muramidase and nitroxyl

Electron Paramagnetic Resonance (EPR) spectroscopy is a powerful method for unraveling structures and dynamics of biomolecules. Out of the EPR tool box, Pulsed Electron-Electron Double Resonance spectroscopy (PELDOR or DEER) enables one to resolve such structures by providing distances between spin centers on the nanometer scale. Most commonly, both spin centers are spin labels or one is a spin label and the other is a paramagnetic metal ion, cluster, amino acid or cofactor radical. Often, the translation of the measured distances into structures is complicated by the long and flexible linker connecting the spin center of the spin label with the biomolecule. Nowadays, this challenge is overcome by computational methods but the currently available approaches have a rather large mean error of roughly 2-3 Å. Here, the new GFN-FF general force-field is combined with the fully automated Conformer Rotamer Ensemble Search Tool (CREST) [P. Pracht et al., Phys. Chem. Chem. Phys., 2020, 22, 7169-7192] to generate conformer ensembles of the R1 side chain (methanthiosulfonate spin label (MTSL) covalently bound to a cysteine) in several cysteine mutants of azurin and T4 lysozyme. In order to determine the Cu2+-R1 and R1-R1 distance distributions, GFN-FF based MD simulations were carried out starting from the most probable R1 conformers found by CREST. The deviation between theory and experiment in mean inter-spin distances was 0.98 Å on average for the mutants of azurin (1.84 Å for T4 lysozyme) and the right modality was obtained. The error of the most probable distances for each mode was only 0.76 Å in the case of azurin. This CREST/MD procedure does thus enable precise distance-to-structure translations and provides a means to disentangle label from protein conformers.

Topics: Azurin; Macromolecular Substances; Models, Molecular; Muramidase; Mutation; Nitrogen Oxides; Protein Conformation; Spin Labels

Direct time-domain simulation of continuous-wave (CW) electron paramagnetic resonance (EPR) spectra from molecular dynamics (MD) trajectories has become increasingly popular, especially for proteins labeled with nitroxide spin labels. Due to the time-consuming nature of simulating adequately long MD trajectories, two approximate methods have been developed to reduce the MD-trajectory length required for modeling EPR spectra: hindered Brownian diffusion (HBD) and hidden Markov models (HMMs). Here, we assess the accuracy of these two approximate methods relative to direct simulations from MD trajectories for three spin-labeled protein systems (a simple helical peptide, a soluble protein, and a membrane protein) and two nitroxide spin labels with differing mobilities (R1 and 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC)). We find that the HMMs generally outperform HBD. Although R1 dynamics partially resembles hindered Brownian diffusion, HMMs accommodate the multiple dynamic time scales for the transitions between rotameric states of R1 that cannot be captured accurately by a HBD model. The MD trajectories of the TOAC-labeled proteins show that its dynamics closely resembles slow multisite exchange between twist-boat and chair ring puckering states. This motion is modeled well by HMM but not by HBD. All MD-trajectory data processing, stochastic trajectory simulations, and CW EPR spectral simulations are implemented in EasySpin, a free software package for MATLAB.

Topics: Calcium-Binding Proteins; Cyclic N-Oxides; Diffusion; Electron Spin Resonance Spectroscopy; Markov Chains; Molecular Dynamics Simulation; Muramidase; Nitrogen Oxides; Peptides; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Interaction Domains and Motifs; Quantum Theory; Software; Spin Labels; Staining and Labeling; Thermodynamics

