ALANINE SCANNING MUTAGENESIS OF THE ALPHA-HELIX-115-123 OF PHAGE-T4 LYSOZYME - EFFECTS ON STRUCTURE, STABILITY AND THE BINDING OF SOLVENT

A series of individual alanine mutations has been constructed in the h elical region 115 to 123 in phage T4 lysozyme in order to evaluate the contribution to protein stability of the different side-chains within this region. Pairwise alanine mutations and a combination mutant with seven alanine substitutions were constructed to evaluate the additive effects upon structure and stability: Only three residues within this region (Ser117, Leu118 and Leu121) have a substantial influence upon stability (change in free energy of unfolding greater than 1.0 kcal/mo l). Replacement of Ser117 with alanine results in an increase in prote in stability of 1.27 kcal/mol, apparently due to the release of strain present in the wild-type protein. Replacement of the buried residues Leu118 and Leu121 is destabilizing. Substitution of the remaining six residues with alanine has relatively Little effect on stability This i s consistent with prior studies showing that only 20 to 30% ofthe resi dues in amphipathic helices in T4 lysozyme are critical for stability. For some of the pairwise alanine mutants the effects on stability are additive. For most of these mutants, however, there is a slight (simi lar to 0.15 to 0.25 kcal/mol) non-additivity such that the double muta nt is more stable than the sum of the constituent single mutants. This effect is consistently observed for residues with positions i, i + 4; i.e. adjacent, but in consecutive turns of the helix, suggesting a we ak but significant interaction between these amino acid residues. A mo re pronounced non-additivity (similar to 0.5 kcal/mol) is seen in the seven-alanine combination mutant. This non-additivity is due to a mode st ''collapse'' or ''repacking'' that occurs for the combination mutan ts (especially the multiple alanine mutant) but is not possible for th e single replacements. The truncation of some side-chains permits an i ncrease in solvent accessibility of main-chain amide and carbonyl grou ps. This effect is most pronounced for the seven-alanine combination m utant, where two solvent molecules, not present in wild-type, hydrogen bond to main-chain carbonyl groups in the middle region of the helix. It has been suggested that the binding of such water molecules might represent the first step in solvent-mediated unfolding of an alpha-hel ix. The appearance of ordered solvent, however, appears to have very l ittle effect on stability (less than or similar to 0.2 kcal/mol).