Mutants Of T4 Lysozyme Research Articles

Site-directed spin labeling of a genetically encoded unnatural amino acid

The traditional site-directed spin labeling (SDSL) method, which utilizes cysteine residues and sulfhydryl-reactive nitroxide reagents, can be challenging for proteins that contain functionally important native cysteine residues or disulfide bonds. To make SDSL amenable to any protein, we introduce an orthogonal labeling strategy, i.e., one that does not rely on any of the functional groups found in the common 20 amino acids. In this method, the genetically encoded unnatural amino acid p-acetyl-L-phenylalanine (p-AcPhe) is reacted with a hydroxylamine reagent to generate a nitroxide side chain (K1). The utility of this scheme was demonstrated with seven mutants of T4 lysozyme, each containing a single p-AcPhe at a solvent-exposed helix site; the mutants were expressed in amounts qualitatively similar to the wild-type protein. In general, the EPR spectra of the resulting K1 mutants reflect higher nitroxide mobilities than the spectra of analogous mutants containing the more constrained disulfide-linked side chain (R1) commonly used in SDSL. Despite this increased flexibility, site dependence of the EPR spectra suggests that K1 will be a useful sensor of local structure and of conformational changes in solution. Distance measurements between pairs of K1 residues using double electron electron resonance (DEER) spectroscopy indicate that K1 will also be useful for distance mapping.

A topical issue: NMR investigations of molecular dynamics

The 20 papers in this topical issue of the Journal of Biomolecular NMR reflect the multitude of NMR techniques and approaches used to investigate conformational dynamics critical for folding and stability, molecular recognition, and catalysis of proteins, nucleic acids, and other biological molecules. Of course, any single issue of a journal cannot include a selection of papers comprehensive enough to truly encompass the diversity of developments and applications in a field that continues to expand as rapidly and creatively as NMR spectroscopy. Many alternative selections of papers could have been made equally well and no doubt, indeed one hopes, the work described in this topical issue will inspire developments of increasingly powerful techniques and applications to increasingly deeper questions in biology. New experimental methods drive biological investigations by NMR spectroscopy and several papers in this topical issue present new techniques or novel combinations of existing methods in solution NMR spectroscopy. The Carr-Purcell-Meiboom-Gill (CPMG) pulse sequence has emerged as a critical method for investigating conformational dynamics in biological macromolecules. Most investigations of conformational dynamics of protein backbones have used IS spin systems, such as H–N or methine H–C, as probes, for example, see the paper by Raleigh, Palmer and coworkers described below. To overcome this limitation, Kay and coworkers introduce new CPMG relaxation dispersion experiments for H and C spins in Gly residues (Vallurupalli et al. 2009). The new methods are applied to a mutant of T4 lysozyme that binds hydrophobic ligands in an internal cavity in the protein. Existing theoretical analyses of relaxation during CPMG pulse trains were derived assuming infinitely short ideal refocusing pulses. Myint and Ishima provide a critical assessment of the effects of evolution and relaxation during real finite-length refocusing pulses of the CPMG pulse train (Myint and Ishima 2009). Field cycling enables measurements site-specific relaxation rate constants at low static magnetic fields with the resolution and sensitivity of a high field NMR spectrometer. Redfield, Kern, and coworkers obtain R1 relaxation rate constants at fields as low as 4 T for the nucleocapsid protein of the SARS coronavirus using a home-built field cycling instrument (Clarkson et al. 2009). Together with conventional high field measurements, these data suggest correlated rigid body motions of structural motifs of the protein. Skrynnikov and coworkers use glycerol to increase solution viscosity and thereby increase the rotational diffusion correlation time of the a-spectrin SH3 domain. The longer rotational correlation time extends the time window to which NMR relaxation methods are sensitive to internal motional processes (Xu et al. 2009). Hydrogen exchange between amide moieties and solvent is a longstanding and powerful approach for studying folding and thermodynamics of proteins. However, for large molecules, even the resolution in H–N two-dimensional correlation spectra becomes limiting and three-dimensional spectra cannot be acquired rapidly enough using conventional Cartesian sampling schemes. Wand and coworkers introduce a new method, called AMORE-HX, that uses radial sampling of a three-dimensional HNCO experiment to determine amide hydrogen exchange rates in real time (Gledhill et al. 2009). They show that radial sampling with four angles (69 , 81 , 10 , 27 ) can resolve 328 of the 334 cross peaks that remain A. G. Palmer III (&) Department of Biochemistry and Molecular Biophysics, Columbia University, 630 West 168th Street, New York, NY 10032, USA e-mail: agp6@columbia.edu

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.

