Mutants Of T4 Lysozyme Research Articles

Single-Molecule Force Spectroscopy on Unfolded and Intrinsically Disordered Proteins

Transcription factors and cell-signaling proteins contain a hyper abundance of domains or segments that are intrinsically disordered (ID) under native conditions. In many cases, intrinsically disordered proteins (IDPs) or ID segments undergo coupled folding and binding and this transition is associated to its biological role. Many techniques for dissecting folding are not readily amenable to IDPs because they are aggregation prone and insoluble. Single-molecule methods provide an avenue to mitigate these issues. We have performed single-molecule force spectroscopy experiments on a Trimethylamine N-oxide (TMAO) refolded IDP and found that this IDP unfolds cooperatively under mechanical force. The data are rich in information and reveal that what appears to be a two-state transition in bulk is actually multi-state at the single-molecule level. We found that the analysis methods typically employed in such experiments may not be applicable because of the high concentrations of TMAO. To address these issues, we have designed unfolded mutants of T4 lysozyme using a random mutagenesis phenotypic screen. We are actively performing both bulk and single-molecule experiments to characterize unfolded mutants of T4 Lysozyme and TMAO refolded IDPs.

Flexibility analysis of activity-enhanced mutants of bacteriophage T4 lysozyme

Abstract Enzymes are essential biological molecules and widely used in industry. Therefore, investigating the mechanisms underlying how enzyme activity is changed by mutations may have widespread clinical and industrial applications as well as understanding of enzyme catalysis. In this paper, various reported mutants of bacteriophage T4 lysozyme were analyzed to investigate the relationship between enzyme activity and flexibility. The activity-enhanced mutants of bacteriophage T4 lysozyme demonstrated a tendency for mutations to localize to the edge of helices and to involve substitutions to flexible amino acids such as glutamic acid and aspartic acid. Additionally, the mutated residues were shown to demonstrate increased flexibility in B-factor. These findings suggest that flexibility modulation may be a feasible means to enhance enzyme activity.

Cryoelectron Microscopy Analysis of Small Heat Shock Protein 16.5 (Hsp16.5) Complexes with T4 Lysozyme Reveals the Structural Basis of Multimode Binding

Small heat shock proteins (sHSPs) are ubiquitous chaperones that bind and sequester non-native proteins preventing their aggregation. Despite extensive studies of sHSPs chaperone activity, the location of the bound substrate within the sHSP oligomer has not been determined. In this paper, we used cryoelectron microscopy (cryoEM) to visualize destabilized mutants of T4 lysozyme (T4L) bound to engineered variants of the small heat shock protein Hsp16.5. In contrast to wild type Hsp16.5, binding of T4L to these variants does not induce oligomer heterogeneity enabling cryoEM analysis of the complexes. CryoEM image reconstruction reveals the sequestration of T4L in the interior of the Hsp16.5 oligomer primarily interacting with the buried N-terminal domain but also tethered by contacts with the α-crystallin domain shell. Analysis of Hsp16.5-WT/T4L complexes uncovers oligomer expansion as a requirement for high affinity binding. In contrast, a low affinity mode of binding is found to involve T4L binding on the outer surface of the oligomer bridging the formation of large complexes of Hsp16.5. These mechanistic principles were validated by cryoEM analysis of an expanded variant of Hsp16.5 in complex with T4L and Hsp16.5-R107G, which is equivalent to a mutant of human αB-crystallin linked to cardiomyopathy. In both cases, high affinity binding is found to involve conformational changes in the N-terminal region consistent with a central role of this region in substrate recognition.

Aryl Azide Photochemistry in Defined Protein Environments

A genetically encoded precursor to an aryl nitrene, para-azidophenylalanine, was introduced site specifically into proteins to deduce if distinct environments were capable of caging a reactive organic intermediate. Following photolysis of mutant T4 lysozyme or green fluorescent proteins, EPR spectra showed, respectively, the presence of a triplet nitrene and an anilino radical.

Molecular dynamics study of an insertion/duplication mutant of bacteriophage T4 lysozyme reveals the nature of α → β transition in full protein context

An α→β transition underlies the first step of disease causing amyloidogenesis in many proteins. In view of this, many studies have been carried out using peptide models to characterize these secondary structural transitions. In this paper we show that an insertion/duplication mutant 'L20' of bacteriophage T4 lysozyme (M. Sagermann, W. A. Baase and B. W. Matthews, Proc. Natl. Acad. Sci. U.S.A., 1999, 96, 6078) displays an α→β transition. We performed molecular dynamics (MD) simulation of L20, using the GROMACS package of programs and united atom GROMOS 53a6 force field for a time period of 600 ns at 300 K, in explicit water. Our MD simulation demonstrated that the transition occurs in a duplicated α-helical region inserted tandemly at the N-terminus of the 'parent' helix. We show that a C-terminal β-sheet anchors the parent helix while the loosely held N-terminal loop in the duplicate region is vulnerable to solvent attack and thus undergoes an α→β transition. Main chain-solvent interactions were seen to stabilize the observed β-structure. Thus L20 serves as a good protein model for characterization of α→β transition in a full length protein.

