Structure Of T4 Lysozyme Research Articles

The investigation of fluorine substitution in difluoroanilines with focus on 2,6-difluoroaniline by spectroscopic methods, density functional theory approach, and molecular docking

In this study, the spectroscopic features and reactive nature of difluoroanilines, with special attention on 2,6-difluoroaniline (2,6-DFA), are explored thoroughly to gain insight into the effect of fluorine substitution in aniline derivatives. The quantum chemically obtained spectral properties including Fourier transform infrared, Raman, ultraviolet, and 1H and 13C nuclear magnetic resonance spectra were obtained by using Density Functional Theory (DFT) calculations at B3LYP/6-311++G(d,p) level of theory, and given along with the ones measured experimentally. The reactivity characteristics such as Fukui descriptor, electrostatic potential, and electronegativity were defined based on electron density generated theoretically. The position of fluorine substitution does not significantly alter the amide group reactivity making all difluoroanilines compatible to each other in terms of the reactivity. The time-dependent DFT was used to calculate the electronic properties such as excitation energies, maximum wavelengths, oscillator strengths, and molecular orbitals energies, and given along with the density of state diagrams. The intramolecular interactions were mapped by reduced density gradient method to reveal possible fluorine influence on the amide group and its reactivity, and the strongest van der Waals interactions in 2,6-DFA were observed. Besides, the thermodynamic and nonlinear optical properties of the title molecule were also investigated. The 2,6-DFA molecule seems to be appealing for multidisciplinary studies about the relevant nonlinear optical properties. To explore the binding capability of 2,6-DFA and see the influence of fluorine substitution in difluoroanilines, molecular docking was performed on a model enzyme structure of T4 lysozyme. Ligand-enzyme binding energies of 2,6-DFA is nearly the same with that of 2,5 DFA and 3,5 DFA at the same binding site which differs from the remaining derivatives.

Optimal Tikhonov regularization for DEER spectroscopy

Tikhonov regularization is the most commonly used method for extracting distance distributions from experimental double electron-electron resonance (DEER) spectroscopy data. This method requires the selection of a regularization parameter, α, and a regularization operator, L. We analyze the performance of a large set of α selection methods and several regularization operators, using a test set of over half a million synthetic noisy DEER traces. These are generated from distance distributions obtained from in silico double labeling of a protein crystal structure of T4 lysozyme with the spin label MTSSL. We compare the methods and operators based on their ability to recover the model distance distributions from the noisy time traces. The results indicate that several α selection methods perform quite well, among them the Akaike information criterion and the generalized cross validation method with either the first- or second-derivative operator. They perform significantly better than currently utilized L-curve methods.

Interaction-component analysis of the effects of urea and its alkylated derivatives on the structure of T4-lysozyme.

The effects of urea and its alkylated derivatives on the structure of T4-lysozyme were analyzed from the standpoint of energetics. Molecular dynamics simulations were conducted with explicit solvent, and the energy-representation method was employed to compute the free energy of transfer of the protein from pure-water solvent to the mixed solvents of water with urea, methylurea, 1,1-dimethylurea, and isopropylurea. Through the decomposition of the transfer free energy into the cosolvent and water contributions, it was observed that the former is partially cancelled by the latter and governs the total free energy of transfer. To determine the interaction component responsible for the transfer energetics, the correlations of the transfer free energy were also examined against the change in the solute-solvent interaction energy upon transfer and the corresponding changes in the electrostatic, van der Waals, and excluded-volume components. It was then found over the set of protein structures ranging from native to (partially) unfolded ones that the transfer free energy changes in parallel with the van der Waals component even when the cosolvent is alkylated. The electrostatic and excluded-volume components play minor roles in the structure modification of the protein, and the denaturing ability of alkylurea is brought by the van der Waals interaction.

Analysis of the solution conformations of T4 lysozyme by paramagnetic NMR spectroscopy.

A large number of crystal structures of bacteriophage T4 lysozyme (T4-L) have shown that it contains two subdomains, which can arrange in a compact conformation (closed state) or, in mutants of T4-L, more extended structures (open state). In solution, wild-type T4-L displays only a single set of nuclear magnetic resonance (NMR) signals, masking any conformational heterogeneity. To probe the conformational space of T4-L, we generated a site-specific lanthanide binding site by attaching 4-mercaptomethyl dipicolinic acid via a disulfide bond to Cys44 in the triple-mutant C54T/C97A/S44C of T4-L and measured pseudocontact shifts (PCS) and magnetically induced residual dipolar couplings (RDC). The data indicate that, in solution and in the absence of substrate, the structure of T4-L is on average more open than suggested by the closed conformation of the crystal structure of wild-type T4-L. A slightly improved fit was obtained by assuming a population-weighted two-state model involving an even more open conformation and the closed state, but paramagnetic relaxation enhancements measured with Gd(3+) argue against such a conformational equilibrium. The fit could not be improved by including a third conformation picked from the hundreds of crystal structures available for T4-L mutants.

