Methionine and alanine substitutions show that the formation of wild-type-like structure in the carboxy-terminal domain of T4 lysozyme is a rate-limiting step in folding.

In an attempt to identify a systematic relation between the structure of a protein and its folding kinetics, the rate of folding was determined for 20 mutants of T4 lysozyme in which a bulky, buried, nonpolar wild-type residue (Leu, Ile, Phe, Val, or Met) was substituted with alanine. Methionine, which approximated the size of the original side chain but which is of different shape and flexibility, was also substituted at most of the same sites. Mutations that substantially destabilize the protein and are located in the carboxy-terminal domain generally slow the rate of folding. Destabilizing mutations in the amino-terminal domain, however, have little effect on the rate of folding. Mutations that have little effect on stability tend to have little effect on the rate, no matter where they are located. These results suggest that, at the rate-limiting step, elements of structure in the C-terminal domain are formed and have a structure similar to that of the fully folded protein. Consistent with this, two variants that somewhat increase the rate of folding (Phe104 --> Met and Val149 --> Met) are located within the carboxy-terminal domain and maintain or improve packing with very little perturbation of the wild-type structure.

Structural analysis of a non-contiguous second-site revertant in T4 lysozyme shows that increasing the rigidity of a protein can enhance its stability.

The mutation Glu108-->Val (E108V) in T4 lysozyme was previously isolated as a second-site revertant that specifically compensated for the loss of function associated with the destabilizing substitution Leu99-->Gly (L99G). Surprisingly, the two sites are 11 A apart, with Leu99 in the core and Glu108 on the surface of the protein. In order to better understand this result we have carried out a detailed thermodynamic, enzymatic and structural analysis of these mutant lysozymes as well as a related variant with the substitution Leu99-->Ala. It was found that E108V does increase the stability of L99G, but it also increases the stability of both the wild-type protein and L99A by essentially equal amounts. The effects of E108V on enzymatic activity are more complicated. The mutation slightly reduces the maximal rate of cell wall hydrolysis of wild-type, L99G and L99A. At the same time, L99G is an unstable protein and rapidly loses activity during the course of the assay, especially at temperatures above 20 degrees C. Thus, even though the double mutant L99G/E108V has a slightly lower maximal rate than L99G, over a period of 20-30 minutes it hydrolyzes more substrate. This decrease in the rate of thermal inactivation appears to be the basis of the action of E108V as a second-site revertant of L99G. Mutant L99A creates a cavity of volume 149 A(3). Instead of enlarging this cavity, mutant L99G results in a 4-5 A displacement of part of helix F (residues 108-113), creating a solvent-accessible declivity. In the double mutant, L99G/E108V, this helix returns to a position akin to wild-type, resulting in a cavity of volume 203 A(3). Whether the mutation Glu108-->Val is incorporated into either wild-type lysozyme, or L99A or L99G, it results in a decrease in crystallographic thermal factors, especially in the helices that include residues 99 and 108. This increase in rigidity, which appears to be due to a combination of increased hydrophobic stabilization plus a restriction of conformational fluctuation, provides a structural basis for the increase in thermostability.

Determination of alpha-helix propensity within the context of a folded protein. Sites 44 and 131 in bacteriophage T4 lysozyme.

To determine the effects of different amino acids on the structure and stability of an alpha-helix in the context of a globular protein, all 19 naturally-occurring amino acids were substituted for Ser44 in phage T4 lysozyme. A more restricted set of nine replacements was also made for Val131. Ser44 and Val131 are two of a very limited number of possible sites in T4 lysozyme that are well within alpha-helices, are solvent-exposed and relatively free of interactions with neighboring residues, and are not involved in crystal contacts. High resolution structures for the majority of the mutants, some of which crystallized non-isomorphously with wild-type, were determined. With the exception of proline, the amino acid substitutions caused little if any perturbation of the alpha-helix backbone. Also the beta-branched residues Thr, Val and Ile show no indication of either side-chain or backbone distortion. Therefore, other than proline, there is no evidence that differences in helix propensities are associated with different amounts of strain introduced into the helix. For reference, and also to allow estimates of side-chain entropy, a survey was made of side-chain conformations in 100 well-refined protein structures. As noted previously all side-chains within alpha-helices strongly avoid the g- conformation (chi 1 approximately 60 degrees). This restricts the beta-branched residues Thr, Val and Ile to a single conformer (g+, chi 1 approximately -60 degrees). Asp, Asn, Met and Ser within helices also overwhelmingly prefer the g+ conformation. For Arg, Cys, Gln, Glu, Leu and Lys the t (chi 1 approximately 180 degrees) and g+ conformers are populated roughly equally. Only the aromatic residues, His, Tyr, Trp and Phe prefer the t conformation. These preferences are the same whether the side-chain is buried or solvent-exposed. In general, the side-chain conformations adopted by the residues substituted at positions 44 and 131 correspond to the most commonly observed conformation for the same amino acid in helices in known protein structures. The changes in protein stability for the replacements at site 131 in general agree well with those at site 44 (correlation r = 0.97), suggesting that these may be representative of substitutions at fully solvent-exposed sites in the middle of alpha-helices. The free energy values also agree quite well with those observed for equivalent replacements in a number of soluble alpha-helical model peptides and with data from "host-guest" studies and statistical surveys (r = 0.69 to 0.93).(ABSTRACT TRUNCATED AT 400 WORDS)

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 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.