Bacteriophage T4 Lysozyme Research Articles

Perturbation of tryptophan residues by point mutations in bacteriophage T4 lysozyme studied by optical detection of triplet-state magnetic resonance spectroscopy.

We have investigated perturbations of the triplet-state properties of Trp residues in bacteriophage T4 lysozyme caused by point mutations using low-temperature phosphorescence and optical detection of triplet-state magnetic resonance (ODMR) spectroscopy. Five temperature-sensitive mutants have been studied in detail. These include lysozymes with the point mutations Gln-105----Ala, Gln-105----Gly, Gln-105----Glu, Ala-146----Thr, and Trp-126----Gln. Changes in phosphorescence 0,0 band wavelength, intensity, the triplet-state zero-field splitting (ZFS), and the wavelength dependence of the ZFS were detected only from Trp-138 in each mutant. In the case of the Q105A mutation, the perturbations on Trp-138 have been ascribed to the combination of an increase in the polarizability of the environment and to the loss of hydrogen bonding of the enamine nitrogen of indole. For the Q105G mutation, we believe that Q is replaced by a solvent molecule in H bonding, leading to relatively small changes. In the Q105E mutation, the perturbation results largely from the introduction of a charged residue. In the case of the mutation A146T, the perturbation is associated with a local conformational change in which Trp-138 is shifted to a more solvent-exposed location. On the other hand, no significant spectroscopic changes in Trp-126 and Trp-158 were found in any of the mutants, suggesting that the perturbations are probably localized near Trp-138 for the mutations of positions 105 and 146. However, in the mutation W126Q, which occurs approximately 16 A away from Trp-138, significant changes of Trp-138 are detected, suggesting that the effects of this mutation are propagated over large distances.

Control of enzyme activity by an engineered disulfide bond.

A novel approach to the control of enzyme catalysis is presented in which a disulfide bond engineered into the active-site cleft of bacteriophage T4 lysozyme is capable of switching the activity on and off. Two cysteines (Thr21----Cys and Thr142----Cys) were introduced by oligonucleotide-directed mutagenesis into the active-site cleft. These cysteines spontaneously formed a disulfide bond under oxidative conditions in vitro, and the catalytic activity of the oxidized (cross-linked) T4 lysozyme was completely lost. On exposure to reducing agent, however, the disulfide bond was rapidly broken, and the reduced (non-cross-linked) lysozyme was restored to full activity. Thus an enzyme has been engineered such that redox potential can be used to control catalytic activity.

Low-temperature unfolding of a mutant of phage T4 lysozyme. 2. Kinetic investigations.

A disulfide-bridged variant of bacteriophage T4 lysozyme has been found to undergo a low- as well as high-temperature unfolding transition in guanidinium chloride [see Chen and Schellman (1989)]. The kinetics for this process have been followed for several temperatures, a range of guanidinium chloride concentrations, and a number of values of pH. Microscopic rate constants for protein unfolding and refolding were extracted from these data to explore the nature of the cold unfolding transition. The data were interpreted using transition-state theory. It was found that the Arrhenius energy is temperature dependent. The transition state is characterized by (1) a high energy and low entropy compared to the native state, (2) a heat capacity which is closer to the native state than to the unfolded state, and (3) a low exposure to solvent compared to the unfolded state, as judged by its interaction with guanidinium chloride. With increasing concentration of guanidinium chloride, the low-temperature unfolding rate increases strongly, and the refolding rate decreases very strongly.

Low-temperature unfolding of a mutant of phage T4 lysozyme. 1. Equilibrium studies.

The mutant protein I3C-C97/C54T of phage T4 lysozyme is free of sulfhydryl groups and has a genetically engineered disulfide bridge between positions 3 and 97 (Perry & Wetzel, 1986). This protein has a maximum stability at 12 degrees C in 3 M guanidinium chloride and undergoes reversible high- and low-temperature melting at 28 and -3 degrees C, respectively, in this medium. The free energy of stabilization of the protein has been studied over a range of temperature that includes both melting transitions. The stability curve fits a constant delta Cp model over the entire range, permitting an unusually complete determination of the thermodynamic parameters of the protein and demonstrating that the low-temperature unfolded form of the protein may be interpreted as an extrapolation with constant delta Cp of the high-temperature unfolded form. The free energy of unfolding is a linear function of guanidinium concentration within experimental error which permits a rough estimate of the stability of the protein at low temperatures and of the differential interaction of the unfolded protein with guanidinium chloride. These equilibrium studies provide a basis for the interpretation of the kinetic studies reported in the following paper.