Temporal Dynamics and FRET Restrained Modeling of an “Invisible” Excited State of T4 Lysozyme

Conformational fluctuations play a central role in enzyme catalysis. However, extracting a 4D view, i.e. structural changes over time, has represented a big challenge in molecular biophysics. For example, in most cases not all conformers of proteins are visible using the standard structural techniques such as X-ray crystallography or nuclear magnetic resonance. In this work, we present our approach using a fluorescence spectroscopic toolbox to resolve three different conformers of the bacteriophage T4 lysozyme (T4L) and their dynamics. We created a set of more than 20 double mutants specifically labeled with a Forster resonance energy transfer (FRET) pair via the insertion of an unnatural amino acid and a single cysteine. Ensemble time correlated single photon counting (eTCSPC) revealed their corresponding population fractions and provides with structural information. Nevertheless, single molecule FRET, in confocal illumination, showed fluorescence lifetime averaging in timescales faster than diffusion time. To fully characterize the dynamics we used filtered fluorescence correlation spectroscopy which combined with eTCSPC represent a time resolution of seven orders of magnitude (ns to ms). In all, we used the measured distance network to generate a FRET restrained model of the three conformers with high precision. The open conformer appears readily available for substrate binding; the close conformer is very similar to the covalent enzyme-substrate adduct in the T26E mutant of T4L; the third conformer appears more compact than the adduct form which, at present, has not been reported in over more than 440 entries in the protein data bank.

CHARMM-GUI Ligand Binder for Absolute Binding Free Energy Calculations and Its Application

Advanced free energy perturbation molecular dynamics (FEP/MD) simulation methods are available to accurately calculate absolute binding free energies of protein-ligand complexes. However, these methods rely on several sophisticated command scripts implementing various biasing energy restraints to enhance the convergence of the FEP/MD calculations, which must all be handled properly to yield correct results. Here, we present a user-friendly Web interface, CHARMM-GUI Ligand Binder ( http://www.charmm-gui.org/input/gbinding ), to provide standardized CHARMM input files for calculations of absolute binding free energies using the FEP/MD simulations. A number of features are implemented to conveniently set up the FEP/MD simulations in highly customizable manners, thereby permitting an accelerated throughput of this important class of computations while decreasing the possibility of human errors. The interface and a series of input files generated by the interface are tested with illustrative calculations of absolute binding free energies of three nonpolar aromatic ligands to the L99A mutant of T4 lysozyme and three FK506-related ligands to FKBP12. Statistical errors within individual calculations are found to be small (~1 kcal/mol), and the calculated binding free energies generally agree well with the experimental measurements and the previous computational studies (within ~2 kcal/mol). Therefore, CHARMM-GUI Ligand Binder provides a convenient and reliable way to set up the ligand binding free energy calculations and can be applicable to pharmaceutically important protein-ligand systems.

Direct Observation of T4 Lysozyme Hinge-Bending Motion by Fluorescence Correlation Spectroscopy

Bacteriophage T4 Lysozyme (T4L) catalyzes the hydrolysis of the peptidoglycan layer of the bacterial cell wall late in the infection cycle. It has long been postulated that equilibrium dynamics enable substrate access to the active site located at the interface between the N- and C-terminal domains. Crystal structures of WT-T4L and point mutants captured a range of conformations that differ by the hinge-bending angle between the two domains. Evidence of equilibrium between open and closed conformations in solution was gleaned from distance measurements between the two domains but the nature of the equilibrium and the timescale of the underlying motion have not been investigated. Here, we used fluorescence fluctuation spectroscopy to directly detect T4L equilibrium conformational fluctuations in solution. For this purpose, Tetramethylrhodamine probes were introduced at pairs of cysteines in regions of the molecule that undergo relative displacement upon transition from open to closed conformations. Correlation analysis of Tetramethylrhodamine intensity fluctuations reveals hinge-bending motion that changes the relative distance and orientation of the N- and C-terminal domains with ≅15 μs relaxation time. That this motion involves interconversion between open and closed conformations was further confirmed by the dampening of its amplitude upon covalent substrate trapping. In contrast to the prevalent two-state model of T4L equilibrium, molecular brightness and number of particles obtained from cumulant analysis suggest that T4L populates multiple intermediate states, consistent with the wide range of hinge-bending angles trapped in the crystal structure of T4L mutants.

