Effect of Internal Cavities on Folding Rates and Routes Revealed by Real-Time Pressure-Jump NMR Spectroscopy

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)The time required to fold proteins usually increases significantly under conditions of high pressure. Taking advantage of this general property of proteins, we combined P-jump experiments with NMR spectroscopy to examine in detail the folding reaction of staphylococcal nuclease (SNase) and of some of its cavity-containing variants. The nearly 100 observables that could be measured simultaneously collectively describe the kinetics of folding as a function of pressure and denaturant concentration with exquisite site specific resolution. SNase variants with cavities in the central core of the protein exhibit a highly heterogeneous transition-state ensemble (TSE) with a smaller solvent-excluded void volume than the TSE of the parent SNase. This heterogeneous TSE experiences Hammond behavior, becoming more native-like (higher molar volume) with increasing denaturant concentration. In contrast, the TSE of the L125A variant, which has a cavity at the secondary core, is only slightly different from that of the parent SNase. Because pressure acts mainly to eliminate solvent-excluded voids, which are heterogeneously distributed throughout structures, it perturbs the protein more selectively than chemical denaturants, thereby facilitating the characterization of intermediates and the consequences of packing on folding mechanisms. Besides demonstrating how internal cavities can affect the routes and rates of folding of a protein, this study illustrates how the combination of P-jump and NMR spectroscopy can yield detailed mechanistic insight into protein folding reactions with exquisite site-specific temporal information.135391461014618Agence National de la Recherche [PiriBio 09-455024]National Science Foundation [MCB-0743422, MCB-0543769, MCB-1050966]Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)French Ministry of Research and Higher EducationFulbright International Graduate FellowshipCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Agence National de la Recherche [PiriBio 09-455024]National Science Foundation [MCB-0743422, MCB-0543769, MCB-1050966