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An engineered variant of T4 lysozyme serves as a model for studying induced remote conformational changes in a full protein context. The design involves a duplicated surface helix, flanked by two loops, that switches between two different conformations spanning about 20 Å. Molecular dynamics simulations of the engineered protein, up to 1 μs, rule out α-helix to β-sheet transitions within the duplicated helix as suggested by others. These simulations highlight how the use of different force fields can lead to radical differences in the structure of the protein. In addition, Markov state modeling and transition path theory were employed to map a 6.6 μs simulation for possible early intermediate states and to provide insights into the onset of the switching motion. The putative intermediates involve the folding of one helical turn in the C-terminal loop through energy driven, sequential rearrangement of nearby salt bridges around the key residue Arg63. These results provide a first step towards understanding the energetics and dynamics of a rather complicated intra-protein motion., (© 2019 The Protein Society.)

Double electron-electron resonance (DEER) is a popular technique that exploits attached spin labels to probe the collective dynamics of biomolecules in a native environment. Like most spectroscopic approaches, DEER detects an ensemble of states accounting for biomolecular dynamics as well as the labels' intrinsic flexibility. Hence, the DEER data alone do not provide high-resolution structural information. To disentangle this variability, we introduce a minimally biased simulation method to sample a structural ensemble that reproduces multiple experimental signals within the uncertainty. In contrast to previous approaches, our method targets the raw data themselves, and thereby it brings forth an unbiased molecular interpretation of the experiments. After validation on the T4 lysozyme, we apply this technique to interpret recent DEER experiments on a membrane transporter binding protein (VcSiaP). The results highlight the large-scale conformational movement that occurs upon substrate binding and reveal that the unbound VcSiaP is more open in solution than the X-ray structure., (Published by Elsevier Ltd.)

Cooperative protein folding requires distant regions of a protein to interact and provide mutual stabilization. The mechanism of this long-distance coupling remains poorly understood. Here, we use T4 lysozyme (T4L*) as a model to investigate long-range communications across two subdomains of a globular protein. T4L* is composed of two structurally distinct subdomains, although it behaves in a two-state manner at equilibrium. The subdomains of T4L* are connected via two topological connections: the N-terminal helix that is structurally part of the C-terminal subdomain (the A-helix) and a long helix that spans both subdomains (the C-helix). To understand the role that the C-helix plays in cooperative folding, we analyzed a circularly permuted version of T4L* (CP13*), whose subdomains are connected only by the C-helix. We demonstrate that when isolated as individual fragments, both subdomains of CP13* can fold autonomously into marginally stable conformations. The energetics of the N-terminal subdomain depend on the formation of a salt bridge known to be important for stability in the full-length protein. We show that the energetic contribution of the salt bridge to the stability of the N-terminal fragment increases when the C-helix is stabilized, such as occurs upon folding of the C-terminal subdomain. These results suggest a model where long-range energetic coupling is mediated by helix stabilization and not specific tertiary interactions., (© 2018 The Protein Society.)

The human mitochondrial amidoxime reducing component (hmARC) is a molybdenum cofactor-dependent enzyme that is involved in the reduction of a diverse range of N-hydroxylated compounds of either physiological or xenobiotic origin. In this study, the use of a fusion-protein approach with T4 lysozyme (T4L) to determine the structure of this hitherto noncrystallizable enzyme by X-ray crystallography is described. A set of four different hmARC-T4L fusion proteins were designed. Two of them contained either an N-terminal or a C-terminal T4L moiety fused to hmARC, while the other two contained T4L as an internal fusion partner tethered to the hmARC enzyme between two predicted secondary-structure elements. One of these internal fusion constructs could be expressed and crystallized successfully. The hmARC-T4L crystals diffracted to 1.7 Å resolution using synchrotron radiation and belonged to space group P2 1 2 1 2 1 with one molecule in the asymmetric unit. Initial attempts to solve the structure by molecular replacement using T4L did not result in electron-density distributions that were sufficient for model building and interpretation of the hmARC moiety. However, this study emphasizes the utility of the T4L fusion-protein approach, which can be used for the crystallization and structure determination of membrane-bound proteins as well as soluble proteins.

According to implicit ligand theory, the standard binding free energy is an exponential average of the binding potential of mean force (BPMF), an exponential average of the interaction energy between the unbound ligand ensemble and a rigid receptor. Here, we use the fast Fourier transform (FFT) to efficiently evaluate BPMFs by calculating interaction energies when rigid ligand configurations from the unbound ensemble are discretely translated across rigid receptor conformations. Results for standard binding free energies between T4 lysozyme and 141 small organic molecules are in good agreement with previous alchemical calculations based on (1) a flexible complex ( R≈0.9 for 24 systems) and (2) flexible ligand with multiple rigid receptor configurations ( R≈0.8 for 141 systems). While the FFT is routinely used for molecular docking, to our knowledge this is the first time that the algorithm has been used for rigorous binding free energy calculations. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

The location and ligand accessibility of internal cavities in cysteine-free wild-type T4 lysozyme was investigated using O 2 gas-pressure NMR spectroscopy and molecular dynamics (MD) simulation. Upon increasing the concentration of dissolved O 2 in solvent to 8.9 mM, O 2 -induced paramagnetic relaxation enhancements (PREs) to the backbone amide and side chain methyl protons were observed, specifically around two cavities in the C-terminal domain. To determine the number of O 2 binding sites and their atomic coordinates from the 1/r 6 distance dependence of the PREs, we established an analytical procedure using Akaike's Information Criterion, in combination with a grid-search. Two O 2 -accessible sites were identified in internal cavities: One site was consistent with the xenon-binding site in the protein in crystal, and the other site was established to be a novel ligand-binding site. MD simulations performed at 10 and 100 mM O 2 revealed dioxygen ingress and egress as well as rotational and translational motions of O 2 in the cavities. It is therefore suggested that conformational fluctuations within the ground-state ensemble transiently develop channels for O 2 association with the internal protein cavities., (© 2017 The Protein Society.)