Site-directed spin labeling in combination with EPR is a powerful method for providing distances on the nm scale in biological systems. The most popular strategy, double electron-electron resonance (DEER), is carried out at cryogenic temperatures (50-80 K) to increase the short spin-spin relaxation time (T2) upon which the technique relies. A challenge is to measure long-range distances (20-60 Å) in proteins near physiological temperatures. Toward this goal we are investigating an alternative approach based on the distance-dependent enhancement of spin-lattice relaxation rate (T1(-1)) of a nitroxide spin label by a paramagnetic metal. With a commonly used nitroxide side chain (R1) and Cu(2+), it has been found that interspin distances ≤25 Å can be determined in this way (Jun et al. Biochemistry 2006, 45, 11666). Here, the upper limit of the accessible distance is extended to ≈40 Å using spin labels with long T1, a high-affinity 5-residue Cu(2+) binding loop inserted into the protein sequence, and pulsed saturation recovery to measure relaxation enhancement. Time-domain Cu(2+) electron paramagnetic resonance, quantum mechanical calculations, and molecular dynamics simulations provide information on the structure and geometry of the Cu(2+) loop and indicate that the metal ion is well-localized in the protein. An important aspect of these studies is that both Cu(2+)/nitroxide DEER at cryogenic temperatures and T1 relaxation measurements at room temperature can be carried out on the same sample, allowing both validation of the relaxation method and assessment of the effect of freezing on protein structure.

Topics: Amino Acid Sequence; Bacteriophage T4; Binding Sites; Copper; Electron Spin Resonance Spectroscopy; Models, Molecular; Muramidase; Mutation; Nitrogen Oxides; Peptides; Protein Structure, Secondary; Proteins; Quantum Theory; Rotation; Spin Labels; Temperature

We present the first example of chemoselective site-specific spin labeling of a monomeric protein with two spectroscopically orthogonal spin labels: a gadolinium(III) chelate complex and a nitroxide radical. A detailed analysis of the performance of two commercially available Gd(III) ligands in the Gd(III)-nitroxide pulse double electron-electron resonance (DEER or PELDOR) experiment is reported. A modification of the flip angle of the pump pulse in the Gd(III)-nitroxide DEER experiment is proposed to optimize sensitivity.

Topics: Bacteriophage T4; Electron Spin Resonance Spectroscopy; Electrons; Gadolinium; Heterocyclic Compounds, 1-Ring; Muramidase; Mutation; Nitrogen Oxides; Spin Labels

Protein oxidation occurs during multiple human pathologies, and protein radicals are known to induce damage to other cell components. Such damage may be modulated by agents that scavenge protein radicals. In this study, the potential protective reactions of the nitroxide TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxyl radical) against Tyr- and Trp-derived radicals (TyrO./TrpN.) have been investigated. Pretreatment of macrophage cells with TEMPO provided protection against photo-oxidation-induced loss of cell viability and Tyr oxidation, with the nitroxide more effective than the hydroxylamine or parent amine. Pulse radiolysis was employed to determine rate constants, k, for the reaction of TEMPO with TyrO. and TrpN. generated on N-Ac-Tyr-amide and N-Ac-Trp-amide, with values of k~10(8) and 7×10(6)M(-1)s(-1), respectively, determined. Analogous studies with lysozyme, chymotrypsin, and pepsin yielded k for TEMPO reacting with TrpN. ranging from 1.5×10(7) (lysozyme) to 1.1×10(8) (pepsin)M(-1)s(-1). Pepsin-derived TyrO. reacted with TEMPO with k~4×10(7)M(-1)s(-1); analogous reactions for lysozyme and chymotrypsin TyrO. were much slower. These data indicate that TEMPO can inhibit secondary reactions of both TyrO. and TrpN., though this is protein dependent. Such protein radical scavenging may contribute to the positive biological effects of nitroxides.

Topics: Animals; Azides; Cell Line; Cell Survival; Chymotrypsin; Cyclic N-Oxides; Electron Spin Resonance Spectroscopy; Free Radical Scavengers; Free Radicals; Hydroxylamine; Kinetics; Macrophages; Mice; Muramidase; Nitrogen Oxides; Oxidants, Photochemical; Oxidation-Reduction; Pepsin A; Pulse Radiolysis; Tryptophan; Tyrosine

