STABILIZING EFFECT OF A MULTIPLE SALT BRIDGE IN A PRENUCLEATED PEPTIDE

The free energy contribution of salt bridges to protein stability rema ins a controversial issue. The role of single salt bridges has been in vestigated in protein and peptide studies; there is no reported value of a multiple salt bridge employing peptide models. We have designed a de novo peptide model to evaluate the strength of a multiple salt bri dge. Measurement of the rate of hydrogen exchange (HX) of the backbone NH groups in ct-helical peptides allows determination of the absolute free energy of individual interactions that participate in helix stab ilization. We apply this procedure here to evaluate the stabilizing co ntribution of a multiple salt bridge involving Glu3, Asp4, and Arg7 in an 11-mer peptide. The peptide is prenucleated by a lactam bridge con necting two side chains spaced four residues apart. Measurement of the HX rates of each NH group in this peptide as a function of pH, togeth er with the pH independent rate of a Gln3 and Asn4 analogue, reveals a substantial favorable contribution to the a-helix from the salt bridg e: Delta G degrees = -1.2 +/- 0.2 kcal/mol. This is the first evaluati on of the strength of an engineered complex salt bridge in a peptide a nd yields a high value, comparable to results of mutation studies on p roteins.