Bacteriophage T4 Lysozyme Research Articles

Molecular basis of thermostability in the lysozyme from bacteriophage T4

MOST proteins are denatured at temperatures above 50–60°C, although some enzymes, especially those from thermophilic organisms, remain active at temperatures up to 80–90 °C. The determination of the three-dimensional structure of the thermostable protease thermolysin showed that heat-stable proteins do not contain unusual structural features absent from less stable proteins1,2. Furthermore, the amino acid sequences of similar proteins from both mesophilic and thermophilic sources have been shown to be homologous, suggesting that the respective structures are similar3,4. Nevertheless, such homologous amino acid sequences also include many differences which obscure those amino acid changes actually responsible for differences in thermostability. We report here the structure of a temperature sensitive (ts) mutant of T4 phage lysozyme. This permits the first direct comparison of two protein structures in which all differences are directly related to a change in thermal stability. It is shown that, except for the replacement of a partially exposed arginine by a histidine, the three-dimensional structure of the ts lysozyme is virtually identical with that of native lysozyme.

The use of deuterium exchange for the study of the distortion of α-helices in proteins

The geometrical distortion of the α-helical structure of the globular proteins sperm-whale myoglobin, bacteriophage T4 lysozyme and hen egg-white lysozyme have been studied by means of deuterium exchange in solution. It was examined with the use of infrared spectroscopy in the region of the amide A band. The parameters of this band are known to be dependent on the length and geometry of the peptide hydrogen bond. In this way an estimation of the structural heterogeneity of the polypeptide backbone of the protein molecule has been achieved by studying the half-width of the amide A band during successive deuteration of the protein in heavy water solution. For all the proteins studied the peptide groups with broad amide A bands were exchanged at the first stage. These groups have been assigned as belonging to the unordered form of the molecule. The α-helical fragments were assigned to have smaller values of half-widths of the amide A band, and these were exchanged at the second stage. From these data α-helical fragments were shown to be characterized by a set of geometrical distortions. The results obtained also disclose a correlation between the degree of geometrical distortion of α-helical structure in the protein molecule and the dynamic accessibility of their peptide groups to a water molecule.

Effect of RNase III on efficiency of translation of bacteriophage T7 lysozyme mRNA

RNase III had no positive effect on the translation of bacteriophage T7 lysozyme mRNA in vivo or in vitro. The time of appearance and quanity of lysozyme in T7-infected E. coli BL107, an RNase III- strain, and T7-infected E. coli BL15, a nearly isogenic RNase III+ strain, were indistinguishable. Nearly identical patterns of lysozyme mRNA activity were obtained when RNA extracted at different times after infection of RNase III+ and RNase III- hosts was translated in cell-free extracts of E. coli containing or lacking RNase III. Exposure of RNA extracted from T7-infected E. coli BL107 (RNase III-) to purified RNase III did not increase the lysozyme mRNA activity of this RNA. The only result that implied that RNase III has a differential effect on the translatability of the lysozyme mRNA was the translation of fractionaed RNA from T7-infected E. coli BL107. Translation of the smallest and largest lysozyme messages, 0.33 x 10(6) and 4 x 10(6) to 5 x 10(6) daltons, was the most inefficient in RNase III- cell-free extracts as compared to RNase III+ cell-free translation. The translation of the most abundant, medium-sized lysozyme mRNA between 0.9 x 10(6) and 1.5 x 10(6) daltons was the least affected by the absence of RNase III. The existence of a lag between the appearance of lysozyme mRNA and the appearance of lysozyme in T7 infection was confirmed. In these studies a very rapid method of RNA extraction was used, eliminating the possibility of continued RNA transcription during cell collection and RNA extraction. With this method of analysis, the length of the lag period was established at about 3 min. The possibility that RNase III is the controlling element of the lag period was eliminated by these investigations.

