Structural and thermodynamic analysis of the binding of solvent at internal sites in T4 lysozyme

To investigate the structural and thermodynamic basis of the binding of solvent at internal sites within proteins a number of mutations were constructed in T4 lysozyme. Some of these were designed to introduce new solvent-binding sites. Others were intended to displace solvent from preexisting sites. In one case Val-149 was replaced with alanine, serine, cysteine, threonine, isoleucine, and glycine. Crystallographic analysis shows that, with the exception of isoleucine, each of these substitutions results in the binding of solvent at a polar site that is sterically blocked in the wild-type enzyme. Mutations designed to perturb or displace a solvent molecule present in the native enzyme included the replacement of Thr-152 with alanine, serine, cysteine, valine, and isoleucine. Although the solvent molecule was moved in some cases by up to 1.7 A, in no case was it completely removed from the folded protein. The results suggest that hydrogen bonds from the protein to bound solvent are energy neutral. The binding of solvent to internal sites within proteins also appears to be energy neutral except insofar as the bound solvent may prevent a loss of energy due to potential hydrogen bonding groups that would otherwise be unsatisfied. The introduction of a solvent-binding site appears to require not only a cavity to accommodate the water molecule but also the presence of polar groups to help satisfy its hydrogen-bonding potential. It may be easier to design a site to accommodate two or more water molecules rather than one as the solvent molecules can then hydrogen-bond to each other. For similar reasons it is often difficult to design a point mutation that will displace a single solvent molecule from the core of a protein.     

Contributions of all 20 amino acids at site 96 to the stability and structure of T4 lysozyme

To try to resolve the loss of stability in the temperature‐sensitive mutant of T4 lysozyme, Arg 96 → His, all of the remaining 18 naturally occurring amino acids were substituted at site 96. Also, in response to suggestions that the charged residues Lys85 and Asp89, which are 5–8 Å away, may have important effects, each of these amino acids was replaced with alanine. Crystal structures were determined for many of the variants. With the exception of the tryptophan and valine mutants R96W and R96V, the crystallographic analysis shows that the substituted side chain following the path of Arg96 in wildtype (WT). The melting temperatures of the variants decrease by up to ～16°C with WT being most stable. There are two site 96 replacements, with lysine or glutamine, that leave the stability close to that of WT. The only element that the side chains of these residues have in common with the WT arginine is the set of three carbon atoms at the C^α, C^β, and C^γ positions. Although each side chain is long and flexible with a polar group at the distal position, the details of the hydrogen bonding to the rest of the protein differ in each case. Also, the glutamine replacement lacks a positive charge. This shows that there is some adaptability in achieving full stabilization at this site. At the other extreme, to be maximally destabilizing a mutation at site 96 must not only eliminate favorable interactions but also introduce an unfavorable element such as steric strain or a hydrogen‐bonding group that remains unsatisfied. Overall, the study highlights the essential need for atomic resolution site‐specific structural information to understand and to predict the stability of mutant proteins. It can be very misleading to simply assume that conservative amino acid substitutions cause small changes in stability, whereas large stability changes are associated with nonconservative replacements.     

Exploring subdomain cooperativity in T4 lysozyme I: structural and energetic studies of a circular permutant and protein fragment

Small proteins are generally observed to fold in an apparent two-state manner. Recently, however, more sensitive techniques have demonstrated that even seemingly single-domain proteins are actually made up of smaller subdomains. T4 lysozyme is one such protein. We explored the relative autonomy of its two individual subdomains and their contribution to the overall stability of T4 lysozyme by examining a circular permutation (CP13*) that relocates the N-terminal A-helix, creating subdomains that are contiguous in sequence. By determining the high-resolution structure of CP13* and characterizing its energy landscape using native state hydrogen exchange (NSHX), we show that connectivity between the subdomains is an important determinant of the energetic cooperativity but not structural integrity of the protein. The circular permutation results in a protein more easily able to populate a partially unfolded form in which the C-terminal subdomain is folded and the N-terminal subdomain is unfolded. We also created a fragment model of this intermediate and demonstrate using X-ray crystallography that its structure is identical to the corresponding residues in the full-length protein with the exception of a small network of hydrophobic interactions. In sum, we conclude that the C-terminal subdomain dominates the energetics of T4 lysozyme folding, and the A-helix serves an important role in coupling the two subdomains.     

