muramidase and methanethiosulfonate

Many extracellular globular proteins have evolved to possess disulphide bonds in their native conformations, which aids in thermodynamic stabilisation. However, disulphide bond breakage by heating leads to irreversible protein denaturation through disulphide-thiol exchange reactions. In this study, we demonstrate that methanethiosulphonate (MTS) specifically suppresses the heat-induced disulphide-thiol exchange reaction, thus improving the heat-resistance of proteins. In the presence of MTS, small globular proteins that contain disulphides can spontaneously refold from heat-denatured states, maintaining wild-type disulphide pairing. Because the disulphide-thiol exchange reaction is triggered by the generation of catalytic amounts of perthiol or thiol, rapid and specific perthiol/thiol protection by MTS reagents prevents irreversible denaturation. Combining MTS reagents with another additive that suppresses chemical modifications, glycinamide, further enhanced protein stabilisation. In the presence of these additives, reliable remnant activities were observed even after autoclaving. However, immunoglobulin G and biotin-binding protein, which are both composed of tetrameric quaternary structures, failed to refold from heat-denatured states, presumably due to chaperon requirements. Elucidation of the chemical modifications involved in irreversible thermoinactivation is useful for the development of preservation buffers with optimum constitutions for specific proteins. In addition, the impact of disulphide bond breakage on the thermoinactivation of proteins can be evaluated using MTS reagents.

Topics: Animals; Carrier Proteins; Cattle; Chickens; Disulfides; Glycine; Hot Temperature; Humans; Hydrolysis; Immunoglobulin G; Lactalbumin; Mesylates; Muramidase; Protein Denaturation; Protein Refolding; Protein Stability; Protein Structure, Quaternary; Ribonuclease, Pancreatic; Solutions; Sulfhydryl Compounds

Topics: Electron Spin Resonance Spectroscopy; Glycerol; Mesylates; Muramidase; Protein Structure, Tertiary; Proteins; Spin Labels; Temperature; Viscosity

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.

Topics: Bacteriophage T4; Electron Spin Resonance Spectroscopy; Mesylates; Models, Molecular; Molecular Dynamics Simulation; Muramidase; Protein Conformation; Proteins; Reproducibility of Results; Software; 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 A and 50 A 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.

Topics: Anisotropy; Bacteriophage T4; Fluorescein; Fluorescence; Fluorescent Dyes; Mesylates; Models, Molecular; Molecular Structure; Muramidase; Mutation; Spin Labels