The goal of the proposed research is to elucidate the effects of amino acid mutations on the fluctuation properties of T4 phage lysozyme (T4L) proximate to the active site cleft. Dr. Hudson will focus on mutations of minimal perturbation as regards size change, and will perform constrained molecular dynamics calculations beginning with either an x-ray or a predicted crystal structure. A component of the research plan involves using the extensive data base of T4L mutant structures to further extend the ability to test structure predictive techniques. The importance of particular networks of nonbonded interactions in determining the overall dynamics of the region proximate to tryptophan 138, the fluorescence probe in the experimental work, as well as that of the active site itself will be probed. The complex decay of the fluorescence of the single tryptophan of T4L mutant forms and other single tryptophan proteins will be analyzed. The development of models will be guided by the results of dynamics simulations. The juxtaposition of experiments and calculations will establish the nature of large amplitude tryptophan dynamics on the interior of a protein. The previous simulation work on T4 phage lysozyme to include a realistic solvent environment will be extended. Finally free energy perturbation methods will be applied to determine the free energy differences for series of common structural/conformational changes observed in several room temperature simulations of mutant T4L. This will provide an enthalpic/entropic decomposition of the thermodynamic driving forces associated with the enhanced fluctuations gating the transition. The nature of nonbonded interactions in mutant proteins will be compared with the thermodynamic properties and fluctuation correlation functions to glean general principles.