Repacking the Core of T4 lysozyme by automated design

Automated protein redesign, as implemented in the program ORBIT, was used to redesign the core of phage T4 lysozyme. A total of 26 buried or partially buried sites in the C-terminal domain were allowed to vary both their sequence and side-chain conformation while the backbone and non-selected side-chains remained fixed. A variant with seven substitutions ("Core-7") was identified as having the most favorable energy. The redesign experiment was repeated with a penalty for the presence of methionine residues. In this case the redesigned protein ("Core-10") had ten amino acid changes. The two designed proteins, as well as the constituent single mutants, and several single-site revertants were over-expressed in Escherichia coli, purified, and subjected to crystallographic and thermal analyses. The thermodynamic and structural data show that some repacking was achieved although neither redesigned protein was more stable than the wild-type protein. The use of the methionine penalty was shown to be effective. Several of the side-chain rotamers in the predicted structure of Core-10 differ from those observed. Rather than changing to new rotamers predicted by the design process, side-chains tend to maintain conformations similar to those seen in the native molecule. In contrast, parts of the backbone change by up to 2.8A relative to both the designed structure and wild-type.Water molecules that are present within the lysozyme molecule were removed during the design process. In the redesigned protein the resultant cavities were, to some degree, re-occupied by side-chain atoms. In the observed structure, however, water molecules were still bound at or near their original sites. This suggests that it may be preferable to leave such water molecules in place during the design procedure. The results emphasize the specificity of the packing that occurs within the core of a typical protein. While point substitutions within the core are tolerated they almost always result in a loss of stability. Likewise, combinations of substitutions may also be tolerated but usually destabilize the protein. Experience with T4 lysozyme suggests that a general core repacking methodology with retention or enhancement of stability may be difficult to achieve without provision for shifts in the backbone.     

Lessons from the lysozyme of phage T4

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°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 Å that apparently has an unfavorable van der Waals contact. Increases in T_m of more than 3–4°C for point mutants are rare, whereas several different types of destabilizing substitutions decrease T_m by 20°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, P2₁2₁2₁ and P2₁ were observed most frequently, consistent with the prediction of Wukovitz and Yeates.     

Alteration of T4 lysozyme structure by second-site reversion of deleterious mutations.

Mutations that suppress the defects introduced into T4 lysozyme by single amino acid substitutions were isolated and characterized. Among 53 primary sites surveyed, 8 yielded second-site revertants; a total of 18 different mutants were obtained. Most of the restorative mutations exerted global effects, generally increasing lysozyme function in a number of primary mutant contexts. Six of them were more specific, suppressing only certain specific deleterious primary substitutions, or diminishing the function of lysozymes bearing otherwise nondeleterious primary substitutions. Some variants of proteins bearing primary substitutions at the positions of Asp 20 and Ala 98 are inferred to have significantly altered structures.     

Neutron structure of the T26H mutant of T4 phage lysozyme provides insight into the catalytic activity of the mutant enzyme and how it differs from that of wild type

T4 phage lysozyme is an inverting glycoside hydrolase that degrades the murein of bacterial cell walls by cleaving the β‐1,4‐glycosidic bond. The substitution of the catalytic Thr26 residue to a histidine converts the wild type from an inverting to a retaining enzyme, which implies that the original general acid Glu11 can also act as an acid/base catalyst in the hydrolysis. Here, we have determined the neutron structure of the perdeuterated T26H mutant to clarify the protonation states of Glu11 and the substituted His26, which are key in the retaining reaction. The 2.09‐Å resolution structure shows that the imidazole group of His26 is in its singly protonated form in the active site, suggesting that the deprotonated N?2 atom of His26 can attack the anomeric carbon of bound substrate as a nucleophile. The carboxyl group of Glu11 is partially protonated and interacts with the unusual neutral state of the guanidine moiety of Arg145, as well as two heavy water molecules. Considering that one of the water‐binding sites has the potential to be occupied by a hydronium ion, the bulk solvent could be the source for the protonation of Glu11. The respective protonation states of Glu11 and His26 are consistent with the bond lengths determined by an unrestrained refinement of the high‐resolution X‐ray structure of T26H at 1.04‐Å resolution. The detail structural information, including the coordinates of the deuterium atoms in the active site, provides insight into the distinctively different catalytic activities of the mutant and wild type enzymes.     

