Hydrophobic packing in T4 lysozyme probed by cavity-filling mutants.

Abstract

To probe the nature of the hydrophobic cores of proteins and to test potential ways of increasing protein thermostability, an attempt was made to improve the packing within T4 bacteriophage lysozyme by engineered amino acid replacements. Two mutations, Vat, which were designed to fil the largest cavities that exist in the folded structure of the native protein, were constructed. The mutant proteins have normal activities and their thermal stabilities are marginally lower than that ofwild-type lysozyme. Crystal structure analysis of the mutant proteins shows that the introduced amino acids are accommodated with very little perturbation of the three-dimensional structure. Incorporation of the more bulky hydrophobic residues within the core of the protein is expected to provide an increase in hydrophobic stabilization, but this is seen to be offset by the introduction of strain. Inspection of the mutant structures shows that in each case the introduced amino acid side chain is forced to adopt a non-optimal dihedral angle yX. Strain is also observed in the form ofbond angle distortion and in unfavorable van der Waals contacts. The results illustrate how the observed core structures of proteins represent a compromise between the hydrophobic effect, which will tend to maximize the core packing density, and the strain energy that would be incurred in eliminating all packing defects. The results also suggest that mutations designed to increase protein stability by filling existing cavities may be effective in some cases but are unlikely to provide a general method for increasing protein stability.It has been anticipated for some time that the folding of proteins is probably dominated by the hydrophobic effect, i.e., by the sequestering of nonpolar side chains away from solvent. Recent studies suggest that alterations in many solvent-exposed residues on the surfaces of proteins have little if any effect on folding and stability (1-4). These results lead one to consider protein folding from a somewhat different perspective. Is it possible that folding is determined not by the whole amino acid sequence but by a small subset of key residues? If so, which are the critical residues and how do they determine three-dimensional structure? In an attempt to address questions such as these, increasing attention, both theoretical (5) and experimental (6)(7)(8), is being directed to the hydrophobic cores of proteins. Such cores have densities comparable to crystals of simple organic molecules (9, 10) but the packing is not perfect and some cavities remain (11). What determines the size of these cavities?In the present study we have used site-directed mutagenesis to probe the nature of the core of T4 bacteriophage lysozyme. Specifically, we have asked whether the hydrophobic packing within the molecule can be improved by appropriate amino acid replacements. The hydrophobic surface area that is buried within a folded protein contributes directly to its free energy of stabilization (12, 13). Therefore, amino acid substitutions t...

Design and structural analysis of an engineered thermostable chicken lysozyme

“…(See Richards and Lim [ 19931 and Matthews [1995] for recent reviews on hydrophobicitylpacking.) Attempts to achieve higher stability by increasing packing density d o not always succeed (e.g., Karpusas et al, 1989). However, the success of this approach is likely to be enhanced if a natural substitution found in a homologous protein is imported, as in the case of position 3 1 in chicken and human lysozymes.…”

“…These include: (1) introducing disulfide bridges (Matsumura et al, 1989b); (2) increasing internal hydrophobic packing (Malcolm et al, 1990); (3) reducing solvent-exposed nonpolar surface area (Pakula & Sauer, 1990); (4) incorporating a metal association site (Kuroki et al, 1989); and (5) adding charged side chains to interact with the a-helix dipoles (Nicholson et al, 1988). However, the above strategies are not always successful and their pursuit has often yielded proteins that are less stable than the WT progenitors for reasons that are not fully understood (e.g., Karpusas et al, 1989;Matsumura et al, 1989a;Eijsink et al, 1992;Leontiev et al, 1993 Abbreviations: hs, a hyperstable variant of chicken lysozyme; GdnHCI, guanidine hydrochloride; T,, the midpoint temperature of the thermal denaturation transition; C,, the midpoint concentration of the GdnHCl denaturation profile; DSC, differential scanning calorimetry; AH,,,, calorimetrically determined enthalpy of denaturation; AC,, the difference between the heat capacities of the native and the denatured states; WT or wt, wild type. …”

The response of T4 lysozyme to large‐to‐small substitutions within the core and its relation to the hydrophobic effect

“…This suggests that some other effect, such as the relaxation of strain, may play a significant role. Strain energy in a given sidechain is expected to range up to about 0.7 kcal/mol in some cases (Karpusas et al, 1989). Also a case was reported in which lysozyme stability was increased by .27 kcal/mol due to the apparent removal of strain from the wild-type protein (Blaber et al, 1995).…”

Stabilization of GroEL minichaperones by core and surface mutations

“…X-ray data collection and re®nement statistics c R merge gives the agreement between intensities of repeated measurements of the same re¯ections and is de®ned as AE(I h,I À hI h i)/AEI h,I , where I h,I are individual values and hI h i is the mean value of the intensity of re¯ection h. more hydrophobic substitution Ala223 3 Val was not anticipated, and most likely results from the movements of the main-chain and side-chain of Thr223 that bring the OG1 atom of Thr223 closer to its hydrogen bonding partners (Figure 1(a)). It is surprising that the hydrophobic core of this protein accommodates a larger side-chain without strain or steric clash, in contrast to many destabilizations caused by small-to-large mutations in protein cores (Karpusas et al, 1989;Daopin et al, 1991). The side-chain of Thr223 in M1 adopts a conformation distinct to that of Val223 in M2 (Figure 1(b)), indicating local¯exibility.…”