Bacteriophage T4 Lysozyme Research Articles

Submicro to Millisecond Conformational Transitions of Bacteriophage T4 Lysozyme with Ångström Accuracy using Multiparameter Fluorescence Detection

We use single molecule FRET and the completeness of Multiparameter Fluorescence Detection to study protein flexibility and dye rigidity from submicro- to millisecond timescales in native and non-native conditions of the bacteriophage T4 Lysozyme (T4L). T4L contains 164-residues and consists of two domains connected by a long alpha helix. The enzyme cleaves the glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine of bacterial cell wall saccharides. From crystalographic and EPR measurement the native enzyme is believed to be in dynamic exchange between various “open” conformations. Most likely a set of hinges is responsible for the dynamic motion of the enzyme. However, the time scale of these hinges is still unknown. To accurately determine protein conformations from single molecule FRET, one needs to consider the dynamic behavior of donor and acceptor fluorophores. To test protein and dye mobility, we used single and double labeled T4L mutants with various fluorophore linkers using site specific mutants. The orthogonal system contains a ketone handle in the N-terminal domain for reaction with a hydroxylamine or hydrazide fluorophore, or a thiol group in the C-terminal domain for reaction with a thiol-specific fluorophore. Furthermore, the double mutant is ideal for site-specific attachment of the donor and acceptor fluorophores. Beside studies on native conformational dynamics we look into non native conformational transitions using chemical denaturants. In addition, we complement our experimental work by modelling the accesible volume (AV) required for each fluorophore using steric interaction with known crystalographic data. The AV model considers all available locations where the dye could be located without causing sterical clashes. This tool in combination with single molecule FRET allows us to quantitatively determine corresponding structural distances from experimental data.

Fractional stochastic description of hinge motions in single protein molecules

In this study, it is demonstrated that the motion of hinges in single protein molecules can be modeled as general fractional Langevin dynamics of harmonically bound particles driven by non-local Gaussian noise with a power-law correlation. This conclusion was justified by comparing the theoretical predictions of the proposed model to the existing molecular dynamics simulations performed on the M6I mutant of phage T4 lysozyme and E. coli ribonuclease H1.

A molecular dynamics study of the interprotein interactions in collagen fibrils

Molecular dynamics simulations of collagen are used to investigate at the atomistic level the nature of the interprotein interactions that are present within a collagen fibril, and which are responsible for the fibril's thermodynamic stability. Simulations both of a collagen fibril and of a fully solvated tropcollagen are compared in order to study the interactions that arise between the proteins upon the process of fibrillogenesis. The interactions studied include direct interprotein hydrogen bonds, water-mediated interprotein hydrogen bonds, and hydrophobic interactions. The simulations are used to quantify the number of interprotein interactions that form; to study which functional groups contribute most towards the interactions; and to study the spatial distribution of interprotein interactions throughout the fibril's D period. The processes of collagen fibrillogenesis and protein folding are then compared with each other, because these two physical processes share many similarities in concept, and the latter has been more widely studied. Molecular dynamics simulations of a bacteriophage T4 lysozyme protein, both in its native state and in and unfolded state, are used as an illustrative example of a typical protein folding process, for direct comparison with the collagen simulations.

Improved Replica Exchange Method for Native-State Protein Sampling.

We present a new replica exchange method, designed for optimal native state protein sampling in explicit solvent, called replica exchange with flexible tempering (REFT). The method was built upon the recently introduced replica exchange with solute tempering (REST). The potential function is adapted to direct the conformational search toward interdomain movements and the flexible portions of the protein. We demonstrate the improved sampling efficiency of REFT compared to the original REST for the bacteriophage T4 lysozyme.

