THE FOLDING OF AN ENZYME .2. SUBSTRUCTURE OF BARNASE AND THE CONTRIBUTION OF DIFFERENT INTERACTIONS TO PROTEIN STABILITY

Barnase is described anatomically in terms of its substructures and their mode of packing. The surface area of hydrophobic residues buried on formation and packing of the structural elements has been calculated. Changes in stability have been measured for 64 mutations, 41 constructed in this study, strategically located over the protein. The purpose is to provide: (1) information on the magnitudes of changes in stabilization energy for mutations of residues that are important in maintaining the structure; and (2) probes for the folding pathway to be used in subsequent studies. The majority of mutations delete functional moieties of side-chains or make isosteric changes. The energetics of the interactions are variable and context-dependent. The following general conclusions may be drawn, however, from this study about the classes of interactions that stabilize the protein. (1) Truncation of buried hydrophobic side-chains has, in general, the greatest effect on stability. For fully buried residues, this averages at 1.5 kcal mol-1 per methylene group with a standard deviation of ±0.6 kcal mol-1. Truncation of partly exposed leucine, isoleucine or valine residues that are in the range of 50 to 80 Å2 of solvent-accessible area (30 to 50 % of the total solvent-accessible area on a Gly-X-Gly tripeptide, i.e. those packed against the surface) has a smaller, but relatively constant effect on stability, at 0.81 kcal mol-1 per methylene group with a statistical standard deviation of±0.18 kcal mol-1. (2) There is a very poor correlation between hydrophobic surface area buried and the free energy change for an extensive data set of hydrophobic mutants. The best correlation is found to be between the free energy change and the number of methylene groups within a 6 Å radius of the hydrophobic groups deleted. (3) Burial of the hydroxyl group of threonine in a pocket that is intended for a γ-methyl group of valine costs 2.5 kcal mol-1, in the range expected for the loss of two hydrogen bonds. In extension of previous studies, it is found that: (1) disruption of surface ion pairs or salt bridges lowers stability by only small amounts (0.3 to 1 kcal mol-1) but disruption of buried pairs, by larger amounts (>3 kcal mol-1); (2) removal of the hydrogen bonding partner of an uncharged group loses 0.5 to 2 kcal mol-1 (the energy of the hydrogen bond plus associated van der Waals' interactions); and (3) removal of the partner of a charged group has similar effects to the disruption of salt bridges. © 1992.