Control of bacteriophage T4 tail lysozyme activity during the infection process

Bacteriophage T4 has an efficient mechanism for injecting the host Escherichiacoli cell with genomic DNA. Its gene product 5 (gp5) has a needle-like structure attached to the end of a tube through which the DNA passes on its way out of the head and into the host. The gp5 needle punctures the outer cell membrane and then digests the peptidoglycan cell wall in the periplasmic space. gp5 is normally post-translationally cleaved between residues 351 and 352. The function of this process in controlling the lysozyme activity of gp5 has now been investigated. When gp5 is over-expressed in E.coli, two mutants (S351H and S351A) showed a reduction of cleavage products and five other mutants (S351L, S351K, S351Y, S351Q, and S351T) showed no cleavage. Furthermore, in a complementation assay at 20 degrees C, the mutants that had no cleavage of gp5 produced a reduced number of plaques compared to wild-type T4. The crystal structure of the non-cleavage phenotype mutant of gp5, S351L, complexed with gene product 27, showed that the 18 residues in the vicinity of the potential cleavage site (disordered in the wild-type structure) had visible electron density. The polypeptide around the potential cleavage site is exposed, thus allowing access for an E.coli protease. The lysozyme activity is inhibited in the wild-type structure by a loop from the adjacent gp5 monomer that binds into the substrate-binding site. The same inhibition is apparent in the mutant structure, showing that the lysozyme is inhibited before gp5 is cleaved and, presumably, the lysozyme is activated only after gp5 has penetrated the outer membrane.     

Subdomain interactions as a determinant in the folding and stability of T4 lysozyme.

The folding of large, multidomain proteins involves the hierarchical assembly of individual domains. It remains unclear whether the stability and folding of small, single-domain proteins occurs through a comparable assembly of small, autonomous folding units. We have investigated the relationship between two subdomains of the protein T4 lysozyme. Thermodynamically, T4 lysozyme behaves as a cooperative unit and the unfolding transition fits a two-state model. The structure of the protein, however, resembles a dumbbell with two potential subdomains: an N-terminal subdomain (residues 13-75), and a C-terminal subdomain (residues 76-164 and 1-12). To investigate the effect of uncoupling these two subdomains within the context of the native protein, we created two circular permutations, both at the subdomain interface (residues 13 and 75). Both variants adopt an active wild-type T4 lysozyme fold. The protein starting with residue 13 is 3 kcal/mol less stable than wild type, whereas the protein beginning at residue 75 is 9 kcal/mol less stable, suggesting that the placement of the termini has a major effect on protein stability while minimally affecting the fold. When isolated as protein fragments, the C-terminal subdomain folds into a marginally stable helical structure, whereas the N-terminal subdomain is predominantly unfolded. ANS fluorescence studies indicate that, at low pH, the C-terminal subdomain adopts a loosely packed acid state. An acid state intermediate is also seen for all of the full-length variants. We propose that this acid state is comprised of an unfolded N-terminal subdomain and a loosely folded C-terminal subdomain.     

A systematic method for analysing the protein hydration structure of T4 lysozyme

A systematic method for the analysis of the hydration structure of proteins is demonstrated on the case study of lysozyme. The method utilises multiple structural data of the same protein deposited in the protein data bank. Clusters of high water occupancy are localised and characterised in terms of their interaction with protein. It is shown that they constitute a network of interconnected hydrogen bonds anchored to the protein molecule. The high occupancy of the clusters does not directly correlate with water–protein interaction energy as was originally hypothesised. The highly occupied clusters rather correspond to the nodes of the hydration network that have the maximum number of hydrogen bonds including both the protein atoms and the surrounding water clusters. Copyright © 2013 John Wiley & Sons, Ltd.     

Guanidinium derivatives bind preferentially and trigger long-distance conformational changes in an engineered T4 lysozyme

The binding of guanidinium ion has been shown to promote a large-scale translation of a tandemly duplicated helix in an engineered mutant of T4 lysozyme. The guanidinium ion acts as a surrogate for the guanidino group of an arginine side chain. Here we determine whether methyl- and ethylguanidinium provide better mimics. The results show that addition of the hydrophobic moieties to the ligand enhances the binding affinity concomitant with reduction in ligand solubility. Crystallographic analysis confirms that binding of the alternative ligands to the engineered site still drives the large-scale conformational change. Thermal analysis and NMR data show, in comparison to guanidinium, an increase in protein stability and in ligand affinity. This is presumably due to the successive increase in hydrophobicity in going from guanidinium to ethylguanidinium. A fluorescence-based optical method was developed to sense the ligand-triggered helix translation in solution. The results are a first step in the de novo design of a molecular switch that is not related to the normal function of the protein.     

Activity losses among T4 lysozyme charge variants after adsorption to colloidal silica.

Enzyme structure and function depend to some extent on enzyme net charge and charge location. Altering the charge of even a single residue may affect the interaction between enzyme and substrate such that all catalytic activity is lost. In this study we investigated the effect of net charge and charge location on the enzymatic activity of synthetic mutants of bacteriophage T4 lysozyme in the presence of colloidal silica. Enzymatic activity decreased upon adsorption, and these changes were variant-specific. Results were interpreted with reference to differences in adsorbed enzyme structure and orientation, and electrostatic effects. By exploring the effects of enzyme charge on adsorption, it may be possible to gain a better understanding of how enzyme structure influences adsorption and function at an interface.     

