Identifying the Structural Determinants of Extreme Folding and Unfolding Barriers
- Author(s): Kelch, Brian A
- Advisor(s): Agard, David A
- et al.
Detailed knowledge of folding intermediate and transition state (TS) structures is critical for understanding protein folding mechanisms. For kinetically-stable proteins such as α-lytic protease (αLP) and its family members, their large free energy barrier to unfolding is central to their biological function. Thus their TS structure plays a crucial role in protein function. However, structural information regarding this important state has been completely lacking, mainly because standard techniques to probe TS structure are not realistically applicable for αLP. Therefore, I used the information embedded in the sequence of homologous proteases to discern the physical mechanisms by which kinetic stability can be modulated. This required experimental validation using various biophysical and biochemical techniques, such as mutagenesis, x-ray crystallography, and detailed kinetic analyses.
From these studies, I have shown that the conserved distortion of a sidechain significantly contributes to the destabilization of the TS for a large sub-class of αLP homologs. The strain from this deformation actually provides a biological advantage in that lifetime is greatly extended. This study was the first that shows that sidechain distortion has been shown to be used for a functional purpose and uncovers an unanticipated challenge for structural biology to identify potentially relevant distortions from high resolution structural studies.
My structural and kinetic analysis of a acid resistant αLP homolog, Nocardiopsis alba Protease (NAPase), identified the physical basis for this proteins acid stability, thus providing crucial structural information about unfolding mechanisms and leading to a model for the TS structure for these proteases. This study provided insight into the evolutionary benefits of kinetic stability as a paradigm for generation of extremophilic behavior.
From a similar study of a thermophilic αLP homolog, Thermobifida fusca Protease A (TFPA), I identified a substructure of these proteases, termed the domain bridge, which is used to modulate the degree of kinetic stability. This study refined our model for the unfolding TS, in which the domain bridge undocks and unfolds allowing the two domains of the protease to separate, with the newly formed crevice filling with solvent. These studies represent the first physical understanding of the structural basis for kinetic stability.