RosettaEPR: Rotamer Library for Spin Label Structure and Dynamics

An increasingly used parameter in structural biology is the measurement of distances between spin labels bound to a protein. One limitation to these measurements is the unknown position of the spin label relative to the protein backbone. To overcome this drawback, we introduce a rotamer library of the methanethiosulfonate spin label (MTSSL) into the protein modeling program Rosetta. Spin label rotamers were derived from conformations observed in crystal structures of spin labeled T4 lysozyme and previously published molecular dynamics simulations. Rosetta’s ability to accurately recover spin label conformations and EPR measured distance distributions was evaluated against 19 experimentally determined MTSSL labeled structures of T4 lysozyme and the membrane protein LeuT and 73 distance distributions from T4 lysozyme and the membrane protein MsbA. For a site in the core of T4 lysozyme, the correct spin label conformation (Χ1 and Χ2) is recovered in 99.8% of trials. In surface positions 53% of the trajectories agree with crystallized conformations in Χ1 and Χ2. This level of recovery is on par with Rosetta performance for the 20 natural amino acids. In addition, Rosetta predicts the distance between two spin labels with a mean error of 4.4 Å. The width of the experimental distance distribution, which reflects the flexibility of the two spin labels, is predicted with a mean error of 1.3 Å. RosettaEPR makes full-atom spin label modeling available to a wide scientific community in conjunction with the powerful suite of modeling methods within Rosetta.

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

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

T4 Lysozyme as a Pac-Man: How Fast Can It Chew?

T4 Lysozyme as a Pac-Man: How Fast Can It Chew?

Room Temperature Electron Spin Resonance Distance Measurements in T4 Lysozyme using Trityl-Based Spin Labels

Pulsed Electron Spin Resonance (ESR) spectroscopy in combination with site-directed spin labeling is unique in providing nanometer- range distances and distributions in biological systems. Distance constraints are valuable for determining structure and structural changes, while distance distributions reflect structural heterogeneity. To date, most of the pulsed ESR techniques require cryogenic temperatures to reduce the rapid electron spin relaxation rate and to prevent averaging of electron-electron dipolar interaction due to the rapid molecular tumbling. To enable measurements at physiological temperatures, we are exploring a trityl-based spin label with a relatively long relaxation time where the protein is immobilized by attachment to a solid support. In this preliminary study, trityl radicals were attached via disulfide linkages to substituted cysteine residues at positions 65 and 80 or at positions 65 and 76 in T4 lysozyme (T4L) immobilized on Sepharose. Double quantum coherence (DQC) was applied to the two spin-labeled double mutants at room temperature. Distances extracted from DQC show a 2.2 nm and a 1.8 nm spin-spin distance, respectively, close to those expected from models of the trityl label in the T4L structure. Narrow distribution of distances was observed in each case, indicating that the trityl spin label is relatively rigid. The results show that, with the use of trityl spin label and DQC, structural constraints can be obtained to study protein structure/dynamics at physiological temperature. Furthermore, as we show, trityl labels enable micromolar sensitivity DQC distance measurements in the temperature range of 60-100 K, since their distant methyl groups only weakly contribute to the relaxation of electron spins. As an additional advantage less intense microwave pulses are needed as compared to nitroxides.

Integrated analysis of the conformation of a protein-linked spin label by crystallography, EPR and NMR spectroscopy

Long-range structural information derived from paramagnetic relaxation enhancement observed in the presence of a paramagnetic nitroxide radical is highly useful for structural characterization of globular, modular and intrinsically disordered proteins, as well as protein–protein and protein-DNA complexes. Here we characterized the conformation of a spin-label attached to the homodimeric protein CylR2 using a combination of X-ray crystallography, electron paramagnetic resonance (EPR) and NMR spectroscopy. Close agreement was found between the conformation of the spin label observed in the crystal structure with interspin distances measured by EPR and signal broadening in NMR spectra, suggesting that the conformation seen in the crystal structure is also preferred in solution. In contrast, conformations of the spin label observed in crystal structures of T4 lysozyme are not in agreement with the paramagnetic relaxation enhancement observed for spin-labeled CylR2 in solution. Our data demonstrate that accurate positioning of the paramagnetic center is essential for high-resolution structure determination.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-011-9471-y) contains supplementary material, which is available to authorized users.