The Role of Adjuvant in Mediating Antigen Structure and Stability

The purpose of this study was to probe the fate of a model antigen, a cysteine-free mutant of bacteriophage T4 lysozyme, to the level of fine structural detail, as a consequence of its interaction with an aluminum (Al)-containing adjuvant. Fluorescence spectroscopy and differential scanning calorimetry were used to compare the thermal stability of the protein in solution versus adsorbed onto an Al-containing adjuvant. Differences in accessible hydrophobic surface areas were investigated using an extrinsic fluorescence probe, 8-Anilino-1-naphthalenesulfonic acid (ANS). As has been observed with other model antigens, the apparent thermal stability of the protein decreased following adsorption onto the adjuvant. ANS spectra suggested that adsorption onto the adjuvant caused an increase in exposure of hydrophobic regions of the protein. Electrostatic interactions drove the adsorption, and disruption of these interactions with high ionic strength buffers facilitated the collection of two-dimensional (15) N heteronuclear single quantum coherence nuclear magnetic resonance data of protein released from the adjuvant. Although the altered stability of the adsorbed protein suggested changes to the protein's structure, the fine structure of the desorbed protein was nearly identical to the protein's structure in the adjuvant-free formulation. Thus, the adjuvant-induced changes to the protein that were responsible for the reduced thermal stability were not observed upon desorption.

Effect of freezing conditions on distances and their distributions derived from Double Electron Electron Resonance (DEER): A study of doubly-spin-labeled T4 lysozyme

Pulsed dipolar ESR spectroscopy, DEER and DQC, require frozen samples. An important issue in the biological application of this technique is how the freezing rate and concentration of cryoprotectant could possibly affect the conformation of biomacromolecule and/or spin-label. We studied in detail the effect of these experimental variables on the distance distributions obtained by DEER from a series of doubly spin-labeled T4 lysozyme mutants. We found that the rate of sample freezing affects mainly the ensemble of spin-label rotamers, but the distance maxima remain essentially unchanged. This suggests that proteins frozen in a regular manner in liquid nitrogen faithfully maintain the distance-dependent structural properties in solution.We compared the results from rapidly freeze-quenched (⩽100μs) samples to those from commonly shock-frozen (slow freeze, 1s or longer) samples. For all the mutants studied we obtained inter-spin distance distributions, which were broader for rapidly frozen samples than for slowly frozen ones. We infer that rapid freezing trapped a larger ensemble of spin label rotamers; whereas, on the time-scale of slower freezing the protein and spin-label achieve a population showing fewer low-energy conformers. We used glycerol as a cryoprotectant in concentrations of 10% and 30% by weight. With 10% glycerol and slow freezing, we observed an increased slope of background signals, which in DEER is related to increased local spin concentration, in this case due to insufficient solvent vitrification, and therefore protein aggregation. This effect was considerably suppressed in slowly frozen samples containing 30% glycerol and rapidly frozen samples containing 10% glycerol. The assignment of bimodal distributions to tether rotamers as opposed to protein conformations is aided by comparing results using MTSL and 4-Bromo MTSL spin-labels. The latter usually produce narrower distance distributions.

Protein Conformational Intermediates Characterized with Novel High Pressure EPR and CD Techniques

Regulation of protein function is often linked to conformational intermediates that exist in equilibrium with the ground state. In many cases, these intermediate states exist as only a small fraction of the total protein conformational ensemble. It has been shown that high hydrostatic pressure is an invaluable tool for populating low-lying, excited states to levels amenable to spectroscopic detection. Here we demonstrate the usefulness of high pressure for populating such states as monitored by two novel techniques: High pressure electron paramagnetic resonance spectroscopy (EPR) and high pressure circular dichroism (CD). High pressure EPR was used in conjunction with site-directed spin labeling to monitor changes in local protein structure with applied pressure (up to 400 MPa). The relative partial molar volume and isothermal compressibility of each conformational substate was determined from the pressure dependence of the equilibrium constant determined from the continuous wave EPR spectra. High pressure CD was employed to detect global changes in secondary and tertiary structure at elevated pressure (up to 200 MPa). The combination of site-specific and global information provided by these techniques provides a more complete description of pressure excited intermediate states. Data from apomyoglobin and a cavity containing mutant (L99A) of T4 lysozyme are presented.

