QUANTITATION OF THE EFFECTS OF AN INTERNAL PROLINE RESIDUE ON INDIVIDUAL HYDROGEN-BOND STABILITIES IN AN ALPHA-HELIX - PH-DEPENDENT AMIDE EXCHANGE IN MELITTIN AND [ALA-14]MELITTIN

pH-Dependent amide exchange rates in methanol have been measured for 21 of the 25 exchangeable backbone amides of [Ala-141 melittin (P14A), a synthetic analogue of bee venom melittin having a Pro to Ala substitution at residue 14. P14A, like melittin, adopts an alpha-helical conformation in methanol. As previously found for melittin [Dempsey, C. E. (1988) Biochemistry 27, 6893], the exchange data could be fit to curves calculated assuming acid and base catalysis by solvent from which residue-specific values of k(H) and k(OMe), the acid- and base-catalyzed exchange rate constants, respectively, were determined. From a comparison of k(min) values, where k(min) is the minimum exchange rate in the curve defining the amide exchange rate as a function of pH, with those previously obtained from melittin, the relative stabilities of individual helical hydrogen bonds in the two peptides were calculated in terms of equilibrium constants for hydrogen-bond-breaking backbone fluctuations. Replacement of P14 in melittin with Ala results in stabilization of amides in a central turn of helix by up to 36-fold (delta-DELTA-G(Pro-Ala) = 9 kJ mol-1) and elsewhere throughout the helix (residues 5-2 1) by 2-1 0- fold. These data indicate a cooperative effect of the Pro to Ala substitution on helix stability and allow a qualitative description of the fluctuational properties of the melittin and P14A helices in methanol. The effects of proline on the properties of the melittin helix are compared with previous theoretical and empirical studies on the effect of proline on the structure and stability of alpha-helices.