蛋白質工学 遺伝子工学とＸ線結晶学の結合 Ｔ４ファージリゾチームにおける研究から

蛋白質工学 遺伝子工学とＸ線結晶学の結合 Ｔ４ファージリゾチームにおける研究から

Comparative triplet-state properties of the three tryptophan residues in bacteriophage T4 lysozyme and in the enzyme complex with methylmercury(II).

Triplet-state energies, zero-field splittings (ZFS), and total decay rate constants of the individual triplet-state sublevels of the tryptophan (Trp) residues located at positions 126, 138, and 158 in bacteriophage T4 lysozyme have been determined by using low-temperature phosphorescence and optical detection of magnetic resonance spectroscopy in zero applied magnetic field. An investigation of spectral and kinetic properties of individual Trp residues was facilitated by measurements on point-mutated proteins containing two Trp----Tyr substitutions. We find that the phosphorescence lifetime of the buried Trp-138 is considerably shorter than those of the solvent-exposed Trp residues. CH3HgII binding to cysteine residues in T4 lysozyme selectively perturbs the triplet state of Trp-158 by means of an external heavy-atom effect. In contrast with the previous observation of selective x-sublevel perturbation in the Trp-CH3Hg complex, the radiative character of the z sublevel (z is the out-of-plane axis) is selectively enhanced due to the heavy-atom perturbation of Trp-158. The observed pattern of radiative and total sublevel decay constants of the perturbed Trp is attributed to a special orientation of the Hg atom with respect to the indole plane.

Optically detected magnetic resonance study of tyrosine residues in point-mutated bacteriophage T4 lysozyme.

Two spectroscopically distinct types of tyrosine (Tyr) residues in triply point mutated bacteriophage T4 lysozyme, which contains no tryptophan (Trp), have been detected by optical detection of triplet-state magnetic resonance (ODMR) spectroscopy. Their triplet states are characterized by similar E but different D values. The Tyr site which exhibits the lower D value and has the red-shifted phosphorescence origin is quenched by energy transfer to Trp and has D and E values comparable to previously studied Tyr residues. The blue-shifted Tyr site, which is not quenched by Trp, exhibits a larger D value that has been found previously. Calculation of energy-transfer efficiencies of Tyr-Trp pairs based on the crystal structure of the native enzyme provides a possible assignment of Tyr sites to the two different spectral types.

Hydrophobic stabilization in T4 lysozyme determined directly by multiple substitutions of Ile 3.

Replacing the isoleucine at amino-acid position three of bacteriophage T4 lysozyme causes changes in the thermodynamic stability of the protein that are directly related to the hydrophobicity of the substituted residue. Structural analysis confirms that the hydrophobic stabilization is proportional to the reduction of the surface area accessible to solvent on folding.

Structural analysis of the temperature-sensitive mutant of bacteriophage T4 lysozyme, glycine 156----aspartic acid.

The structure of the mutant of bacteriophage T4 lysozyme in which Gly-156 is replaced by aspartic acid is described. The lysozyme was isolated by screening for temperature-sensitive mutants and has a melting temperature at pH 6.5 that is 6.1 degrees C lower than wild type. The mutant structure is destabilized, in part, because Gly-156 has conformational angles (phi, psi) that are not optimal for a residue with a beta-carbon. High resolution crystallographic refinement of the mutant structure (R = 17.7% at 1.7 A resolution) shows that the Gly----Asp substitution does not significantly alter the configurational angles (phi, psi) but forces the backbone to move, as a whole, approximately 0.6 A away from its position in wild-type lysozyme. This induced strain weakens a hydrogen bond network that exists in the wild-type structure and also contributes to the reduced stability of the mutant lysozyme. The introduction of an acidic side chain reduces the overall charge on the molecule and thereby tends to increase the stability of the mutant structure relative to wild type. However, at neutral pH this generalized electrostatic stabilization is offset by specific electrostatic repulsion between Asp-156 and Asp-92. The activity of the mutant lysozyme is approximately 50% that of wild-type lysozyme. This reduction in activity might be due to introduction of a negative charge and/or perturbation of the surface of the molecule in the region that is assumed to interact with peptidoglycan substrates.

Relative efficiency of long range nonradiative energy transfer among tryptophan residues in bacteriophage T4 lysozyme

The three tryptophan (trp) residues located at positions 126, 138, and 158 in wild-type bacteriophage T4 lysozyme have been investigated by low temperature phosphorescence and ODMR spectroscopy. Assignment of spectral properties to individual trp residues was facilitated by measurements on mutant proteins containing one or two trp→tyr (tyrosine) substitutions. It has been observed that (i) the triplet state energies (ET) of the trp residues lie in the order: ET(126)>ET(158)>ET(138) and (ii) S→S nonradiative energy transfer takes place predominantly from trp 126 to trp 158. Calculated orientation factors in Förster’s equation using crystal structure data support this result, suggesting a specific direction for the S1←S0 transition moment of trp.