Improving Prediction Accuracy via Subspace Modeling in a Statistical Geometry Based Computational Protein Mutagenesis

A computational mutagenesis is detailed whereby each single residue substitution in a protein chain of primary sequence length N is represented as a sparse N-dimensional feature vector, whose M << N nonzero components locally quantify environmental perturbations occurring at the mutated position and its neighbors in the protein structure. The methodology makes use of both the Delaunay tessellation algorithm for representing protein structures, as well as a four-body, knowledge based, statistical contact potential. Feature vectors for each subset of mutants due to all possible residue substitutions at a particular position cohabit the same M-dimensional subspace, where the value of M and the identities of the M nonzero components are similarly position dependent. The approach is used to characterize a large experimental dataset of single residue substitutions in bacteriophage T4 lysozyme, each categorized as either unaffected or affected based on the measured level of mutant activity relative to that of the native protein. Performance of a single classifier trained with the collective set of mutants in N-space is compared to that of an ensemble of position-specific classifiers trained using disjoint mutant subsets residing in significantly smaller subspaces. Results suggest that significant improvements can be achieved through subspace modeling.

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 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.

Specific Structural Features of the N-Acetylmuramoyl- l-Alanine Amidase AmiD from Escherichia coli and Mechanistic Implications for Enzymes of This Family

AmiD is the fifth identified N-acetylmuramoyl- l-alanine zinc amidase of Escherichia coli. This periplasmic lipoprotein is anchored in the outer membrane and has a broad specificity. AmiD is capable of cleaving the intact peptidoglycan (PG) as well as soluble fragments containing N-acetylmuramic acid regardless of the presence of an anhydro form or not, unlike the four other amidases, AmiA, AmiB, AmiC, and AmpD, which have some specificity. AmiD function is, however, not clearly established but it could be part of the enzymatic machinery involved in the PG turnover in E. coli. We solved three structures of the E. coli zinc amidase AmiD devoid of its lipidic anchorage: the holoenzyme, the apoenzyme in complex with the substrate anhydro- N-acetylmuramic-acid- l-Ala-γ- d-Glu- l-Lys, and the holoenzyme in complex with the l-Ala-γ- d-Glu- l-Lys peptide, the product of the hydrolysis of this substrate by AmiD. The AmiD structure shows a relatively flexible N-terminal extension that allows an easy reach of the PG by the enzyme inserted into the outer membrane. The C-terminal domain provides a potential extended geometrical complementarity to the substrate. AmiD shares a common fold with AmpD, the bacteriophage T7 lysozyme, and the PG recognition proteins, which are receptor proteins involved in the innate immune responses of a wide range of organisms. Analysis of the different structures reveals the similarity between the catalytic mechanism of zinc amidases of the AmiD family and the thermolysin-related zinc peptidases.

Antimicrobial peptides derived from goose egg white lysozyme

Peptide fragments possessing antimicrobial activity were obtained by protease digestion of goose egg white lysozyme. Digested peptide purified from RP-HPLC which showed no lysozyme activity exhibited bactericidal activity toward Gram-negative and Gram-positive bacteria. LC/MS–MS and automated Edman degradation revealed the amino acid sequence to be Thr-Ala-Lys-Pro-Glu-Gly-Leu-Ser-Tyr. This sequence corresponds to amino acid positions 20–28, located at the N-terminal outer part of goose lysozyme. The peptide acted on bacterial membrane as shown by scanning electron microscopy. The mechanism of action could be explained from a helical structure that may be formed by the centered Pro residue and the terminal Lys residue after the peptide attaches to a cell membrane. This is the first study to report that a peptide derived from the protease digests of G-type lysozyme possesses antimicrobial activity with broad spectrum activity. Our result is comparative to the previous reports of Chicken lysozyme and T4 phage lysozyme, which showed antimicrobial activity after digestion with protease. These results might contribute to the usage of antimicrobial peptides engineered by genetic or chemical synthesis.