The structure of phage T4 lysozyme in solution noticeably differs from its crystalline structure

In the preceding paper [l] it was shown by the method of large-angle diffuse X-ray scattering [2,3] that the structure of bovine pancreatic ribonuclease and that of its complex with an inhibitor in solution coincide with their crystalline structures(on a distance scale 2 10 A). Also it was shown [2] that hen egg white lysozyme structure in solution differs from its crystalline structure by a small opening of the substrate-binding cleft, the binding of the competitive inhibitor stabilizing the closed state of the cleft observed in the crystalline structure. Sperm whale myoglobin structure in solution also differs from its crystalline structure, the differences being interpreted as changes of the GH hairpin position relative to the remaining part of the molecule [4] . In all probability in both cases we are dealing with fluctuations in relative positions of large masses of a protein molecule which are frozen in the crystalline structure but which manifest themselves in solution. The possibility of such fluctuations was demonstrated by direct model calculations for hen egg-white lysozyme [2]. Here the above-mentioned method has been used to study phage T4 lysozyme. The crystalline structure of this protein is characterized by the closed substratebinding cleft, therefore ‘in order to allow the substrate to enter, the enzyme would have to undergo a fairly substantial conformational change’ [6] . Our results provide evidence of a noticeable difference between

Prediction methods for equipment reliability evaluation : A. R. Eames. Radio Electron. Engr48, (7/8) 333 (July/August 1978)

The antibacterial working range of six lysozymes was tested under ambient and high pressure, on a panel of five gram-positive (Enterococcus faecalis, Bacillus subtilis, Listeria innocua, Staphylococcus aureus and Micrococcus lysodeikticus) and five gram-negative bacteria (Yersinia enterocolitica, Shigella flexneri, Escherichia coli O157:H7, Pseudomonas aeruginosa and Salmonella typhimurium). The lysozymes included two that are commercially available (hen egg white lysozyme or HEWL, and mutanolysin from Streptomyces globisporus or M1L), and four that were chromatographically purified (bacteriophage λ lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). T4L, LaL and GEWL were highly pure as evaluated by silver staining of SDS-PAGE gels and zymogram analysis while CFL was only partially pure.At ambient pressure each gram-positive test organism displayed a specific pattern of sensitivity to the six lysozymes, but none of the gram-negative bacteria was sensitive to any of the lysozymes. High pressure treatment (130–300 MPa, 25 °C, 15 min) sensitised several gram-positive and gram-negative bacteria for one or more lysozymes. M. lysodeikticus and P. aeruginosa became sensitive to all lysozymes under high pressure, S. typhimurium remained completely insensitive to all lysozymes, and the other bacteria showed sensitisation to some of the lysozymes. The possible applications of the different lysozymes as biopreservatives, and the possible reasons for the observed differences in bactericidal specificity are discussed.

Stability of phage T4 lysozymes I. Native properties and thermal stability of wild type and two mutant lysozymes

Two mutants of phage T4 lysozyme were prepared and characterized. One mutation substituted a tyrosine residue for tryptophan at position 138. The other substituted tyrosines at all three tryptophan positions of the wild type molecule (126, 138, 158). Comparative studies of the physical properties (absorption, flourescence, circular dichroism) of the three enzymes were performed as a function of pH. Also, the proteins were reversibly melted as a function of pH. Since the unfolding reaction appeared to be a two-state process for all these proteins, the data were analyzed by the van 't Hoff procedure. The changes in stability and activity produced by substitution of Trp 138 were especially significant. The other substitutions were neutral. See the end of the paper for a summary of conclusions. In the appendix the appropriate thermodynamic relations are developed for a constant ΔC P transition. Parts of this material were presented at the meeting of the Society of Biological Chemists in San Francisco in June, 1976 and at the International Conference on Biomolecules at Regensburg, G.F.R. in October, 1976.

Functional modifications of the translational system in Bacillus subtilis during sporulation

Extracts of sporulating cells were found to be defective in vitro translation of phage SP01 ribonucleic acid (RNA) and vegetative Bacillus subtilis RNA. The activity of washed ribosomes from sporulating cells was very similar to that of washed ribosomes from vegetative cells in translating polyuridylic acid, SP01 RNA, and vegetative RNA. The S-150 fraction from either vegetative or sporulating cells grown in Difco sporulation medium contained an apparent inhibitor of protein synthesis. The crude initiation factor fraction from ribosomes of sporulating cells was defective in promoting the initiation factor-dependent translation of SP01 RNA. The crude initiation factor preparations from sporulating cells were as active as the corresponding preparations from vegetative cells in promoting the initiation factor-dependent translation of either phage Qbeta or phage T4 RNA by washed Escherichia coli ribosomes. The crude initiation factors from sporulating cells were perhaps more active than those from vegetative cells in promoting the initiation factor-dependent synthesis of phage T4 lysozyme by E. coli ribosomes. The crude initiation factor preparations from either vegetative or stationary-phase cells of an asporogenous mutant showed similar ability to promote the in vitro translation of SP01 RNA.