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.

CPMG relaxation dispersion NMR experiments measuring glycine 1Hα and 13Cα chemical shifts in the ‘invisible’ excited states of proteins

Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR experiments are extremely powerful for characterizing millisecond time-scale conformational exchange processes in biomolecules. A large number of such CPMG experiments have now emerged for measuring protein backbone chemical shifts of sparsely populated (>0.5%), excited state conformers that cannot be directly detected in NMR spectra and that are invisible to most other biophysical methods as well. A notable deficiency is, however, the absence of CPMG experiments for measurement of (1)H(alpha) and (13)C(alpha) chemical shifts of glycine residues in the excited state that reflects the fact that in this case the (1)H(alpha), (13)C(alpha) spins form a three-spin system that is more complex than the AX (1)H(alpha)-(13)C(alpha) spin systems in the other amino acids. Here pulse sequences for recording (1)H(alpha) and (13)C(alpha) CPMG relaxation dispersion profiles derived from glycine residues are presented that provide information from which (1)H(alpha), (13)C(alpha) chemical shifts can be obtained. The utility of these experiments is demonstrated by an application to a mutant of T4 lysozyme that undergoes a millisecond time-scale exchange process facilitating the binding of hydrophobic ligands to an internal cavity in the protein.

Calculation of the EPR Spectrum of a Small Nitroxide from Molecular Dynamics Simulations

Algorithms have been developed for calculating the continuous wave electron paramagnetic resonance (EPR) spectrum of a nitroxide spin label from a free-induction decay (FID) derived from a molecular dynamics (MD) simulation. These algorithms have been successfully used to simulate the EPR spectra of two spin-labeled mutants of T4 lysozyme, T4L F153R1 and T4L K65R1 [DeSensi et al., Biophys. J. 94, 3798-3809 (2008)]. In this previous work, MD simulations were performed using the AMBER suite of programs, the all-atom AMBER99 force field, the particle mesh Ewald (PME) method for treating long-range electrostatic interactions, and the SPC/E water model. In order to test the effect of the water model used in the AMBER-based molecular dynamics simulations, calculations for a small nitroxide [3-hydroxymethyl-(1-oxy-2,2,5,5-tetramethylpyrroline) or 3MeOHSL] are being performed using a variety of different water models (SPC/E, TIP3P, TIP4P). The EPR spectra calculated from these molecular dynamics simulations of 3MeOHSL will be compared to experimental data.

Structural and Thermodynamic Characterization of T4 Lysozyme Mutants and the Contribution of Internal Cavities to Pressure Denaturation

Structural and Thermodynamic Characterization of T4 Lysozyme Mutants and the Contribution of Internal Cavities to Pressure Denaturation

Free-solution label-free detection of alpha-crystallin chaperone interactions by back-scattering interferometry.

We report the quantitative, label-free analysis of protein-protein interactions in free solution within picoliter volumes using backscatter interferometry (BSI). Changes in the refractive index are measured for solutions introduced on a PDMS microchip allowing determination of forward and reverse rate constants for two-mode binding. Time-dependent BSI traces are directly fit using a global analysis approach to characterize the interaction of the small heat-shock protein alpha-Crystallin with two substrates: destabilized mutants of T4 lysozyme and the in vivo target betaB1-Crystallin. The results recapitulate the selectivity of alphaB-Crystallin differentially binding T4L mutants according to their free energies of unfolding. Furthermore, we demonstrate that an alphaA-Crystallin mutant linked to hereditary cataract has activated binding to betaB1-Crystallin. Binding isotherms obtained from steady-state values of the BSI signal yielded meaningful dissociation constants and establishes BSI as a novel tool for the rapid identification of molecular partners using exceedingly small sample quantities under physiological conditions. This work demonstrates that BSI can be extended to screen libraries of disease-related mutants to quantify changes in affinity and/or kinetics of binding.

Computations of standard binding free energies with molecular dynamics simulations.

