Journal article
Structure of Spin-Labeled Methylmethanethiolsulfonate in Solution and Bound to TEM-1 β-Lactamase Determined by Electron Nuclear Double Resonance Spectroscopy
Biochemistry (Easton), Vol.41(3), pp.797-808
01/22/2002
DOI: 10.1021/bi010539p
PMID: 11790101
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.
Details
- Title: Subtitle
- Structure of Spin-Labeled Methylmethanethiolsulfonate in Solution and Bound to TEM-1 β-Lactamase Determined by Electron Nuclear Double Resonance Spectroscopy
- Creators
- Devkumar MUSTAFI - To whom correspondence should be addressed. Phone: 702-1667. Fax: 702-0439. E-mail: dmustafi@midway.uchicago.eduAlejandro SOSA-PEINADO - Present address: Department of Biochemistry, School of Medicine,National Autonomous University of Mexico, Mexico D.F. 04510Vanita GUPTADavid J GORDONMarvin W MAKINEN
- Resource Type
- Journal article
- Publication Details
- Biochemistry (Easton), Vol.41(3), pp.797-808
- Publisher
- American Chemical Society
- DOI
- 10.1021/bi010539p
- PMID
- 11790101
- ISSN
- 0006-2960
- eISSN
- 1520-4995
- Language
- English
- Date published
- 01/22/2002
- Academic Unit
- Stead Family Department of Pediatrics; Hematology/Oncology
- Record Identifier
- 9984093465502771
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