Substrate-induced evolution of lysozymes

Digestion by five different lysozymes (mucopeptide N-acetylmuramoylhydrolases, EC 3.2.1.17) of a soluble uncross-linked peptidoglycan secreted by Micrococcus luteus (Mirelman, D., Bracha, R. and Sharon N. (1974) Biochemistry 13, 5045–5053) was investigated. Hen egg-white and human lysozymes converted the peptidoglycan into the disaccharide GlcNAc- β(1 → 4)- N-acetylmuramic acid (-MurNAc), the tetrasaccharide (GlNAc- β(1 → 4)-MurNAc) 2 and the disaccharide-hexapeptide: ▪ With goose egg-white lysozyme, mainly the disaccharide and the disaccharidehexapeptide were produced, in approximately equimolar amounts. Papaya lysozyme afforded mainly products without peptide substitution, such as the disaccharide and tetrasaccharide, while T4 phage lysozyme gave only the disaccharide-hexapeptide. Lysozymes from different sources thus differ in their specificity requirements with respect to peptide substitution on the lactyl moiety of N-acetylmuramic acid. This pattern of specificity is discussed in terms which correlate it with the differences in the structures of their presumed natural substrates. We postulate that evolutionary changes in the substrate structure may have influenced the development of the active site of lysozyme so that it could function most efficiently with the particular natural substrate encountered by each species.

Coding properties of an ochre-suppressing derivative of Escherichia coli tRNA ITyr

The ability of an ochre-suppressing derivative of Escherichia coli tRNATyrI to suppress amber and ochre mutations in the same codon (homotopic mutations) at several sites in three different genes (E. coli β-galactosidase, phage T4 rIIB cistron and T4 lysozyme) was tested. This suppressor transfer RNA nearly always suppresses the amber mutation better than the ochre mutation. The ratio of ochre codon to amber codon suppression efficiency in homotopic pairs is different at the different sites tested. Thus, reading context must play a role in suppression. However, the influence of context in the genes tested is different on suppressor tRNAs other than su+oc-A2 indicating that parts of tRNAs other than the anticodon sequence UUA must also participate in the suppression process. At some sites nucleotides adjacent to the nonsense codon influenced suppression in a systematic fashion but no general mechanism to explain these effects is yet apparent.

Exploring structural homology of proteins

A method for systematically comparing the folding of the three-dimensional structures of proteins has been developed. A search function, plotted in terms of three Eulerian angles, represents the number of sequentially equivalenced amino acids. For each orientation one protein structure is rotated about its center of mass with respect to the other and probabilities are calculated which estimate the degree of structural parallelism. The structurally equivalent residues with highest probabilities are then selected for the best common topology. It was observed that, when structures containing about 150 residues were compared, the random background had a mean value of around 14 residues and the standard deviation was approximately nine residues. The method has been shown to be successful in determining the similarity of the NAD binding domains of lactate dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase, and in comparing the heme binding fold of cytochrome b 5 with the globins. Application of the method to compare hen egg white lysozyme and T4 phage lysozyme led to a single significant peak of 62 residues. The structural homology indicated by this peak showed that the substrate, as bound to hen egg white lysozyme, has a corresponding binding site in the large cleft of the phage lysozyme. The predicted binding site of N-acetyl glucosamine at position C compares well with an N-acetyl glucosamine center observed to bind to crystalline phage lysozyme (B. W. Matthews, personal communication). Some results for the comparison of the two Fe-S cage binding domains of ferredoxin are also presented.