An extensive set of electron spin resonance spectra was obtained over a wide range of frequencies (9, 95, 170, and 240 GHz) and temperatures (2 to 32 degrees C) to explore the dynamic modes of nitroxide-labeled T4 lysozyme in solution. A commonly used nitroxide side chain (R1), or a methylated analogue with hindered internal motion (R2), was substituted for the native side chain at solvent-exposed helical sites, 72 or 131. The spectra at all four frequencies were simultaneously fit with the slowly relaxing local structure (SRLS) model. Good fits were achieved at all the temperatures. Two principle dynamic modes are included in the SRLS model, the global tumbling of the protein and the internal motion consisting of backbone fluctuations and side chain isomerizations. Three distinct spectral components were required for R1 and two for R2 to account for the spectra at all temperatures. One is a highly ordered and slow motional component, which is observed in the spectra of both R1 and R2; it may correspond to conformers stabilized by interaction with the protein surface. The fraction of this component decreases with increasing temperature and is more populated in the R2 spectra, possibly arising from stronger interaction of the nitroxide ring with the protein surface due to the additional methyl group. The other two components of R1 and the second component of R2 are characterized by fast anisotropic diffusion and relatively low ordering, most likely corresponding to conformers having little or no interactions with nearby residues. Ficoll of different concentrations was added to increase the solution viscosity, thereby slowing down the global tumbling of the protein. A significant effect of Ficoll on the internal motion of an immobilized component was apparent in R2 but not in R1. The ability of such multifrequency studies to separate the effects of faster internal modes of motion from slower overall motions is clearly demonstrated, and its utility in future studies is considered.

Topics: Bacteriophage T4; Electron Spin Resonance Spectroscopy; Molecular Weight; Motion; Muramidase; Mutagenesis, Site-Directed; Nitrogen Oxides; Protein Conformation; Spin Labels; Sucrose; Temperature; Water

Site-directed spin labeling provides a means for exploring structure and dynamics in proteins. To interpret the complex EPR spectra that often arise, it is necessary to characterize the rotamers of the spin-labeled side chain and the interactions they make with the local environment in proteins of known structure. For this purpose, crystal structures have been determined for T4 lysozyme bearing a nitroxide side chain (R1) at the solvent-exposed helical sites 41 and 44 in the B helix. These sites are of particular interest in that the corresponding EPR spectra reveal two dynamic states of R1, one of which is relatively immobilized suggesting interactions of the nitroxide with the environment. The crystal structures together with the effect of mutagenesis of nearest neighbors on the motion of R1 suggest intrahelical interactions of 41R1 with the i + 4 residue and of 44R1 with the i + 1 residue. Such interactions appear to be specific to particular rotamers of the R1 side chain.

Topics: Bacteriophage T4; Crystallography, X-Ray; Electron Spin Resonance Spectroscopy; Models, Molecular; Muramidase; Mutant Proteins; Nitrogen Oxides; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Spin Labels; Thermodynamics

Two single cysteine substitution mutants at helix surface sites in T4 lysozyme (D72C and V131C) have been modified with a series of nitroxide methanethiosulfonate reagents to investigate the structural and dynamical origins of their electron paramagnetic resonance spectra. The novel reagents include 4-substituted derivatives of either the pyrroline or pyrrolidine series of nitroxides. The spectral line shapes were analyzed as a function of side chain structure and temperature using a simulation method with a single order parameter and diffusion rates about three orthogonal axes as parameters. Taken together, the results provide strong support for an anisotropic motional model of the side chain, which was previously proposed from qualitative features of the spectra and crystal structures of spin labeled T4 lysozyme. Site-specific differences in apparent order parameter are interpreted in terms of backbone dynamics modes with characteristic correlation times in the nanosecond or faster time scale. The saturated 4-substituted pyrrolidine nitroxides are shown to be a suitable template for novel "functionalized" side chains designed to mimic salient features of the native side chains they replace.

Topics: Amino Acid Substitution; Anisotropy; Arginine; Aspartic Acid; Bacteriophage T4; Cysteine; Electron Spin Resonance Spectroscopy; Free Radicals; Models, Molecular; Muramidase; Mutagenesis, Site-Directed; Nitrogen Oxides; Protein Conformation; Protein Structure, Secondary; Spin Labels; Spin Trapping; Temperature; Valine