title: Structure of spin-labeled methylmethanethiolsulfonate in solution and bound to TEM-1 beta-lactamase determined by electron nuclear double resonance spectroscopy.
abstract: Site-directed spin-labeling of proteins whereby the spin-label methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)methanethiolsulfonate (SLMTS) is reacted with the -SH groups of cysteinyl residues incorporated into a protein by mutagenesis has been successfully applied to investigate secondary structure and conformational transitions of proteins. In these studies, it is expected that the spin-label moiety adopts different conformations dependent on its local environment. To determine the conformation of SLMTS in solution reacted with L-cysteine (SLMTCys) and bound in the active site of the Glu240Cys mutant of TEM-1 beta-lactamase, we have synthesized SLMTS both of natural abundance isotope composition and in site-specifically deuterated forms for electron nuclear double resonance (ENDOR) studies. ENDOR-determined electron-proton distances from the unpaired electron of the nitroxyl group of the spin-label to the methylene and methyl protons of SLMTS showed three conformations of the oxypyrrolinyl ring with respect to rotation around the S-S bond dependent on the solvent dielectric constant. For SLMTCys, two conformations of the molecule were compatible with the ENDOR-determined electron-nucleus distances to the side-chain methylene protons and to H(alpha) and H(beta1,2) of cysteine. To determine SLMTS conformation reacted with the Glu240Cys mutant of TEM-1 beta-lactamase, enzyme was overexpressed in both ordinary and perdeuterated minimal medium. Resonance features of H(alpha) and H(beta1,2) of the Cys240 residue of the mutant and of the side-chain methylene protons within the spin-label moiety yielded electron-proton distances that sterically accommodated the two conformations of free SLMTCys in solution.
authors.affiliation: Department of Biochemistry and Molecular Biology, The University of Chicago, Cummings Life Science Center, 920 East 58th Street, Chicago, Illinois 60637, USA. dmustafi@midway.uchicago.edu
authors.forename: Devkumar
authors.initials: D
authors.lastname: Mustafi
authors.forename: Alejandro
authors.initials: A
authors.lastname: Sosa-Peinado
authors.forename: Vanita
authors.initials: V
authors.lastname: Gupta
authors.forename: David J
authors.initials: DJ
authors.lastname: Gordon
authors.forename: Marvin W
authors.initials: MW
authors.lastname: Makinen
mesh_terms: D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D008741:Methyl Methanesulfonate / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012997:Solvents; D013113:Spin Labels / Q000138:chemical synthesis; D013499:Surface Properties; D001618:beta-Lactamases / Q000737:chemistry*
chemical_list: D012997:Solvents; D013113:Spin Labels; D008741:Methyl Methanesulfonate; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1; D003545:Cysteine