The Specificity Requirements of Bacteriophage T4 Lysozyme

A series of bacterial cell wall glycopeptides of low molecular weight and cell wall nucleotide precursors have been tested for their inhibitory action on the digestion by T4 lysozyme of a radioactively labeled linear uncrosslinked peptidoglycan. The disaccharide-peptides GlcNAc-MurNAc-l-Ala-D-Glu(A2pm) (C5) and GlcNAc-MurNAc-L-Ala-D-Glu(A2pm-D-Ala) (C6) as well as the monosaccharide-peptide MurNAc-L-Ala-D-Glu(A2pm) were found to be good competitive inhibitors (with similar Ki values) whereas the disaccharide-pentapeptide GlcNAcMurNAc-L-Ala-DGlu-Gly-L-Lys-D-Ala was a poor inhibitor. T4 lysozyme did not catalyse transglycosylation reactions from Escherichia coli B peptidoglycan to the disaccharide-peptide C6. No changes were seen in the circular dichroism spectra (200-250 nm) or fluorescence emmission spectra upon binding of the good inhibitors. The results obtained indicate that T4 lysozyme has a small active site capable of recognizing a unit consisting of MurNAc-L-Ala-D-Glu(A2pm).

Effect of neighboring nucleotide sequences on suppression efficiency in amber mutants of T4 phage lysozyme.

Variations in suppression efficiency were observed among nonsense mutations at different locations within the lysozyme gene (e) of T4 phage. The present experiments using three amber mutants in lysozyme gene indicate such variations presumably depend upon the base sequences neighboring to the nonsense mutations.

Comparison of the predicted and observed secondary structure of T4 phage lysozyme

Predictions of the secondary structure of T4 phage lysozyme, made by a number of investigators on the basis of the amino acid sequence, are compared with the structure of the protein determined experimentally by X-ray crystallography. Within the amino terminal half of the molecule the locations of helices predicted by a number of methods agree moderately well with the observed structure, however within the carboxyl half of the molecule the overall agreement is poor. For eleven different helix predictions, the coefficients giving the correlation between prediction and observation range from 0.14 to 0.42. The accuracy of the predictions for both β-sheet regions and for turns are generally lower than for the helices, and in a number of instances the agreement between prediction and observation is no better than would be expected for a random selection of residues. The structural predictions for T4 phage lysozyme are much less successful than was the case for adenylate kinase (Schulz et al. (1974) Nature 250, 140–142). No one method of prediction is clearly superior to all others, and although empirical predictions based on larger numbers of known protein structure tend to be more accurate than those based on a limited sample, the improvement in accuracy is not dramatic, suggesting that the accuracy of current empirical predictive methods will not be substantially increased simply by the inclusion of more data from additional protein structure determinations.

Studies on the role of bacteriophage T7 lysozyme during phage infection

Bacteriophage T7 lysozyme is not required for lysis of the host but instead is involved in DNA metabolism after infection. The lysozyme apparently causes release of host DNA from its membrane complex, but this release is not needed for efficient degradation of host DNA. Therefore, the effects of lysozyme deficiency on DNA synthesis cannot be explained as a reduction in the availability of precursors. T7 lysozyme also causes release of newly made T7 DNA (or phage particles) from the membrane and this may be its primary role in T7 infection

Studies on the specificity of action of bacteriophage T4 lysozyme.

Lysozyme from bacteriophage T4 was found to digest a soluble, uncrosslinked peptidoglycan which is secreted by cells of Micrococcus luteus when incubated in the presence of penicillin G. Analysis of the enzymatic degradation products shows that T4 acts as an endo-acetylmuramidase capable of cleaving glycosidic bonds only at muramic acid residues that are substituted with peptide side-chains. The results indicate that the secreted peptidoglycan may consist of a mixture of chains, approximately half of which are substituted by peptide side chains on most of their muramic acid residues, while the other half is made up of chains in which the muramic acid moieties are unsubstituted.

Logical analysis of the mechanism of protein folding III. Prediction of the strong long-range interactions.