Abstract: 126 ABDOMINAL OBESITY AND METABOLIC SYNDROME: IMPLICATIONS FOR ASSESSMENT AND MANAGEMENT OF GLOBAL CARDIOMETABOLIC RISK

Ion channel-coupled receptor (ICCR) is a recent technology based on the fusion of G protein-coupled receptors (GPCRs) to an ion channel. Binding of ligands on the GPCR triggers conformational changes of the receptor that are mechanically transmitted to the ion channel gates, generating an electrical signal easily detectable with conventional electrophysiological techniques. ICCRs are heterologously expressed in Xenopus oocytes and offers several advantages such as: (i) real-time recordings on single cells, (ii) standard laboratory environment and inexpensive media for Xenopus oocytes maintenance, (iii) absence of protein purification steps, (iv) sensitivity to agonists and antagonists in concentration-dependent manner, (v) compatibility with a Gi/o protein activation assay based on Kir3.x channels, and (vi) ability to detect receptor activation independently of intracellular effectors. This last characteristic of ICCRs led to the development of a functional assay for G protein-“uncoupled” receptors such as GPCRs optimized for crystallization by alteration of their third intracellular (i3) loop. One of the most widely used approaches consists in replacing the i3 loop with the T4 phage lysozyme (T4L) domain that obstructs the access of G proteins to their binding site. We recently demonstrated that the ICCR technology can functionally characterize GPCRs(T4L). Two-electrode voltage-clamp (TEVC) recordings revealed that apparent affinities and sensitivities to ligands are not affected by T4L insertion, while ICCRs(T4L) displayed a partial agonist phenotype upon binding of full agonists, suggesting that ICCRs could detect intermediate-active states. This chapter aims to provide exhaustive details from molecular biology steps to electrophysiological recordings for the design and the characterization of ICCRs and ICCRs(T4L).

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 A resolution one of the bond angles at the site of substitution appeared to be distorted by about 11( degrees ), 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 A resolution. The high-resolution refinement of the structure of R96H does not support the bond angle distortion seen in the 1.9 A 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.

Halogenated Benzenes Bound within a Non-polar Cavity in T4 Lysozyme Provide Examples of I⋯S and I⋯Se Halogen-bonding

We showed earlier that the mutation of Leu99 to alanine in bacteriophage T4 lysozyme creates an internal cavity of volume ∼ 150 Å 3 that binds benzene and a variety of other ligands. As such, this cavity provides an excellent target to study protein-ligand interaction. Here, we use low-temperature crystallography and related techniques to analyze the binding of halogen-incorporated benzenes typified by C 6F 5X, where X = H, F, Cl, Br or I, and C 6H 5X, where X = H or I was also studied. Because of the increased electron density of fluorine relative to hydrogen, the geometry of binding of the fluoro compounds can often be determined more precisely than their hydrogen-containing analogs. All of the ligands bind in essentially the same plane but the center of the phenyl ring can translate by up to 1.2 Å. In no case does the ligand rotate freely within the cavity. The walls of the cavity consist predominantly of hydrocarbon atoms, and in several cases it appears that van der Waals interactions define the geometry of binding. In comparing the smallest with the largest ligand, the cavity volume increases from 181 Å 3 to 245 Å 3. This shows that the protein is flexible and adapts to the size and shape of the ligand. There is a remarkably close contact of 3.0 Å between the iodine atom on C 6F 5I and the sulfur or selenium atom of Met or SeMet102. This interaction is 1.0 Å less than the sum of the van der Waals radii and is a clear example of a so-called halogen bond. Notwithstanding this close approach, the increase in binding energy for the halogen bond relative to a van der Waals contact is estimated to be only about 0.5–0.7 kcal/mol.

Coarse-Graining Protein Structures With Local Multivariate Features from Molecular Dynamics

A multivariate statistical theory, local feature analysis (LFA), extracts functionally relevant domains from molecular dynamics (MD) trajectories. The LFA representations, like those of principal component analysis (PCA), are low dimensional and provide a reduced basis set for collective motions of simulated proteins, but the local features are sparsely distributed and spatially localized, in contrast to global PCA modes. One key problem in the assignment of local features is the coarse-graining of redundant LFA output functions by means of seed atoms. One can solve the combinatorial problem by adding seed atoms one after another to a growing set, minimizing a reconstruction error at each addition. This allows for an efficient implementation, but the sequential algorithm does not guarantee the optimal mutual correlation of the sequentially assigned features. Here, we present a novel coarse-graining algorithm for proteins that directly minimizes the mutual correlation of seed atoms by Monte Carlo (MC) simulations. Tests on MD trajectories of two biological systems, bacteriophage T4 lysozyme and myosin II motor domain S1, demonstrate that the new algorithm provides statistically reproducible results and describes functionally relevant dynamics. The well-known undersampling of large-scale motion by short MD simulations is apparent also in our model, but the new coarse-graining offers a major advantage over PCA; converged features are invariant across multiple windows of the trajectory, dividing the protein into converged regions and a smaller number of localized, undersampled regions. In addition to its use in structure classification, the proposed coarse-graining thus provides a localized measure of MD sampling efficiency.

