Exhaustive mutagenesis in silico: Multicoordinate free energy calculationson proteins and peptides

We have extended and applied a multicoordinate free energy method, chemical Monte Carlo/Molecular Dynamics (CMC/MD), to calculate the relative free en ergies of different amino acid side-chains. CMC/MD allows the calculation o f the relative free energies for many chemical species from a single free e nergy calculation. We have previously shown its utility in host:guest chemi stry (Pitera and Kollman, J Am Chem Soc 1998;120:7557-7567)(1) and ligand d esign (Eriksson et al,, J Med Chem 1999;42:868-881)(2), and here demonstrat e its utility in calculations of amino acid properties and protein stabilit y. We first study the relative solvation free energies of N-methylated and acetylated alanine, valine, and serine amino acids. With careful inclusion of rotameric states, internal energies, and both the solution and vacuum st ates of the calculation, we calculate relative solvation free energies in g ood agreement with thermodynamic integration (TI) calculations. Interesting ly, we find that a significant amount of the unfavorable solvation of valin e seen in prior work (Sun et al,, J Am Chem Soc 1998;114:6798-6801)(3) is c aused by restraining the backbone in an extended conformation. In contrast, the solvation free energy of serine is calculated to be less favorable tha n expected from experiment, due to the formation of a favorable intramolecu lar hydrogen bond in the vacuum state. These monomer calculations emphasize the need to accurately consider all significant conformations of flexible molecules in free energy calculations. This development of the CMC/MD metho d paves the way for computations of protein stability analogous to the bioc hemical technique of "exhaustive mutagenesis." We have carried out just suc h a calculation at position 133 of T4 lysozyme, where we use CMC/MD to calc ulate the relative stability of eight different side-chain mutants in a sin gle free energy calculation. Our T4 calculations show good agreement with t he prior free energy calculations of Veenstra et al, (Prot Eng 1997;10:789- 807)(4) and excellent agreement with the experiments of Mendel et al, (Scie nce 1992;256:1798-1802),(5) (C) 2000 Wiley-Liss, Inc.