Structure of Spin-Labeled Methylmethanethiolsulfonate in Solution and Bound to TEM-1 #beta#-Lactamase Determined by Electron Nuclear Double Resonance Spectroscopy

摘要

Site-directed spin-labeling of proteins whereby the spin-label methyl 3-(2,2,5,5-tetramethyl-1-oxypyrrolinyl)methyanethiolsulfonate (SLMTS) is reacted with the -SH groups of cysteinyl residues incorporated into a protein by mutagenesis has been successfully aplied 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 is solution reacted with L-cysteine (SLMTCys) and bound int he 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 conformatins of the molecule were compatible with the ENDOR-determined electron-nucleus distances to the side-chain methylene protons and to H~(#alpha#) and H~(#beta#1,2) of cysteine. To detemine 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~(#beta#1,2) of for 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.