Structural Factors Contributing to the Hydrophobic Effect: The Partly Exposed Hydrophobic Minicore in Chymotrypsin Inhibitor

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DOI

  • Daniel E. Otzen
  • Michael Rheinnecker, Cambridge University
  • ,
  • Alan R. Fersht, Cambridge University

The structural basis for the stability of a partly solvent-exposed hydrophobic minicore, formed by the residues Leu51, Val57, and Phe69 in the reactive loop of the serine protease inhibitor chymotrypsin inhibitor 2 (CI2), was analyzed from the stability of 17 mutant proteins, in which side chain methylene groups were deleted or rearranged. The mutations destabilize the protein by 0.3-4.8 kcal/mol, an average of 0.6 kcal per removed methylene group. Double mutant cycles show significant interaction between individual pairs of side chains. There is an excellent linear correlation (r = 0.995) between the free energy of unfolding relative to wild type (∆∆Gu-f) and the packing density nc (the number of methyl and methylene groups within 6 A of the removed atoms). ∆∆Gu-f correlates more weakly with changes in solvent-accessible surface area upon mutation. The correlation improves when the change in solventaccessible surface are upon mutation is separated into distinct contributions from polar atoms that are able to hydrogen bond to solvent (∆AHB) and from nonpolar atoms (∆AHP). There is also a correlation, however, between packing density and changes in surface area. Elsewhere in CI2, ∆∆Gu-f for mutations in the buried hydrophobic core correlates best with packing density, whereas in the exposed surface of the a-helix, the best correlation is with change in surface area. The results of hydrophobic changes at the surface of the protein are consistent with the classical model of the hydrophobic effect, whereas in the core, interactions are dominated by van der Waals' term, since van der Waals' interactions are additive; therefore, the higher the packing density, the stronger the van der Waals' interactions. The interdependence of the three structural parameters nC, ∆AHP, and ∆AHB in the minicore suggests that interactions in partly exposed parts of proteins are governed by a mixture of the classical hydrophobic effect and van der Waals' interactions.

Original languageEnglish
JournalBiochemistry
Volume34
Issue40
Pages (from-to)13051-13058
Number of pages8
ISSN0006-2960
DOIs
Publication statusPublished - 1 Jan 1995

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