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Mutagenesis reveals that R261, a conserved active site arginine, stabilizes the active site environment enabling β-lactone formation. Our results have implications not only for OXA-24/40, but the larger family of CHDLs now challenging clinical settings on a global scale.After decades of progress in computational protein design, the design of proteins folding and functioning in lipid membranes appears today as the next frontier. Some notable successes in the de novo design of simplified model membrane protein systems have helped articulate