The main objective of this proposal is to further develop a novel method established in the first proposal, proposal 1, for calculating the absolute entropy, S and the Helmholtz free energy, F (F=E-TS where E is the potential energy and 7 the absolute temperature). This method - the hypothetical scanning Monte Carlo (molecular dynamics) HSMC(MD) is an important ingredient in our approach for treating flexibility in biological macromolecules which also includes development of conformational search techniques and simplified solvation models; thus, HSMC(MD) will also be applied to problems treated within the framework of this approach. The main advantages of HSMC(MD) are: (i) Free energy differences between two microstates m and n (e.g., a helix and a hairpin of a peptide) or between two ligands bound to an active site of an enzyme can be obtained by carrying out only two different simulations from which Fm and Fn are obtained leading to AFmn = Fm -Fn without the need to resort to thermodynamic integration, (ii) The method is exact in the sense that all interactions are considered and the only approximation is due to insufficient sampling, (iii) Rigorous lower and upper bounds for F are provided. HSMC(MD) was developed initially for peptides, water, and self-avoiding walks. In this proposal (2) we seek to extend it to chain segments in proteins such as side chains, surface loops or ligands solvated by explicit water. Thus, the method will be used for studying structural preferences in mobile loops that play an important role in enzyme function (of the enzymes: a-amylase, triose phosphate isomerase (TIM), streptavidin, and acetylcholinesterase). We shall also calculate the relative free energy of binding of biotin and iminobiotin to streptovidin and their absolute free energies of binding, and binding free energy of amino acids to aspartyl-tRNA synthetase, comparing our results to the experiment and to previous computational work. We shall also predict loop structures in the CASP competition collaborating with Dr. Troy Wymore. Lattice model are used extensively for synthetic polymers and for studying protein folding. Thus, we shall improve HSMC for lattice chain models and apply it to several such models, in particular to study the population of microstates visited during conformational transitions in a model of the protein calmodulin. The new programs will be posted on the World Wide Web. ? ? ?
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