Lessons from the lysozyme of phage T4

Abstract

An overview is presented of some of the major insights that have come from studies of the structure, stability, and folding of T4 phage lysozyme. A major purpose of this review is to provide the reader with a complete tabulation of all of the variants that have been characterized, including melting temperatures, crystallographic data, Protein Data Bank access codes, and references to the original literature. The greatest increase in melting temperature (T(m)) for any point mutant is 5.1 degrees C for the mutant Ser 117 --> Val. This is achieved in part not only by hydrophobic stabilization but also by eliminating an unusually short hydrogen bond of 2.48 A that apparently has an unfavorable van der Waals contact. Increases in T(m) of more than 3-4 degrees C for point mutants are rare, whereas several different types of destabilizing substitutions decrease T(m) by 20 degrees C or thereabouts. The energetic cost of cavity creation and its relation to the hydrophobic effect, derived from early studies of "large-to-small" mutants in the core of T4 lysozyme, has recently been strongly supported by related studies of the intrinsic membrane protein bacteriorhodopsin. The L99A cavity in the C-terminal domain of the protein, which readily binds benzene and many other ligands, has been the subject of extensive study. Crystallographic evidence, together with recent NMR analysis, suggest that these ligands are admitted by a conformational change involving Helix F and its neighbors. A total of 43 nonisomorphous crystal forms of different monomeric lysozyme mutants were obtained plus three more for synthetically-engineered dimers. Among the 43 space groups, P2(1)2(1)2(1) and P2(1) were observed most frequently, consistent with the prediction of Wukovitz and Yeates.

Figure 1

Backbone of T4 lysozyme showing its two-domain structure. The polypeptide chain is colored following the spectrum, from blue at the N-terminus to red at the opposite end.

Figure 2

Figure illustrating the tolerance of T4 lysozyme to point mutation (following Ref. 4). Substitutions at the green-colored sites have little if any effect on folding or activity. One or more substitutions at the red sites compromise folding and/or activity. These locations, typically in the core or the active site cleft, are the least tolerant sites. The yellow sites are tolerant, but less so than the green ones (see text for details).

Figure 3

Histograms summarizing how known mutations in T4 lysozyme (Table S1) affect stability. (a) Histogram showing the distribution of ΔT_m for 312 known point mutants. The most stabilizing and least stabilizing variants are discussed in the text. Roughly speaking, a change of 3°C in melting temperature corresponds to a change in the free energy of stabilization (ΔΔG) of about 1 kcal/mol. (b) Histogram summarizing the changes in melting temperature for all of the nonsingle site mutations in Table S1. This includes a total of 199 multiple mutants, insertion and deletion mutants and mutations that introduce disulfide bridges. Supporting Information Figures S1a and S1b are similar to Figure 3a and b but plot ΔΔG rather than ΔT_m.

Figure 4

Sketch illustrating two different ways in which a protein could respond to an insertion within the body of an α-helix. The site at which the insertion is to be made is indicated at the top of the figure. The presence of the insertion can result in looping out (left), which is rare for T4 lysozyme, or translocation (right), which is common.

Figure 5

In mutant A73-[AAA] the insertion of three alanines (colored in yellow) within the long helix (red) connecting the lower N-terminal domain and the upper C-terminal domain causes large-scale reorganization that is highly unusual for any mutant structure. In particular, the blue and green helices, which are at an angle of 105° in wild-type (left) reorganize to form a single, straight helix in the mutant (right).

Figure 6

Comparison of the energetic effects of core mutations in T4 lysozyme (blue) and bacteriorhodopsin (red). Dependence of protein stability on cavity volume is shown on the left, and on surface area on the right. From Joh et al. with permission.

Figure 7

The blue-colored surface shows the cavity in the core of T4 lysozyme resulting from the mutation of leucine 99 to alanine. For clarity, part of the cavity surface has been removed to show the location occupied by benzene, one of many ligands that can bind within this cavity.