Structural and Dynamic Response to Core Repacking Substitutions in T4 Lysozyme Detected by Site-Directed Spin Labeling

Proteins can evolve to gain new functionality via mutations that decrease stability, and presumably, increase flexibility. Particularly interesting examples are mutations that modify the hydrophobic core packing and create new functions, e.g. novel ligand binding sites created by cavity-forming mutants. Many core repacking mutants of T4 Lysozyme (T4L) have been characterized, some of which bind non-polar ligands. Overall, the crystal structures of these mutants are very similar to the WT and offer little insight into the mechanism whereby the ligand reaches the cavity. Site-Directed Spin Labeling (SDSL) has been applied to investigate the structural and dynamical response of T4L in solution to 28 core repacking mutations, many of which have known crystal structures. Remarkably, the most common response in the cavity-creating susbtitutions is the appearance of a second conformational substate, with populations as high as 70 %, showing that the cavities allows for an alternative protein fold, at least locally. Osmotic perturbation shows that the conformations are in slow exchange on the EPR time scale. Thus, the cavity mutations excite molecular flexibility, with a characteristic time scale > 100 ns, probably in the μs-ms range. Interspin distance measurements carried out for two of the cavity mutants to study the magnitude and nature of the conformational rearrangement suggest the interesting possibility that the alternative states are also present in the WT protein, but at a different ratio.In some cases, ns backbone modes were also excited by the core mutation, but the effects were subtle. Remarkably, the type and magnitude of the changes in the protein flexibility were not necessarily correlated with the extent of destabilization by the substitution. The relationship of the cavity-excited states to the mechanism of ligand entry is under study.

Geofold: Topology‐based protein unfolding pathways capture the effects of engineered disulfides on kinetic stability

Protein unfolding is modeled as an ensemble of pathways, where each step in each pathway is the addition of one topologically possible conformational degree of freedom. Starting with a known protein structure, GeoFold hierarchically partitions (cuts) the native structure into substructures using revolute joints and translations. The energy of each cut and its activation barrier are calculated using buried solvent accessible surface area, side chain entropy, hydrogen bonding, buried cavities, and backbone degrees of freedom. A directed acyclic graph is constructed from the cuts, representing a network of simultaneous equilibria. Finite difference simulations on this graph simulate native unfolding pathways. Experimentally observed changes in the unfolding rates for disulfide mutants of barnase, T4 lysozyme, dihydrofolate reductase, and factor for inversion stimulation were qualitatively reproduced in these simulations. Detailed unfolding pathways for each case explain the effects of changes in the chain topology on the folding energy landscape. GeoFold is a useful tool for the inference of the effects of disulfide engineering on the energy landscape of protein unfolding.

Improved Binding Free Energy Predictions from Single-Reference Thermodynamic Integration Augmented with Hamiltonian Replica Exchange.

Reliable predictions of relative binding free energies are essential in drug discovery, where chemists modify promising compounds with the aim of increasing binding affinity. Conventional Thermodynamic Integration (TI) approaches can estimate corresponding changes in binding free energies, but suffer from inadequate sampling due to ruggedness of the molecular energy surfaces. Here, we present an improved TI strategy for computing relative binding free energies of congeneric ligands. This strategy employs a specific, unphysical single-reference (SR) state and Hamiltonian replica exchange (HREX) to locally enhance sampling. We then apply this strategy to compute relative binding free energies of twelve ligands in the L99A mutant of T4 Lysozyme. Besides the ligands, our approach enhances hindered rotations of the important V111, as well as V87 and L118 sidechains. Concurrently, we devise practical strategies to monitor and improve HREX-SRTI efficiency. Overall, the HREX-SRTI results agree well (R(2) = 0.76, RMSE = 0.3 kcal/mol) with available experimental data. When optimized for efficiency, the HREX-SRTI precision matches that of experimental measurements.

Solution structure of a minor and transiently formed state of a T4 lysozyme mutant

Proteins are inherently plastic molecules, whose function often critically depends on excursions between different molecular conformations (conformers). However, a rigorous understanding of the relation between a protein's structure, dynamics and function remains elusive. This is because many of the conformers on its energy landscape are only transiently formed and marginally populated (less than a few per cent of the total number of molecules), so that they cannot be individually characterized by most biophysical tools. Here we study a lysozyme mutant from phage T4 that binds hydrophobic molecules and populates an excited state transiently (about 1 ms) to about 3% at 25 °C (ref. 5). We show that such binding occurs only via the ground state, and present the atomic-level model of the 'invisible', excited state obtained using a combined strategy of relaxation-dispersion NMR (ref. 6) and CS-Rosetta model building that rationalizes this observation. The model was tested using structure-based design calculations identifying point mutants predicted to stabilize the excited state relative to the ground state. In this way a pair of mutations were introduced, inverting the relative populations of the ground and excited states and altering function. Our results suggest a mechanism for the evolution of a protein's function by changing the delicate balance between the states on its energy landscape. More generally, they show that our approach can generate and validate models of excited protein states.