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

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

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

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

Structural Changes of T4 Lysozyme upon Adsorption to Silica Nanoparticles Measured by Circular Dichroism

The change in the secondary structure of T4 lysozyme upon adsorption to silica particles was studied with circular dichroism. Two different mutants of the protein along with the wild type were investigated. The mutants differ from wild type by substitution of isoleucine for cysteine or tryptophan at position 3 and were chosen to represent a range of stability as quantified by their energies of thermal unfolding. The mutants differ in ΔG, at 65°C and pH 6.5, compared to the wild-type enzyme with -2.8 and 1.2 kcal/mol for the tryptophan and cysteine mutants, respectively. After adsorption to 9-nm silica nanoparticles for 90 min, a large change in the spectrum was observed for the less stable tryptophan mutant, while the changes were smaller for the wild type and the cysteine mutant. The spectral changes before and after adsorption corresponded to a calculated loss of α-helix of 12% for the wild type, 9% for the cysteine mutant, and 29% for the tryptophan mutant. Structural changes during adsorption of the proteins were also followed kinetically at 222 nm. The rate of conformational change differed among the three proteins and was fastest for the tryptophan mutant. In the case of the tryptophan mutant the time required for half of the measured change to occur was approximately 5 min, while for the cysteine mutant and the wild-type T4 lysozyme more than 10 min was required.

CD and NMR determination of the solution structure of a peptide corresponding to T4 lysozyme residues 38–51

Solid phase methods have been used to synthesise a peptide corresponding to residues 38–51 of T4 lysozyme. The peptide, LYS(38–51), encompasses helix B in the crystal structure of T4 lysozyme. CD and 1H-NMR analysis showed that the peptide was unstructured in aqueous solution but adopted a helical conformation in the more hydrophobic environment provided by 50% TFE and SDS micelles. The solution structure derived from the NMR data was similar to that of the helix in the X-ray structure, although there was some fraying at the N-terminus.

Genetic analysis of bacteriophage T4 lysozyme structure and function

Genetic analysis of bacteriophage T4 lysozyme structure and function

Stability and solvation of Thr/Ser to Ala and Gly mutations at the N-cap of α-helices

The solvation of polar groups at the N-terminal end of α-helices was studied by comparing the crystal structures of T4 lysozyme, barley chymotrypsin inhibitor 2 (CI2), barnase and their respective N-cap mutants. Whether or not the N3 residue is solvated on mutating the N-cap Thr/Ser to Ala or Gly appears to be related to the identities and the side-chain conformations of the N2 and N3 residues. When these two residues are alanines, as is in the pseudo-wild-type CI2 (E33A/E34A), the main-chain NH at the N3 position is exposed to the solvent and can be solvated. If the N2 residue is an Asp or a Glu, it is more likely that the side-chain of these residues will form a surrogate N-cap with the amide NH at N3 to compensate for the lost -OH group. In this case, no additional solvation will be observed. In general, Gly can be more stable than Ala at the N-cap because its small side-chain allows nearby polar groups to form hydrogen bonds with optimal geometry with solvent molecules or other polar groups.

Systematic mutation of bacteriophage T4 lysozyme

Amber mutations were introduced into every codon (except the initiating AUG) of the bacteriophage T4 lysozyme gene. The amber alleles were introduced into a bacteriophage P22 hybrid, called P22 e416, in which the normal P22 lysozyme gene is replaced by its T4 homologue, and which consequently depends upon T4 lysozyme for its ability to form a plaque. The resulting amber mutants were tested for plaque formation on amber suppressor strains of Salmonella typhimurium. Experiments with other hybrid phages engineered to produce different amounts of wild-type T4 lysozyme have shown that, to score as deleterious, a mutation must reduce lysozyme activity to less than 3% of that produced by wild-type P22 e416. Plating the collection of amber mutants covering 163 of the 164 codons of T4 lysozyme, on 13 suppressor strains that each insert a different amino acid residue in response to the amber codon, tests the effects of multiple single amino acid substitutions at every position in the protein (except the first). Of the resulting 2015 single amino acid substitutions in T4 lysozyme, 328 were found to be sufficiently deleterious to inhibit plaque formation. More than half (55%) of the positions in the protein tolerated all substitutions examined. Among (N-terminal) amber fragments, only those of 161 or more residues are active. The effects of many of the deleterious substitutions are interpretable in light of the known structure of T4 lysozyme. Residues in the molecule that are refractory to replacements generally have solvent-inaccessible side-chains; the catalytic Glu11 and Asp20 residues are notable exceptions. Especially sensitive sites include residues involved in buried salt bridges near the catalytic site (Asp10, Arg145 and Arg148) and a few others that may have critical structural roles (Gly30, Trp138 and Tyr161).