A novel thermophilic lysozyme from bacteriophage φIN93

The lysozyme of bacteriophage φIN93 was purified to apparent homogeneity with Carboxymethyl Sepharose and Hydroxyapatie columns from lysates of the phage grown on Thermus aquaticus TZ2. The enzyme is a single polypeptide chain with a molecular weight of 33,000. From the determined N-terminal amio acids of the enzyme, the locus of the gene was specified on a φIN93 genome. The enzyme was not similar to egg white lysozyme, T4 phage lysozyme, or lambda phage lysozyme. The enzyme, φIN93 lysozyme, was found to be a novel type of thermophilic lysozyme, which lyses specifically Thermus sp. cells, and exhibited conspicuous thermal stability at 95 °C for 1 h in the presence of β-mercaptoethanol.

Combining Elastic Network Analysis and Molecular Dynamics Simulations by Hamiltonian Replica Exchange.

Coarse-grained elastic network models (ENM) of proteins can be used efficiently to explore the global mobility of a protein around a reference structure. A new Hamiltonian-replica exchange molecular dynamics (H-RexMD) method has been designed that effectively combines information extracted from an ENM analysis with atomic-resolution MD simulations. The ENM analysis is used to construct a distance-dependent penalty (flooding or biasing) potential that can drive the structure away from its current conformation in directions compatible with the ENM model. Various levels of the penalty or biasing potential are added to the force field description of the MD simulation along the replica coordinate. One replica runs at the original force field. By focusing the penalty potential on the relevant soft degrees of freedom the method avoids the rapid increase of the replica number with increasing system size to cover a desired temperature range in conventional (temperature) RexMD simulations. The application to domain motions in lysozyme of bacteriophage T4 and to peptide folding indicates significantly improved conformational sampling compared to conventional MD simulations.

Construction of New Generation Shuttle Vectors Harboring Bacteriophage T4 Lysozyme Gene (gene e)

Construction of New Generation Shuttle Vectors Harboring Bacteriophage T4 Lysozyme Gene (gene e)

A finite element framework for computation of protein normal modes and mechanical response

A computational framework based on the Finite Element Method is presented to calculate the normal modes and mechanical response of proteins and their supramolecular assemblies. Motivated by elastic network models, proteins are treated as continuum elastic solids with molecular volume defined by their solvent-excluded surface. The discretized Finite Element representation is obtained using a surface simplification algorithm that facilitates the generation of models of arbitrary prescribed spatial resolution. The procedure is applied to a mutant of T4 phage lysozyme, G-actin, syntenin, cytochrome-c', beta-tubulin, and the supramolecular assembly filamentous actin (F-actin). Equilibrium thermal fluctuations of alpha-carbon atoms and their inter-residue correlations compare favorably with all-atom-based results, the Rotational-Translational Block procedure, and experiment. Additionally, the free vibration and compressive buckling responses of F-actin are in quantitative agreement with experiment. The proposed methodology is applicable to any protein or protein assembly and facilitates the incorporation of specific atomic-level interactions, including aqueous-electrolyte-mediated electrostatic effects and solvent damping. The procedure is equally applicable to proteins with known atomic coordinates as it is to electron density maps of proteins, protein complexes, and supramolecular assemblies of unknown atomic structure.