Context-dependent protein stabilization by methionine-to-leucine substitution shown in T4 lysozyme.

The substitution of methionines with leucines within the interior of a protein is expected to increase stability both because of a more favorable solvent transfer term as well as the reduced entropic cost of holding a leucine side chain in a defined position. Together, these two terms are expected to contribute about 1.4 kcal/mol to protein stability for each Met --> Leu substitution when fully buried. At the same time, this expected beneficial effect may be offset by steric factors due to differences in the shape of leucine and methionine. To investigate the interplay between these factors, all methionines in T4 lysozyme except at the amino-terminus were individually replaced with leucine. Of these mutants, M106L and M120L have stabilities 0.5 kcal/mol higher than wild-type T4 lysozyme, while M6L is significantly destabilized (-2.8 kcal/mol). M102L, described previously, is also destabilized (-0.9 kcal/mol). Based on this limited sample it appears that methionine-to-leucine substitutions can increase protein stability but only in a situation where the methionine side chain is fully or partially buried, yet allows the introduction of the leucine without concomitant steric interference. The variants, together with methionine-to-lysine substitutions at the same sites, follow the general pattern that substitutions at rigid, internal sites tend to be most destabilizing, whereas replacements at more solvent-exposed sites are better tolerated.     

Metal ions‐binding T4 lysozyme as an intramolecular protein purification tag compatible with X‐ray crystallography

Phage T4 lysozyme is a well folded and highly soluble protein that is widely used as an insertion tag to improve solubility and crystallization properties of poorly behaved recombinant proteins. It has been used in the fusion protein strategy to facilitate crystallization of various proteins including multiple G protein‐coupled receptors, lipid kinases, or sterol binding proteins. Here, we present a structural and biochemical characterization of its novel, metal ions‐binding mutant (mbT4L). We demonstrate that mbT4L can be used as a purification tag in the immobilized‐metal affinity chromatography and that, in many respects, it is superior to the conventional hexahistidine tag. In addition, structural characterization of mbT4L suggests that mbT4L can be used as a purification tag compatible with X‐ray crystallography.     

Evaluation at atomic resolution of the role of strain in destabilizing the temperature‐sensitive T4 lysozyme mutant Arg 96 → His

Mutant R96H is a classic temperature‐sensitive mutant of bacteriophage T4 lysozyme. It was in fact the first variant of the protein to be characterized structurally. Subsequently, it has been studied extensively by a variety of experimental and computational techniques, but the reasons for the loss of stability of the mutant protein remain controversial. In the crystallographic refinement of the mutant structure at 1.9 Å resolution one of the bond angles at the site of substitution appeared to be distorted by about 11^°, and it was suggested that this steric strain was one of the major factors in destabilizing the mutant. Different computationally‐derived models of the mutant structure, however, did not show such distortion. To determine the geometry at the site of mutation more reliably, we have extended the resolution of the data and refined the wildtype (WT) and mutant structures to be better than 1.1 Å resolution. The high‐resolution refinement of the structure of R96H does not support the bond angle distortion seen in the 1.9 Å structure determination. At the same time, it does confirm other manifestations of strain seen previously including an unusual rotameric state for His96 with distorted hydrogen bonding. The rotamer strain has been estimated as about 0.8 kcal/mol, which is about 25% of the overall reduction in stability of the mutant. Because of concern that contacts from a neighboring molecule in the crystal might influence the geometry at the site of mutation we also constructed and analyzed supplemental mutant structures in which this crystal contact was eliminated. High‐resolution refinement shows that the crystal contacts have essentially no effect on the conformation of Arg96 in WT or on His96 in the R96H mutant.     

Crystal structure of a mutant human lysozyme with a substituted disulfide bond

The three-dimensional structure of a mutant human lysozyme, W64CC65A, in which a non-native disulfide bond Cys64--Cys81 is substituted for the Cys65--Cys81 of the wild type protein by replacing Trp64 and Cys65 with Cys and Ala, respectively, was determined by X-ray crystallography and refined to an R-value of 0.181, using 33,187 reflections at 1.87-A resolution. The refined model of the W64CC65A protein consisted of four molecules, which were related by two noncrystallographic twofold axes and a translation vector. Although no specific structural differences could be observed among these four molecules, the overall B-factors of each molecule were quite different. The overall structure of W64CC65A, especially in the alpha-helical domain, was found to be quite similar to that of the wild type protein. Moreover, the side-chain conformation of the newly formed Cys64--Cys81 bond was quite similar to that of the Cys65--Cys81 bond of the wild-type protein. However, in the beta-sheet domain, the main-chain atoms of the loop region from positions 66-75 could not be determined, and significant structural changes due to the formation of the non-native disulfide bond could be observed. From these results, it is clear that the loop region of the mutant protein does not fold with the specific folding as observed in the wild-type protein.     