An increasing number of studies have reported computations of the standard (absolute) binding free energy of small ligands to proteins using molecular dynamics (MD) simulations and explicit solvent molecules that are in good agreement with experiments. This encouraging progress suggests that physics-based approaches hold the promise of making important contributions to the process of drug discovery and optimization in the near future. Two types of approaches are principally used to compute binding free energies with MD simulations. The most widely known is the alchemical double decoupling method, in which the interaction of the ligand with its surroundings are progressively switched off. It is also possible to use a potential of mean force (PMF) method, in which the ligand is physically separated from the protein receptor. For both of these computational approaches, restraining potentials may be activated and released during the simulation for sampling efficiently the changes in translational, rotational, and conformational freedom of the ligand and protein upon binding. Because such restraining potentials add bias to the simulations, it is important that their effects be rigorously removed to yield a binding free energy that is properly unbiased with respect to the standard state. A review of recent results is presented, and differences in computational methods are discussed. Examples of computations with T4-lysozyme mutants, FKBP12, SH2 domain, and cytochrome P450 are discussed and compared. Remaining difficulties and challenges are highlighted.

Progress in ab initio QM/MM free-energy simulations of electrostatic energies in proteins: accelerated QM/MM studies of pKa, redox reactions and solvation free energies.

Hybrid quantum mechanical/molecular mechanical (QM/MM) approaches have been used to provide a general scheme for chemical reactions in proteins. However, such approaches still present a major challenge to computational chemists, not only because of the need for very large computer time in order to evaluate the QM energy but also because of the need for proper computational sampling. This review focuses on the sampling issue in QM/MM evaluations of electrostatic energies in proteins. We chose this example since electrostatic energies play a major role in controlling the function of proteins and are key to the structure-function correlation of biological molecules. Thus, the correct treatment of electrostatics is essential for the accurate simulation of biological systems. Although we will be presenting different types of QM/MM calculations of electrostatic energies (and related properties) here, our focus will be on pKa calculations. This reflects the fact that pKa's of ionizable groups in proteins provide one of the most direct benchmarks for the accuracy of electrostatic models of macromolecules. While pKa calculations by semimacroscopic models have given reasonable results in many cases, existing attempts to perform pKa calculations using QM/MM-FEP have led to discrepancies between calculated and experimental values. In this work, we accelerate our QM/MM calculations using an updated mean charge distribution and a classical reference potential. We examine both a surface residue (Asp3) of the bovine pancreatic trypsin inhibitor and a residue buried in a hydrophobic pocket (Lys102) of the T4-lysozyme mutant. We demonstrate that, by using this approach, we are able to reproduce the relevant side chain pKa's with an accuracy of 3 kcal/mol. This is well within the 7 kcal/mol energy difference observed in studies of enzymatic catalysis, and is thus sufficient accuracy to determine the main contributions to the catalytic energies of enzymes. We also provide an overall perspective of the potential of QM/MM calculations in general evaluations of electrostatic free energies, pointing out that our approach should provide a very powerful and accurate tool to predict the electrostatics of not only solution but also enzymatic reactions, as well as the solvation free energies of even larger systems, such as nucleic acid bases incorporated into DNA.

Calculation of absolute protein-ligand binding free energy using distributed replica sampling

Distributed replica sampling [T. Rodinger et al., J. Chem. Theory Comput. 2, 725 (2006)] is a simple and general scheme for Boltzmann sampling of conformational space by computer simulation in which multiple replicas of the system undergo a random walk in reaction coordinate or temperature space. Individual replicas are linked through a generalized Hamiltonian containing an extra potential energy term or bias which depends on the distribution of all replicas, thus enforcing the desired sampling distribution along the coordinate or parameter of interest regardless of free energy barriers. In contrast to replica exchange methods, efficient implementation of the algorithm does not require synchronicity of the individual simulations. The algorithm is inherently suited for large-scale simulations using shared or heterogeneous computing platforms such as a distributed network. In this work, we build on our original algorithm by introducing Boltzmann-weighted jumping, which allows moves of a larger magnitude and thus enhances sampling efficiency along the reaction coordinate. The approach is demonstrated using a realistic and biologically relevant application; we calculate the standard binding free energy of benzene to the L99A mutant of T4 lysozyme. Distributed replica sampling is used in conjunction with thermodynamic integration to compute the potential of mean force for extracting the ligand from protein and solvent along a nonphysical spatial coordinate. Dynamic treatment of the reaction coordinate leads to faster statistical convergence of the potential of mean force than a conventional static coordinate, which suffers from slow transitions on a rugged potential energy surface.

