ENERGETIC COST AND STRUCTURAL CONSEQUENCES OF BURYING A HYDROXYL GROUP WITHIN THE CORE OF A PROTEIN DETERMINED FROM ALA-]SER AND VAL-]THR SUBSTITUTIONS IN T4 LYSOZYME

In order to determine the thermodynamic cost of introducing a polar gr oup within the core of a protein, a series of nine Ala --> Ser and 3 V al --> Thr substitutions was constructed in T4 lysozyme. The sites wer e all within alpha-helices but ranged from fully solvent-exposed to to tally buried. The range of destabilization incurred by the Ala --> Ser substitutions was found to be very similar to that for the Val --> Th r replacements. For the solvent-exposed and partly exposed sites the d estabilization was modest (less than or similar to 0.5 kcal/mol). For the completely buried sites the destabilization was larger, but variab le (approximately 1-3 kcal/mol). Crystal structure determinations show ed that the Ala --> Ser mutant structures were, in general, very simil ar to their wild-type counterparts, even though the replacements intro duce a hydroxyl group. This is in part because the introduced serines are all within alpha-helices and at congested sites can avoid steric c lashes with surrounding atoms by making a hydrogen bond to a backbone carbonyl oxygen in the preceding turn of the helix. The three substitu ted threonine side chains essentially superimpose on their valine coun terparts but display somewhat larger conformational adjustments. The r esults illustrate how a protein structure will adapt in different ways to avoid the presence of an unsatisfied hydrogen bond donor or accept or. In the most extreme case, Val 149 --> Thr, which is also the most destabilizing variant (DELTADELTAG = 2.8 kcal/mol), a water molecule i s incorporated in the mutant structure in order to provide a hydrogen- bonding partner. The results are consistent with the view that many hy drogen bonds within proteins contribute only marginally to stability b ut that noncharged polar groups that lack a hydrogen-bonding partner a re very destabilizing (DELTADELTAG greater than or similar to 3 kcal/m ol). Supportive of other studies, the alpha-helix propensity of alanin e is seen to be higher than that of serine (DELTADELTAG = 0.46 +/- 0.0 4 kcal/mol), while threonine and valine are similar in alpha-helix pro pensity.