Energetic cost and structural consequences of burying a hydroxyl group within the core of a protein determined from Ala .fwdarw. Ser and Val .fwdarw. Thr substitutions in T4 lysozyme

Abstract

In order to determine the thermodynamic cost of introducing a polar group within the core of a protein, a series of nine Ala-->Ser and 3 Val-->Thr substitutions was constructed in T4 lysozyme. The sites were all within alpha-helices but ranged from fully solvent-exposed to totally buried. The range of destabilization incurred by the Ala-->Ser substitutions was found to be very similar to that for the Val-->Thr replacements. For the solvent-exposed and partly exposed sites the destabilization was modest (approximately less than 0.5 kcal/mol). For the completely buried sites the destabilization was larger, but variable (approximately 1-3 kcal/mol). Crystal structure determinations showed that the Ala-->Ser mutant structures were, in general, very similar to their wild-type counterparts, even though the replacements introduce a hydroxyl group. This is in part because the introduced serines are all within alpha-helices and at congested sites can avoid steric clashes with surrounding atoms by making a hydrogen bond to a backbone carbonyl oxygen in the preceding turn of the helix. The three substituted threonine side chains essentially superimpose on their valine counterparts but display somewhat larger conformational adjustments. The results illustrate how a protein structure will adapt in different ways to avoid the presence of an unsatisfied hydrogen bond donor or acceptor. In the most extreme case, Val 149-->Thr, which is also the most destabilizing variant (delta delta G = 2.8 kcal/mol), a water molecule is incorporated in the mutant structure in order to provide a hydrogen-bonding partner. The results are consistent with the view that many hydrogen bonds within proteins contribute only marginally to stability but that noncharged polar groups that lack a hydrogen-bonding partner are very destabilizing (delta delta G approximately greater than 3 kcal/mol). Supportive of other studies, the alpha-helix propensity of alanine is seen to be higher than that of serine (delta delta G = 0.46 +/- 0.04 kcal/mol), while threonine and valine are similar in alpha-helix propensity.

Energetics of hydrogen bonding in proteins: A model compound study

“…These results indicate the stabilizing contribution that the amide-hydroxyl hydrogen bond can make to the enthalpy. Blaber et al (1993) conclude that the introduction of hydrogen bonds into T4 lysozyme does not increase the stability of the protein. However, the destabilization is generally entropic, as indicated by the positive values of AAH" in the table.…”

“…Blaber et al (1993) have studied the effects of introducing polar groups within T4 lysozyme in order to determine the energetics and structural consequences of burying a hydroxyl group. From their data and the AC, of the wildtype protein (10.5 kJ K" mol-' [Zhang et al, 1991]), we have calculated AH" of unfolding at the T, of the wild-type protein (51.55 "C) and the difference in the unfolding AH" (AAH" = AH&,,,,,, -AH$i/d.,,,pe) ( Table 7).…”

“…The majority of hydrogen bonds in globular proteins are amide-amide hydrogen bonds involved in regular, secondary structure (Baker & Hubbard, 1984). However, site-directed mutagenesis experiments aimed at understanding the contribution of hydrogen bonding to protein stability (Shirley et al, 1992;Blaber et al, 1993) can only investigate the contribution of hydrogen bonds involving side chains (see below). Although providing important data, mutagenesis studies are unable to address the contribution of backbone hydrogen bonding, which is the most relevant to understanding protein stability.…”

A High Performing Tool for Residue Solvent Accessibility Prediction

“…The following examples clearly show how powerful our tool might be: our prediction for the protein chain 119LA [35] in the NM215 dataset (with default parameters) reaches accuracy 93% and correlation 0.86, compared to accuracy and correlation results, respectively, of 77% and 0.58 for SABLE [13], 83% and 0.62 for RSA-PRP [20], 80% and 0.56 for SARPRED [14]. Even better, our prediction for the protein chain 1bmv 1 [36] in the RS126 dataset (with default parameters) reaches accuracy 97% and correlation 0.95, compared to accuracy and correlation results, respectively, of 74% and 0.52 for SABLE, 70% and 0.42 for RSA-PRP, 69% and 0.37 for SARPRED.…”

Using affinity chromatography to engineer and characterize pH‐dependent protein switches

“…Matthews and coworkers, for example, have shown that specifically engineered polar and charged residues in the core of T4 lysozyme dramatically deteriorate the protein's stability and function. 20,23 Even though the overall fold of the enzyme was maintained, the protein underwent a substantial loss of function as a result. Similarly, the engineering of small-to-large mutations into the core of a protein also resulted in severely destabilized protein structures.…”