The analysis of T4 lysozyme mutants to get an insight of activity enhancement

The analysis of T4 lysozyme mutants to get an insight of activity enhancement

Structural and Thermodynamic Characterization of T4 Lysozyme Mutants and the Contribution of Internal Cavities to Pressure Denaturation

Using small-angle X-ray scattering (SAXS) and tryptophan fluorescence spectroscopy, we have identified multiple compact denatured states of a series of T4 lysozyme mutants that are stabilized by high pressures. Recent studies imply that the mechanism of pressure denaturation is the penetration of water into the protein rather than the transfer of hydrophobic residues into water. To investigate water penetration and the volume change associated with pressure denaturation, we studied the solution behavior of four T4 lysozyme mutants having different cavity volumes at low and neutral pH up to a pressure of 400 MPa (0.1 MPa = 0.9869 atm). At low pH, L99A T4 lysozyme expanded from a compact folded state to a partially unfolded state with a corresponding change in radius of gyration from 17 to 32 A. The volume change upon denaturation correlated well with the total cavity volume, indicating that all of the molecule's major cavities are hydrated with pressure. As a direct comparison to high-pressure crystal structures of L99A T4 lysozyme solved at neutral pH [Collins, M. D., Hummer, G., Quillin, M. L., Matthews, B. W., and Gruner, S. M. (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 16668-16671], pressure denaturation of L99A and the structurally similar L99G/E108V mutant was studied at neutral pH. The pressure-denatured state at neutral pH is even more compact than at low pH, and the small volume changes associated with denaturation suggest that the preferential filling of large cavities is responsible for the compactness of the pressure-denatured state. These results confirm that pressure denaturation is characteristically distinct from thermal or chemical denaturation.

Use of experimental crystallographic phases to examine the hydration of polar and nonpolar cavities in T4 lysozyme

There is conflicting evidence as to whether cavities in proteins that are nonpolar and large enough to accommodate solvent are empty or are occupied by disordered water molecules. Here, we use multiple-wavelength x-ray data collected from crystals of the selenomethionine-substituted L99A/M102L mutant of T4 lysozyme to obtain a high-resolution electron density map free of bias that is unavoidably associated with conventional model-based structure determination and refinement. The mutant, L99A/M102L, has four cavities, two being polar in character and the other two nonpolar. Cavity 1 (polar, volume 45.2 A(3)) was expected to contain two well ordered water molecules, and this is confirmed in the experimental electron density map. Likewise, cavity 2 (polar, 16.9 A(3)) is confirmed to contain a single water molecule. Cavity 3 (nonpolar, 21.4 A(3)) was seen to be empty in conventional x-ray refinement, and this is confirmed in the experimental map. Unexpectedly, however, cavity 4 (nonpolar, volume 133.5 A(3)) was seen to contain diffuse electron density equivalent to approximately 1.5 water molecules. Although cavity 4 is largely nonpolar, it does have some polar character, and this apparently contributes to the presence of solvent. The cavity is large enough to accommodate four to five water molecules, and it appears that a hydrogen-bonded chain of three or more solvent molecules could occupy the cavity at a given time. The results are consistent with theoretical predictions that cavities in proteins that are strictly nonpolar will not contain solvent until the volume is large enough to permit mutually satisfying water-water hydrogen bonds.

Simulation of Nitroxide Electron Paramagnetic Resonance Spectra from Brownian Trajectories and Molecular Dynamics Simulations

A simulated continuous wave electron paramagnetic resonance spectrum of a nitroxide spin label can be obtained from the Fourier transform of a free induction decay. It has been previously shown that the free induction decay can be calculated by solving the time-dependent stochastic Liouville equation for a set of Brownian trajectories defining the rotational dynamics of the label. In this work, a quaternion-based Monte Carlo algorithm has been developed to generate Brownian trajectories describing the global rotational diffusion of a spin-labeled protein. Also, molecular dynamics simulations of two spin-labeled mutants of T4 lysozyme, T4L F153R1, and T4L K65R1 have been used to generate trajectories describing the internal dynamics of the protein and the local dynamics of the spin-label side chain. Trajectories from the molecular dynamics simulations combined with trajectories describing the global rotational diffusion of the protein are used to account for all of the dynamics of a spin-labeled protein. Spectra calculated from these combined trajectories correspond well to the experimental spectra for the buried site T4L F153R1 and the helix surface site T4L K65R1. This work provides a framework to further explore the modeling of the dynamics of the spin-label side chain in the wide variety of labeling environments encountered in site-directed spin labeling studies.

