure, and local minima and maxima respectively to folding intermediates and transition states. The energy landscape determines a protein's folding and dynamic behaviors, and thus function. My research focuses on understanding general properties of the energy landscape and their sequence determinants. I used Borrelia OspA as a model system. This protein contains two globular domains connected with a unique single-layer beta-sheet, providing a relatively simplified system to study complex folding.;First, I developed a method based on amide hydrogen exchange to determine both thermodynamics and kinetics for the formation of multiple folding intermediates. With this method, I dissected OspA into five folding units, identified and characterized four folding intermediates, capturing the global features of the OspA energy landscape.;Second, I applied systematic mutagenesis coupled with a novel Bronsted-like analysis to quantify conformational heterogeneity in the energy landscape. This method also addressed the inherent ambiguity of the phi-value analysis, the most commonly used approach to characterize folding transition states. I resolved a previously identified, apparently single intermediate into two distinct species, and demonstrated that fractional phi-values, which are often observed in phi-value analysis, more likely result from conformational heterogeneity than from partial structure formation as generally presumed. |