Aims: Bacterial infection is a major challenge in wound care. Antimicrobial wound dressings are of great value for treating wound infections. Endolysins are evolving as a new class of antimicrobials with multiple applications. This study describes the production and evaluation of T4 lysozyme (T4Lyz), product of gene e of the T4 bacteriophage, fused with Cellulose Binding Module (CBM) for facile immobilization to cellulosic wound dressing., Methods and Results: Genes encoding T4Lyz-CBM and T4Lyz were cloned and expressed in Escherichia coli and the enzymes were purified by cation exchange chromatography. While the CBM tag did not alter the optimum pH and stability features of T4Lyz, the lytic activity of the fusion protein was lowered. The bactericidal activity of T4Lyz-CBM, determined by viable count plating assay after 1 h incubation with Micrococcus lysodeikticus was 97·5% with 10 μg ml(-1) , and 99·96% and 95% for E. coli and Pseudomonas mendocina, respectively, with 200 μg ml(-1) enzyme. T4Lyz-CBM was immobilized to wound dressing gauze with a capacity of 5·5 μg mg(-1) matrix, whereas the unmodified T4Lyz did not exhibit any binding. The immobilized protein retained its bactericidal activity against Gram-positive and Gram-negative bacteria. Both free and immobilized T4Lyz-CBM, after heat denaturation, retained their bactericidal activities against Gram-negative bacteria only. The immobilized enzyme exhibited higher stability than the free enzyme when stored in dry form or in the presence of polyol stabilizers., Conclusion: Tagging T4Lyz with CBM provides a facile, irreversible binding to cellulosic wound dressing while retaining its activity. This approach may be suitable even for other antimicrobial enzymes and -peptides., Significance and Impact of the Study: The spread of antibiotic resistance requires innovative strategies for discovery and development of effective antimicrobial alternatives. This report presents a novel strategy for producing antimicrobial wound dressing materials., (© 2016 The Society for Applied Microbiology.)

Conformational change in protein-ligand complexes is widely modeled, but the protein accommodation expected on binding a congeneric series of ligands has received less attention. Given their use in medicinal chemistry, there are surprisingly few substantial series of congeneric ligand complexes in the Protein Data Bank (PDB). Here we determine the structures of eight alkyl benzenes, in single-methylene increases from benzene to n-hexylbenzene, bound to an enclosed cavity in T4 lysozyme. The volume of the apo cavity suffices to accommodate benzene but, even with toluene, larger cavity conformations become observable in the electron density, and over the series two other major conformations are observed. These involve discrete changes in main-chain conformation, expanding the site; few continuous changes in the site are observed. In most structures, two discrete protein conformations are observed simultaneously, and energetic considerations suggest that these conformations are low in energy relative to the ground state. An analysis of 121 lysozyme cavity structures in the PDB finds that these three conformations dominate the previously determined structures, largely modeled in a single conformation. An investigation of the few congeneric series in the PDB suggests that discrete changes are common adaptations to a series of growing ligands. The discrete, but relatively few, conformational states observed here, and their energetic accessibility, may have implications for anticipating protein conformational change in ligand design.

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.

Several variants of T4 lysozyme have been identified that sequester small organic ligands in cavities or clefts. To evaluate potential binding sites for non-polar molecules, we screened a number of hydrophobic large-to-small mutants for stabilization in the presence of benzene. In addition to Leu99-->Ala, binding was indicated for at least five other mutants. Variants Met102-->Ala and Leu133-->Gly, and a crevice mutant, Phe104-->Ala, were further characterized using X-ray crystallography and thermal denaturation. As predicted from the shape of the cavity in the benzene complex, mutant Leu133-->Gly also bound p-xylene. We attempted to enlarge the cavity of the Met102-->Ala mutant into a deep crevice through an additional substitution, but the double mutant failed to bind ligands because an adjacent helix rearranged into a non-helical structure, apparently due to the loss of packing interactions. In general, the protein structure contracted slightly to reduce the volume of the void created by truncating substitutions and expanded upon binding the non-polar ligand, with shifts similar to those resulting from the mutations.A polar molecule binding site was also created by truncating Arg95 to alanine. This creates a highly complementary buried polar environment that can be utilized as a specific "receptor" for a guanidinium ion. Our results suggest that creating a deficiency through truncating mutations of buried residues generates "binding potential" for ligands with characteristics similar to the deleted side-chain. Analysis of complex and apo crystal structures of binding and non-binding mutants suggests that ligand size and shape as well as protein flexibility and complementarity are all determinants of binding. Binding at non-polar sites is governed by hydrophobicity and steric interactions and is relatively permissive. Binding at a polar site is more restrictive and requires extensive complementarity between the ligand and the site.

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).