Replacements of Pro86 in phage T4 lysozyme extend an alpha-helix but do not alter protein stability.

To investigate the relation between protein stability and the predicted stabilities of individual secondary structural elements, residue Pro86 in an alpha-helix in phage T4 lysozyme was replaced by ten different amino acids. The x-ray crystal structures of seven of the mutant lysozymes were determined at high resolution. In each case, replacement of the proline resulted in the formation of an extended alpha-helix. This involves a large conformational change in residues 81 to 83 and smaller shifts that extend 20 angstroms across the protein surface. Unexpectedly, all ten amino acid substitutions marginally reduce protein thermostability. This insensitivity of stability to the amino acid at position 86 is not simply explained by statistical and thermodynamic criteria for helical propensity. The observed conformational changes illustrate a general mechanism by which proteins can tolerate mutations.

Contributions of hydrogen bonds of Thr 157 to the thermodynamic stability of phage T4 lysozyme.

Measurements of changes in structure and stability caused by 13 different substitutions for threonine 157 in phage T4 lysozyme show that the most stable lysozyme variants contain hydrogen bonds analogous to those in the wild-type enzyme and that structural adjustments allow the protein to be surprisingly tolerant of amino-acid substitutions.

Enhanced protein thermostability from site-directed mutations that decrease the entropy of unfolding.

It is proposed that the stability of a protein can be increased by selected amino acid substitutions that decrease the configurational entropy of unfolding. Two such substitutions, one of the form Xaa----Pro and the other of the form Gly----Xaa, were constructed in bacteriophage T4 lysozyme at sites consistent with the known three-dimensional structure. Both substitutions stabilize the protein toward reversible and irreversible thermal denaturation at physiological pH. The substitutions have no effect on enzymatic activity. High-resolution crystallographic analysis of the proline-containing mutant protein (Ala-82----Pro) shows that its three-dimensional structure is essentially identical with the wild-type enzyme. The overall structure of the other mutant enzyme (Gly-77----Ala) is also very similar to wild-type lysozyme, although there are localized conformational adjustments in the vicinity of the altered amino acid. The combination of a number of such amino acid replacements, each of which is expected to contribute approximately 1 kcal/mol (1 cal = 4.184 J) to the free energy of folding, may provide a general strategy for substantial improvement in the stability of a protein.

Mutations in an upstream regulatory sequence that increase expression of the bacteriophage T4 lysozyme gene.

A P22 hybrid phage bearing the bacteriophage T4 lysozyme gene (e), as well as T4 sequences upstream from the lysozyme gene, was constructed. Amber mutations were introduced into gene e in the hybrid phage, and the resulting mutant phages were tested for the ability to form plaques on amber suppressor strains. Revertant phages that were able to form plaques on amber suppressors that did not suppress the parent amber mutant phages were isolated following UV mutagenesis. Secondary site pseudorevertants were identified among the revertants by a genetic test. Four of the suppressing secondary site mutations were mapped and sequenced. They were found to consist of small sequence alterations immediately upstream from gene e, all of which would tend to destabilize potential base-pairing interactions in the transcript. The mutations were shown to increase lysozyme expression when introduced into an otherwise wild-type hybrid phage, but were found to have little effect on transcription of the lysozyme gene.

Structural studies of mutants of the lysozyme of bacteriophage T4: The temperature-sensitive mutant protein Thr157 → Ile

To understand the roles of individual amino acids in the folding and stability of globular proteins, a systematic structural analysis of mutants of the lysozyme of bacteriophage T4 has been undertaken. The isolation, characterization, crystallographic refinement and structural analysis of a temperature-sensitive lysozyme in which threonine 157 is replaced by isoleucine is reported here. This mutation reduces the temperature of the midpoint of the reversible thermal denaturation transition by 11 deg.C at pH 2.0. Electron density maps showing differences between the wild-type and mutant X-ray crystal structures have obvious features corresponding to the substitution of threonine 157 by isoleucine. There is little difference electron density in the remainder of the molecule, indicating that the structural changes are localized to the site of the mutation. Highresolution crystallographic refinement of the mutant lysozyme structure confirms that it is very similar to wild-type lysozyme. The largest conformational differences are in the γ-carbon of residue 157 and in the side-chain of Asp159, which shift 1.0 Å and 1.1 Å, respectively. In the wild-type enzyme, the γ-hydroxyl group of Thr157 participates in a network of hydrogen bonds. Substitution of Thr157 with an isoleucine disrupts this set of hydrogen bonds. A water molecule bound in the vicinity of Thr155 partially restores the hydrogen bond network in the mutant structure, but the buried main-chain amide of Asp159 is not near a hydrogen bond acceptor. This unsatisfied hydrogen-bonding potential is the most obvious reason for the reduction in stability of the temperature-sensitive mutant protein.