Comparison of the crystal structure of bacteriophage T4 lysozyme at low, medium, and high ionic strengths

Crystals of bacteriophage T4 lysozyme used for structural studies are routinely grown from concentrated phosphate solutions. It has been found that crystals in the same space group can also be grown from solutions containing 0.05 M imidazole chloride, 0.4 M sodium choride, and 30% polyethylene glycol 3500. These crystals, in addition, can also be equilibrated with a similar mother liquor in which the sodium chloride concentration is reduced to 0.025 M. The availability of these three crystal variants has permitted the structure of T4 lysozyme to be compared at low, medium, and high ionic strength. At the same time the X-ray structure of phage T4 lysozyme crystallized from phosphate solutions has been further refined against a new and improved X-ray diffraction data set. The structures of T4 lysozyme in the crystals grown with polyethylene glycol as a precipitant, regardless of the sodium chloride concentration, were very similar to the structure in crystals grown from concentrated phosphate solutions. The main differences are related to the formation of mixed disulfides between cysteine residues 54 and 97 and 2-mercaptoethanol, rather than to the differences in the salt concentration in the crystal mother liquor. Formation of the mixed disulfide at residue 54 resulted in the displacement of Arg-52 and the disruption of the salt bridge between this residue and Glu-62. Other than this change, no obvious alterations in existing salt bridges in T4 lysozyme were observed. Neither did the reduction in the ionic strength of the mother liquor result in the formation of new salt bridge interactions. These results are consistent with the ideas that a crystal structure determined at high salt concentrations is a good representation of the structure at lower ionic strengths, and that models of electrostatic interactions in proteins that are based on crystal structures determined at high salt concentrations are likely to be relevant at physiological ionic strengths.

Assignment of the backbone proton and nitrogen-15 NMR resonances of bacteriophage T4 lysozyme

The proton and nitrogen (15NH-H alpha-H beta) resonances of bacteriophage T4 lysozyme were assigned by 15N-aided 1H NMR. The assignments were directed from the backbone amide 1H-15N nuclei, with the heteronuclear single-multiple-quantum coherence (HSMQC) spectrum of uniformly 15N enriched protein serving as the master template for this work. The main-chain amide 1H-15N resonances and H alpha resonances were resolved and classified into 18 amino acid types by using HMQC and 15N-edited COSY measurements, respectively, of T4 lysozymes selectively enriched with one or more of alpha-15N-labeled Ala, Arg, Asn, Asp, Gly, Gln, Glu, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val. The heteronuclear spectra were complemented by proton DQF-COSY and TOCSY spectra of unlabeled protein in H2O and D2O buffers, from which the H beta resonances of many residues were identified. The NOE cross peaks to almost every amide proton were resolved in 15N-edited NOESY spectra of the selectively 15N enriched protein samples. Residue specific assignments were determined by using NOE connectivities between protons in the 15NH-H alpha-H beta spin systems of known amino acid type. Additional assignments of the aromatic proton resonances were obtained from 1H NMR spectra of unlabeled and selectively deuterated protein samples. The secondary structure of T4 lysozyme indicated from a qualitative analysis of the NOESY data is consistent with the crystallographic model of the protein.

Structure of phage P22 gene 19 lysozyme inferred from its homology with phage T4 lysozyme: Implications for lysozyme evolution

The amino acid sequence of the lysozyme from phage P22 is shown to be homologous (26% identity) with the lysozyme from bacteriophage T4. The sequence correspondence suggests that the structure of P22 lysozyme is similar to the known structure of T4 lysozyme within the “core” of the molecule, including the active site cleft. However, P22 lysozyme appears to lack two surface loops present in T4 lysozyme. It is possible that P22 lysozyme may provide an “evolutionary link” between the phage-type lysozymes and the goose-type lysozymes.

Mutations and protein stability.

AbstractThe comparative study of proteins which differ in primary structure by point mutations permits one to use thermodynamic experiments to obtain information about the role of specific amino acids in determining protein structure and stability. We have now determined the thermodynamic changes induced in six mutants of T4 lysozyme and have compared the results with the wildtype enzyme. Our work is in collaboration with B. Matthews and his colleagues, who have determined the crystal structure of T4 lysozyme and have obtained difference Fourier maps for four of the mutants. The ultimate aim is to correlate changes in protein stability with changes in the detailed structure of the protein. This paper discusses the thermodynamic results obtained from the mutants studied. All the mutants have a lower Tm than the wild‐type enzyme and changes in the enthalpy of denaturation are sometimes extraordinarily large. Changes in ΔH of denaturation are usually accompanied by compensating changes in ΔS. The general question of protein stability and the manner in which it varies with temperature and mutations is discussed.