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.

Applications of Genetically Encoded Unnatural Amino Acids for Protein Structure Mapping by Site-Directed Spin Labeling

Site-directed spin labeling (SDSL) is an important method for obtaining structural information on proteins that are refractory to other techniques. In this method, interspin distances are used as structural constraints for protein structure mapping, and here we present two applications of genetically encoded unnatural amino acids for such studies. One application is orthogonal labeling, in which an unnatural amino acid bearing a unique functional group is used to specifically introduce spin labels into proteins that are not amenable to the traditional, thiol-based SDSL method. Recently, a strategy based on p-acetyl-L-phenylalanine was reported [PNAS 2009 106(51):21637], in which the ketone was used to generate a ketoxime-linked spin label (K1). Here, we present an analogous strategy based on p-azido-L-phenylalanine, which can be specifically modified using “click” chemistry by a cyclooctyne-nitroxide reagent to generate a triazole-linked nitroxide side chain (C1). In contrast to the previous orthogonal labeling strategy, the azide-cyclooctyne reaction proceeds uncatalyzed at pH 7, and spectra of C1 mutants of T4 lysozyme (T4L) reflect a more ordered nitroxide motion than the analogous K1 mutant. A second application involves the Cu(II) binding unnatural amino acid (2,2’-bipyridin-5-yl)alanine (BpyAla), which enhances the longitudinal relaxation rate (1/T1) of a nearby nitroxide when copper is bound, thereby allowing average interspin distances to be measured at ambient temperature using saturation recovery EPR. In contrast to other methods for paramagnetic metal introduction, BpyAla can be introduced via a single mutation into any protein topology. Studies of T4L mutants containing BpyAla and a disulfide-linked nitroxide indicate that interspin distances of up to ∼30Å are accessible to T1 based EPR methods compared to ∼20Å for traditional T2 based methods. Both applications are expected to enhance distance mapping capability by EPR spectroscopy.

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.

High-pressure EPR reveals conformational equilibria and volumetric properties of spin-labeled proteins.

Identifying equilibrium conformational exchange and characterizing conformational substates is essential for elucidating mechanisms of function in proteins. Site-directed spin labeling has previously been employed to detect conformational changes triggered by some event, but verifying conformational exchange at equilibrium is more challenging. Conformational exchange (microsecond-millisecond) is slow on the EPR time scale, and this proves to be an advantage in directly revealing the presence of multiple substates as distinguishable components in the EPR spectrum, allowing the direct determination of equilibrium constants and free energy differences. However, rotameric exchange of the spin label side chain can also give rise to multiple components in the EPR spectrum. Using spin-labeled mutants of T4 lysozyme, it is shown that high-pressure EPR can be used to: (i) demonstrate equilibrium between spectrally resolved states, (ii) aid in distinguishing conformational from rotameric exchange as the origin of the resolved states, and (iii) determine the relative partial molar volume (ΔV°) and isothermal compressibility (Δβ(T)) of conformational substates in two-component equilibria from the pressure dependence of the equilibrium constant. These volumetric properties provide insight into the structure of the substates. Finally, the pressure dependence of internal side-chain motion is interpreted in terms of volume fluctuations on the nanosecond time scale, the magnitude of which may reflect local backbone flexibility.

Rotamer libraries of spin labelled cysteines for protein studies

Studies of structure and dynamics of proteins using site-directed spin labelling rely on explicit modelling of spin label conformations. The large computational effort associated with such modelling with molecular dynamics (MD) simulations can be avoided by a rotamer library approach based on a coarse-grained representation of the conformational space of the spin label. We show here that libraries of about 200 rotamers, obtained by iterative projection of a long MD trajectory of the free spin label onto a set of canonical dihedral angles, provide a representation of the underlying trajectory adequate for EPR distance measurements. Rotamer analysis was performed on selected X-ray structures of spin labelled T4 lysozyme mutants to characterize the spin label rotamer ensemble on a single protein site. Furthermore, predictions based on the rotamer library approach are shown to be in nearly quantitative agreement with electron paramagnetic resonance (EPR) distance data on the Na(+)/H(+) antiporter NhaA and on the light-harvesting complex LHCII whose structures are known from independent cryo electron microscopy and X-ray studies, respectively. Suggestions for the selection of labelling sites in proteins are given, limitations of the approach discussed, and requirements for further development are outlined.