Abstract It has been found that strong long-range interactions occur in regions having large β-structural potentials. As has been described previously (Nagano, 1974), interactions among regions having both helical and β-structural potentials (αβ-gaβ interactions) are also very important. Accordingly, an idea is presented in this paper that the relative stability of a protein conformation could be estimated by a relatively simple mathematical function of sequence and conformation. The function P(p,q) is called the non-energy part of pseudo-free energy, because minimization of the sum of P(p,q) and energy functions (cf. Levitt, 1974; Warme & Scheraga, 1974) can be expected to lead to a plausible model of a protein. A merit of the function is that it can help us decide which way to go in manipulating a temporarily built model, e.g. towards a helix-rich protein or towards a β-structure-rich protein. The estimation of P(p,q) as an artificial potential does not use much computer time because only the co-ordinates of the β-carbon atoms (α-carbon atoms if the residue is Gly) are used. It is composed of terms of the long-range interactions PL and short-range interactions PS. The term PL represents the relative strength of helix-helix interactions, helix-β-candidate interactions and β-candidate-β-candidate interactions. It is assumed that both helical and β-structural potentials can be measured as the differences between the predicted function for helix and β-structure, respectively, as defined previously (Nagano, 1973), and the corresponding largest values ever found. A hypothesis that two residues distantly separated in the primary sequence contribute less to the stability of the whole molecule is finally discarded because the true conformation of concanavalin A becomes very unstable compared with its false conformation folded like the main part of subtilisin. The parameters thus determined indicate that the helix-β-candidate interactions are almost as important as the β-candidate-β-candidate interactions. Both helix and loop prediction functions are combined to give the short-range interactions term, PS, according to whether the region is really helical or not, and to whether it is really looped or not. The function P(p,q) can be used as a criterion for judging whether the predicted conformation is realistic or false, because the parameters can be adjusted to give, within limits, reasonable values of −10 kcal/residue for true conformations and higher than −5 kcal/residue for false conformations. As an application of the present theory of protein folding, the tertiary structure of bacteriophage T4 lysozyme is predicted and presented in Figure 1, prior to the X-ray structure becoming available.

Phage T4 lysozyme Physical properties and reversible unfolding

Phage T4 lysozyme has been used extensively in studies of the genetic code. However, little work has been done on the characterization of the purified enzyme. Therefore, we determined the spectral properties of native T4 lysozyme and used these properties to follow the unfolding transition. The ultraviolet absorption spectrum and solvent perturbation difference spectrum indicate that the aromatic amino acids are extensively exposed to solvent. The CD and ORD spectra are characteristic of a high fraction of helix. Guanidine hydrochloride denaturation results show that over a T4 lysozyme concentration range of 0.07–1 g/l the c m = 2.7 M guanidine hydrochloride at pH 5 and that the transition is 100% reversible as judged by enzymatic assay and four different spectrophotometric criteria: CD at 295 nm, CD at 223 nm, fluorescence intensity at 350 nm and wavelength of maximum fluorescence. Guanidine hydrochloride denaturation at pH 2.5 was followed using fluorescence emission and has a c m = 1.7 M guanidine hydrochloride, indicating a strong pH dependence of chemical unfolding. Reversible thermal denaturation conditions were located at acid pH, 0.2 M NaCl, 10 −4 M dithiothreitol and 10 −6 M T4 lysozyme. The CD signal at 223 nm was used to measure the unfolding. Thermodynamic analysis of the thermal data showed an increase in T m, ΔH unf and ΔS unf with increasing pH.

Troponin and parvalbumin calcium binding regions predicted in myosin light chain and T4 lysozyme.

A computer search of available protein sequences and structures suggests that bacteriophage T4 lysozyme contains one region and that rabbit myosin light chains contain three regions similar, and supposedly homologous, to the calcium binding region of carp muscle calcium binding parvalbumin.

The amino acid substitution tyrosine 88 → histidine in the lysozyme of phage T4: The molecular cause of the cold-sensitive phenotype of the mutant cseBU56

The amino acid substitution tyrosine 88 → histidine in the lysozyme of phage T4: The molecular cause of the cold-sensitive phenotype of the mutant cseBU56

Preparative polyacrylamide gel electrophoresis of ribonucleic acid. Identification of multiple molecular species of bacteriophage T7 lysozyme messenger ribonucleic acid.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPreparative polyacrylamide gel electrophoresis of ribonucleic acid. Identification of multiple molecular species of bacteriophage T7 lysozyme messenger ribonucleic acidFrederick S. Hagen and Elton T. YoungCite this: Biochemistry 1974, 13, 16, 3394–3400Publication Date (Print):July 1, 1974Publication History Published online1 May 2002Published inissue 1 July 1974https://doi.org/10.1021/bi00713a033RIGHTS & PERMISSIONSArticle Views114Altmetric-Citations32LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts