STRUCTURE/STABILITY OF AN EXTREME THERMOPHILE PROTEIN

作为长期结构稳定性研究的一部分

As part of a long range study of structure and stability of extreme thermophile proteins, we propose a thorough, quantitative study of the structure, stability, and DNA-binding of the extremely thermostable Sac7d protein from Sulfolobus acidocaldarius, a thermophile which grows up to 92C. Extreme thermophile proteins are highly optimized systems that are expected to contain information useful for efficient rational protein engineering in medicine and biotechnology. The Sac7d protein provides an amenable, well-behaved compact system for probing the physical basis of protein stability. It is the smallest, most stable protein known which unfolds reversibly and lacks disulfide linkage and cofactors. Preliminary data (NMR, DSC, partial specific volume) indicate that the protein folds with significant secondary structure (an a-helix and five strands of B-sheet) and a well packed core. The project will be divided into three parts: First, NMR will be used to obtain a high resolution structure of the wild type protein in solution using distance geometry, restrained molecular dynamics, and a full-relaxation matrix refinement. In addition to traditional methods, a new Monte Carlo method will be used for defining the precision and accuracy of the NMR structure. The reliability of a rigid model will be tested by comparison of the standard deviation of the fit to the standard error of the NOESY data. The imprecision with respect to a rigid model will be investigated as a possible measure of flexibility. The hydrogen exchange kinetics of the slow exchanging core amide hydrogens of the protein will be determined and compared with those obtained for well characterized mesophile proteins as well as Sac7d mutants proteins. Third, site-directed mutagenesis in conjunction with DSC and NMR will be used to probe the stability of the protein. In addition to specific interactions indicated by the NMR structure to be potentially important in stabilizing the protein, the contribution of packing density will be investigated. The operating hypothesis to be tested is that efficient packing and optima van der Waals contacts can be an important factor in conferring enhanced stability on an extreme thermophile protein. The contribution of optimum packing of the core will be investigate by truncating leucine, isoleucine and valine residues using site directed mutagenesis. The change in free energy of folding per methyl/methylene/methine group removed will be compared to that observed in mesophile proteins. The packing density of the protein will be calculated using Richard's Voronoi polyhedra method. Confidence limits for the calculated packing density based on the NMR structure will be determined using the Monte Carlo precision. In addition, the partial specific volume of the wild type and mutant proteins will be measured.