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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 A2 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 A radius of the hydrophobic groups deleted. (3)
Burial of the hydroxyl group of threonine in a pocket that is intended for a
gamma-methyl group of valine costs 2.5 kcal mol-1, in the range expected for the
loss of two hydrogen bonds.(ABSTRACT TRUNCATED AT 400 WORDS)
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