Protein Science, Vol 1, Issue 5 641-653, Copyright © 1992 by Cold Spring Harbor Laboratory Press

ARTICLE

Thermodynamics of melittin tetramerization determined by circular dichroism and implications for protein folding

W. WILCOX and D. EISENBERG
Present address: Medical Genetics Division, Department of Pediatrics, Cedars-Sinai Medical Center, Los Angeles, California.

The tetramerization of melittin, a 26-amino acid peptide from Apis mellifera bee venom, has been studied as a model for protein folding. Melittin converts from a monomeric random coil to an {alpha}-helical tetramer as the pH is raised from 4.0 to 9.5, as ionic strength is increased, as temperature is raised or lowered from about 37{deg}C, or as phosphate is added. The thermodynamics of this tetramerization (termed ``folding'') are explored using circular dichroism. The melittin tetramer has two pK(a) values of 7.5 and 8.5 corresponding to protonation of the N-terminus and Lys 23, respectively. pK(a) values calculated with the program DelPhi (Gilson, M.K., Sharp, K.A., & Honig, B.H., 1987, J. Comp. Chem. 9, 327-335; Gilson, M.K. & Honig, B.H., 1988a, Proteins 3, 32-52; Gilson, M.K. & Honig, B.H., 1988b, Proteins 4, 7-18) agree with experimental titration data. Greater electrostatic repulsion of these protonated groups destabilizes the tetramer by 3.6 kcal/mol at pH 4.0 compared to pH 9.5. Increasing the concentration of NaCl in the solution from 0 to 0.5 M stabilizes the tetramer by 5-6 kcal/mol at pH 4.0. The effect of NaCl is modeled with a ligand-binding approach. The melittin tetramer is found to have a temperature of maximum stability ranging from 35.5 to 43{deg}C depending on the pH, unfolding above and below that temperature. {delta}C(0)(p) for folding ranges from -0.085 to -0.102 cal g(-1) K(-1), comparable to that of other small globular proteins (Privalov, P.L., 1979, Adv. Protein Chem. 33, 167-241). {delta}H(0) and {delta}S(0) are found to decrease with temperature, presumably due to the hydrophobic effect (Kauzmann, W., 1959, Adv. Protein Chem. 14, 1-63). Phosphate is found to perturb the equilibrium substantially with a maximal effect at 150 mM, stabilizing the tetramer at pH 7.4 and 25{deg}C by 4.6 kcal/mol. The enthalpy change due to addition of phosphate (-7.5 kcal/mol at 25{deg}C) can be accounted for by simple dielectric screening. Both circular dichroism and crystallographic results suggest that phosphate may bind Lys 23 at the ends of the elongated tetramer. These detailed measurements give insight into the relative importance of various forces for the stability of melittin in the folded form and may provide an experimental standard for future tests of computational energetics on this simple protein system.
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