Eglin c is a small protease inhibitor whose structural and thermodynamic properties have been well studied. Previous thermodynamic measurements on mutants at solvent-accessible positions in the protein's helix have shown the unexpected result that the data could be best fit by the inclusion of residue- and position-specific parameters to the model. To explore the origins of this surprising result, long molecular dynamics simulations in explicit solvent have been performed. These simulations indicate specific long-range interactions between the solvent-exposed residues in the eglin c alpha-helix and binding loop, an unexpected observation for such a small protein. The residues involved in the interaction are on opposite sides of the protein, about 25 A apart. Simulations of alanine substitutions at the solvent-exposed helix positions, arginine 22, glutamic acid 23, threonine 26, and leucine 27, show both small and large perturbations of eglin c dynamics. Two mutations exhibit large impacts on the long-range helix-loop interactions. Previous stability measurements (Yi et al., Biochemistry 2003;42:7594-7603) had indicated that an alanine substitution at position 27 was less stabilizing than at other solvent-exposed positions in the helix. The L27A mutation effects observed in these simulations suggest that the position-dependent loss of stability measured in wet bench experiments is derived from changes in dynamics that involve long-range interactions; thus, these simulations support the hypothesis that solvent-exposed positions in helices are not always equivalent.
2005 Wiley-Liss, Inc.