Correlations between structure and thermodynamic stability of phage T4 lysozyme

Correlations between structure and thermodynamic stability of phage T4 lysozyme

An efficient general-purpose least-squares refinement program for macromolecular structures

A package of programs has been developed for efficient restrained least-squares refinement of macromolecular crystal structures. The package has been designed to be as flexible and general purpose as possible. The process of refinement is divided into basic units and an independent computer program handles each task. Each functional unit communicates with other programs in the package by way of files of well defined format. To modify or replace any program, the user need only understand the function of that particular element. Stereochemical restraints are defined in a general way that can be applied to proteins, nucleic acids, prosthetic groups, solvent atoms and so on. Guide values for bond lengths and bond angles are specified in a straightforward direct manner. Designated groups of atoms can be held constant or constrained to behave as a rigid body during refinement. In order to make the package as efficient as possible, the fast Fourier transform algorithm is used for all the crystallographic transformations. To highlight potential errors in the refined structure the user can list those atoms that have the worst bond lengths and angles, or have the largest positional, temperature-factor or occupancy gradients. It is also possible to check that protein and solvent atoms do not sterically clash with symmetry-related neighbors. Applications of the program package to a bacteriochlorophyll-containing protein, thermolysin-inhibitor complexes and mutants of bacteriophage T4 lysozyme are described.

Temperature-sensitive mutations of bacteriophage T4 lysozyme occur at sites with low mobility and low solvent accessibility in the folded protein.

Twenty-five different temperature-sensitive point mutations at 20 sites in the lysozyme gene of bacteriophage T4 have been identified. All of the mutations alter amino acid side chains that have lower than average crystallographic thermal factors and reduced solvent accessibility in the folded protein. This suggests that the amino acids with well-defined conformations can form specific intramolecular interactions that make relatively large contributions to the thermal stability of the protein. Residues with high mobility or high solvent accessibility are much less susceptible to destabilizing substitutions, suggesting that, in general, such amino acids contribute less to protein stability. The pattern of the sites of ts substitutions observed in the folded conformation of T4 lysozyme suggests that severe destabilizing mutations that primarily affect the free energy of the unfolded state are rare. These results indicate that proteins can be stabilized by adding new interactions to regions that are rigid or buried in the folded conformation.

Proton NMR measurements of bacteriophage T4 lysozyme aided by 15N isotopic labeling: structural and dynamic studies of larger proteins.

A strategy for resolution and assignment of single proton resonances in proteins of molecular mass up to at least 40 kDa is presented. This approach is based on 15N (or 13C) labeling of selected residues in a protein. The resonances from protons directly bonded to labeled atoms are detected in a two-dimensional 1H-15N (or 13C) spectrum. The nuclear Overhauser effects from isotopically tagged protons are selectively observed in one-dimensional isotope-directed measurements. Using this approach, we have observed approximately 160 resonances from 15N-bonded protons in the backbone and sidechains of uniformly 15N-labeled T4 lysozyme (molecular mass = 18.7 kDa). Partial proton-deuterium exchange can be used to simplify the 1H-15N spectrum of this protein. These resonances are identified by amino acid class using selective incorporation of 15N-labeled amino acids and are assigned to specific residues by mutational substitution, multiple 15N and 13C labeling, and isotope-directed nuclear Overhauser effect measurements. For example, using a phenyl[15N]alanine-labeled lysozyme variant containing two consecutive phenylalanine residues in an alpha-helical region, we observe an isotope-directed nuclear Overhauser effect from the amide proton of Phe-66 to that of Phe-67.

27] Structure and thermal stability of phage T4 lysozyme

Publisher Summary This chapter discusses the phage T4 lysozyme system. Methods of rapidly generating mutants, characterizing their thermal stability, and accurately determining their X-ray crystal structures are discussed. Results and insights into the structural basis of protein thermal stability are summarized. Phage T4 lysozyme provides a powerful model system for studying the structural basis of protein thermal stability. By comparing the properties of mutant and wild-type proteins, the contributions of individual chemical groups can be estimated. The diversity of substitutions in the collection of mutant lysozymes indicates that many types of interactions contribute to stability. Despite great advances in the technology of generating and studying mutants, the fundamental problem of assigning the observed effects to the energy of the folded or unfolded state remains unsolved. One approach to overcome this limitation is to look for structural patterns in the native conformation that are associated with thermal stability.