Expression of the bacteriophage T4 lysozyme gene in tall fescue confers resistance to gray leaf spot and brown patch diseases

Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species worldwide. Fungal diseases present a major limitation in the maintenance of tall fescue lawns, landscapes, and forage fields. Two severe fungal diseases of tall fescue are brown patch, caused by Rhizoctonia solani, and gray leaf spot, caused by Magnaporthe grisea. These diseases are often major problems of other turfgrass species as well. In efforts to obtain tall fescue plants resistant to these diseases, we introduced the bacteriophage T4 lysozyme gene into tall fescue through Agrobacterium-mediated genetic transformation. In replicated experiments under controlled environments conducive to disease development, 6 of 13 transgenic events showed high resistance to inoculation of a mixture of two M. grisea isolates from tall fescue. Three of these six resistant plants also displayed significant resistance to an R. solani isolate from tall fescue. Thus, we have demonstrated that the bacteriophage T4 lysozyme gene confers resistance to both gray leaf spot and brown patch diseases in transgenic tall fescue plants. The gene may have wide applications in engineered fungal disease resistance in various crops.

An approach to crystallizing proteins by synthetic symmetrization

Previous studies of symmetry preferences in protein crystals suggest that symmetric proteins, such as homodimers, might crystallize more readily on average than asymmetric, monomeric proteins. Proteins that are naturally monomeric can be made homodimeric artificially by forming disulfide bonds between individual cysteine residues introduced by mutagenesis. Furthermore, by creating a variety of single-cysteine mutants, a series of distinct synthetic dimers can be generated for a given protein of interest, with each expected to gain advantage from its added symmetry and to exhibit a crystallization behavior distinct from the other constructs. This strategy was tested on phage T4 lysozyme, a protein whose crystallization as a monomer has been studied exhaustively. Experiments on three single-cysteine mutants, each prepared in dimeric form, yielded numerous novel crystal forms that cannot be realized by monomeric lysozyme. Six new crystal forms have been characterized. The results suggest that synthetic symmetrization may be a useful approach for enlarging the search space for crystallizing proteins.

Comparison of the internal dynamics of globular proteins in the microcrystalline and rehydrated lyophilized states

Natural abundance solid-state 13C-NMR spin-lattice relaxation experiments in the laboratory ( T 1) and off-resonance rotating ( T 1ρ) frames were applied for qualitative comparison of the internal molecular dynamics of barstar, hen egg white lysozyme and bacteriophage T4 lysozyme in both the microcrystalline and the rehydrated (water content is 50% of the protein mass) lyophilized states. The microcrystalline state of proteins provides a better spectral resolution; however, less is known about the local structure and dynamics in the different states. We found by visual comparison of both T 1 and T 1ρ relaxation decays of various resonance bands of the CPMAS spectra that within the ns–μs range of correlation times there is no appreciable difference in the internal dynamics between rehydrated lyophilized and crystalline states for all three proteins tested. This suggests that the internal conformational dynamics depends weakly if at all on inter-protein interactions in the solid state. Hence, physical properties of globular proteins in a fully hydrated solid state seem to be similar to those in solution. This result at least partly removes concerns about biological relevance of studies of globular proteins in the solid state.

Cell wall substrate specificity of six different lysozymes and lysozyme inhibitory activity of bacterial extracts

We have investigated the specificity of six different lysozymes for peptidoglycan substrates obtained by extraction of a number of gram-negative bacteria and Micrococcus lysodeikticus with chloroform/Tris-HCl buffer (chloroform/buffer). 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 lambda lysozyme or LaL, bacteriophage T4 lysozyme or T4L, goose egg white lysozyme or GEWL, and cauliflower lysozyme or CFL). HEWL was much more effective on M. lysodeikticus than on any of the gram-negative cell walls, while the opposite was found for LaL. Also the gram-negative cell walls showed remarkable differences in susceptibility to the different lysozymes, even for closely related species like Escherichia coli and Salmonella Typhimurium. These differences could not be due to the presence of lysozyme inhibitors such as Ivy from E. coli in the cell wall substrates because we showed that chloroform extraction effectively removed this inhibitor. Interestingly, we found strong inhibitory activity to HEWL in the chloroform/buffer extracts of Salmonella Typhimurium, and to LaL in the extracts of Pseudomonas aeruginosa, suggesting that other lysozyme inhibitors than Ivy exist and are probably widespread in gram-negative bacteria.