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

To further examine the structural and thermodynamic basis of hydrophobic stabilization in proteins, all of the bulky non-polar residues that are buried or largely buried within the core of T4 lysozyme were substituted with alanine. In 25 cases, including eight reported previously, it was possible to determine the crystal structures of the variants. The structures of four variants with double substitutions were also determined. In the majority of cases the "large-to-small" substitutions lead to internal cavities. In other cases declivities or channels open to the surface were formed. In some cases the structural changes were minimal (mainchain shifts < or = 0.3 A); in other cases mainchain atoms moved up to 2 A. In the case of Ile 29 --> Ala the structure collapsed to such a degree that the volume of the putative cavity was zero. Crystallographic analysis suggests that the occupancy of the engineered cavities by solvent is usually low. The mutants Val 149 --> Ala (V149A) and Met 6 --> Ala (M6A), however, are exceptions and have, respectively, one and two well-ordered water molecules within the cavity. The Val 149 --> Ala substitution allows the solvent molecule to hydrogen bond to polar atoms that are occluded in the wild-type molecule. Similarly, the replacement of Met 6 with alanine allows the two solvent molecules to hydrogen bond to each other and to polar atoms on the protein. Except for Val 149 --> Ala the loss of stability of all the cavity mutants can be rationalized as a combination of two terms. The first is a constant for a given class of substitution (e.g., -2.1 kcal/mol for all Leu --> Ala substitutions) and can be considered as the difference between the free energy of transfer of leucine and alanine from solvent to the core of the protein. The second term can be considered as the energy cost of forming the cavity and is consistent with a numerical value of 22 cal mol(-1) A(-3). Physically, this term is due to the loss of van der Waal''s interactions between the bulky sidechain that is removed and the atoms that form the wall of the cavity. The overall results are consistent with the prior rationalization of Leu --> Ala mutants in T4 lysozyme by Eriksson et al. (Eriksson et al., 1992, Science 255:178-183).     

Exploring subdomain cooperativity in T4 lysozyme II: uncovering the C-terminal subdomain as a hidden intermediate in the kinetic folding pathway

Intermediates along a protein's folding pathway can play an important role in its biology. Previous kinetics studies have revealed an early folding intermediate for T4 lysozyme, a small, well-characterized protein composed of an N-terminal and a C-terminal subdomain. Pulse-labeling hydrogen exchange studies suggest that residues from both subdomains contribute to the structure of this intermediate. On the other hand, equilibrium native state hydrogen experiments have revealed a high-energy, partially unfolded form of the protein that has an unstructured N-terminal subdomain and a structured C-terminal subdomain. To resolve this discrepancy between kinetics and equilibrium data, we performed detailed kinetics analyses of the folding and unfolding pathways of T4 lysozyme, as well as several point mutants and large-scale variants. The data support the argument for the presence of two distinct intermediates, one present on each side of the rate-limiting transition state barrier. The effects of circular permutation and site-specific mutations in the wild-type and circular permutant background, as well as a fragment containing just the C-terminal subdomain, support a model for the unfolding intermediate with an unfolded N-terminal and a folded C-terminal subdomain. Our results suggest that the partially unfolded form identified by native state hydrogen exchange resides on the folded side of the rate-limiting transition state and is, therefore, under most conditions, a "hidden" intermediate.     

Alanine-scanning mutagenesis of the beta-sheet region of phage T4 lysozyme suggests that tertiary context has a dominant effect on beta-sheet formation

In general, alpha-helical conformations in proteins depend in large part on the amino acid residues within the helix and their proximal interactions. For example, an alanine residue has a high propensity to adopt an alpha-helical conformation, whereas that of a glycine residue is low. The sequence preferences for beta-sheet formation are less obvious. To identify the factors that influence beta-sheet conformation, a series of scanning polyalanine mutations were made within the strands and associated turns of the beta-sheet region in T4 lysozyme. For each construct the stability of the folded protein was reduced substantially, consistent with removal of native packing interactions. However, the crystal structures showed that each of the mutants retained the beta-sheet conformation. These results suggest that the structure of the beta-sheet region of T4 lysozyme is maintained to a substantial extent by tertiary interactions with the surrounding parts of the protein. Such tertiary interactions may be important in determining the structures of beta-sheets in general.