CONTRIBUTIONS OF A HYDROGEN-BOND SALT BRIDGE NETWORK TO THE STABILITYOF SECONDARY AND TERTIARY STRUCTURE IN LAMBDA-REPRESSOR

In the N-terminal domain of lambda repressor, the Asp 14 side chain fo rms an intrahelical, hydrogen bond/salt bridge with the Arg 17 side ch ain and a tertiary hydrogen bond with the Ser 77 side chain. By measur ing the stabilities to urea denaturation of the wild-type N-terminal d omain and variants containing single, double, and triple alanine subst itutions at positions 14, 17, and 77, the side-chain interaction energ ies, the coupling energy between interactions, and the intrinsic effec ts of each wild-type side chain on protein stability have been estimat ed. These studies indicate that the Asp 14-Arg 17 and Asp 14-Ser 77 in teractions are stabilizing by roughly 0.8 and 1.5 kcal/mol, respective ly, but that Asp 14, by itself, is destabilizing by roughly 0.9 kcal/m ol. We also show that a peptide model of alpha-helix 1, which contains Asp 14 and Arg 17, forms a reasonably stable, monomeric helix in solu tion and responds to alanine mutations at positions 14 and 17 in the f ashion expected from the intact protein studies. These studies suggest that it is possible to view the stability effects of mutations in int act proteins in a hierarchical fashion, with the stability of units of secondary structure being distinguishable from the stability of terti ary structure.