1P-039 3次元RISM理論に基づくT4リゾチーム変異体におけるベンゼン結合部位の理論的同定(蛋白質・構造機能相関(1),第46回日本生物物理学会年会)

1P-039 3次元RISM理論に基づくT4リゾチーム変異体におけるベンゼン結合部位の理論的同定(蛋白質・構造機能相関(1),第46回日本生物物理学会年会)

Structure and Orientation of T4 Lysozyme Bound to the Small Heat Shock Protein α-Crystallin

We have determined the structural changes that accompany the formation of a stable complex between a destabilized mutant of T4 lysozyme (T4L) and the small heat shock protein α-crystallin. Using pairs of fluorescence or spin label probes to fingerprint the T4L tertiary fold, we demonstrate that binding disrupts tertiary packing in the two domains as well as across the active-site cleft. Furthermore, increased distances between i and i + 4 residues of helices support a model in which the bound structure is not native-like but significantly unfolded. In the confines of the oligomer, T4L has a preferential orientation with residues in the more hydrophobic C-terminal domain sequestered in a buried environment, while residues in the N-terminal domain are exposed to the aqueous solvent. Furthermore, electron paramagnetic resonance spectral line shapes of sites in the N-terminal domain are narrower than in the folded, unbound T4L reflecting an unstructured backbone and an asymmetric pattern of contacts between T4L and α-crystallin. The net orientation is not affected by the location of the destabilizing mutation consistent with the notion that binding is not triggered by recognition of localized unfolding. Together, the structural and thermodynamic data indicate that the stably bound conformation of T4L is unfolded and support a model in which the two modes of substrate binding originate from two discrete binding sites on the chaperone.

Accurate prediction of enzyme mutant activity based on a multibody statistical potential

An important area of research in biochemistry and molecular biology focuses on characterization of enzyme mutants. However, synthesis and analysis of experimental mutants is time consuming and expensive. We describe a machine-learning approach for inferring the activity levels of all unexplored single point mutants of an enzyme, based on a training set of such mutants with experimentally measured activity. Based on a Delaunay tessellation-derived four-body statistical potential function, a perturbation vector measuring environmental changes relative to wild type (wt) at every residue position uniquely characterizes each enzyme mutant for model development and prediction. First, a measure of model performance utilizing area (AUC) under the receiver operating characteristic (ROC) curve surpasses 0.83 and 0.77 for data sets of experimental HIV-1 protease and T4 lysozyme mutants, respectively. Additionally, a novel method is introduced for evaluating statistical significance associated with the number of correct test set predictions obtained from a trained model. Third, 100 stratified random splits of the protease and T4 lysozyme mutant data sets into training and test sets achieve 77.0% and 80.8% mean accuracy, respectively. Next, protease and T4 lysozyme models trained with experimental mutants are used to predict activity levels for all remaining mutants; a subsequent search for publications reporting on dozens of these test mutants reveals that experimental results are matched by 79% and 86% of predictions, respectively. Finally, learning curves for each mutant enzyme system indicate the influence of training set size on model performance. Prediction databases at http://proteins.gmu.edu/automute/

Distance Measurements by Fluorescence Energy Homotransfer: Evaluation in T4 Lysozyme and Correlation with Dipolar Coupling between Spin Labels

We demonstrate the feasibility and practical limitations of using steady-state anisotropy to determine distances from fluorescence homotransfer in the context of a protein of known crystal structure. Eight double mutants of T4 lysozyme spanning the distance range between 20 Å and 50 Å were labeled with a methanethiosulfonate derivative of fluorescein. The measured distances in liquid solution are in agreement with those determined from dipolar coupling between spin labels in the frozen state. They can be interpreted in the context of the crystal structure after accounting for the probe linking arm. Overall, the results establish the necessary calibration for this spectroscopic ruler. The measurement of similar distance trends using independent probes sets the stage for the complementary use of homotransfer and dipolar coupling in the determination of static structures and detection of conformational changes.

2P035 キャビティに基づく構造揺らぎの検出 : T4リゾチーム変異体の可変圧力NMR(蛋白質(構造・構造機能相関),ポスター発表,第45回日本生物物理学会年会)

2P035 キャビティに基づく構造揺らぎの検出 : T4リゾチーム変異体の可変圧力NMR(蛋白質(構造・構造機能相関),ポスター発表,第45回日本生物物理学会年会)