Identification of the SecB residue that is labeled by methanethiosulfonate
EMSL Project ID
5391
Abstract
Aim: To identify the residue of SecB that is labeled by methanethiosulfonate (MTSL), a stable nitroxide spin label.SecB, a small tetrameric cytosolic chaperone in E.coli, facilitates the export of precursor polypeptides by maintaining them in a nonnative conformation and passing them to SecA, which is a peripheral member of the membrane-bound translocation apparatus. To elucidate the interaction between SecB and unfolded precursor we are mapping the molecular path that precursor takes when it binds SecB. The use of site-directed mutagenesis to systematically replace single residues with cysteine coupled with electron paramagnetic resonance (EPR) allows us to scan through SecB identifying sites of interaction. EPR technology requires an unpaired electron, which we provide by labeling the introduced cysteinyl residue in SecB with methanethiosulfonate, a stable nitroxide spin label. Ideally in our experimental approach one would remove all native cysteines and introduce a single cysteine at the site of interest. However, we have been unable to replace two of the four native cysteines and maintain the protein in an active form. Thus each of our constructs has three cysteines. It is imperative that we know confidently that we quantitatively label the introduced cysteine and not the native cysteines.
In order to confidently identify labeled residue position further experiments will be performed in EMSL High performance mass spectrometry facility: Molecular mass of intact proteins will be measured on 7T FTICRMS in order to confirm number of labels per protein. Further, proteins will be digested by trypsin to peptide level. LC-MS and LC-MS/MS experiments on LCQ ion trap and 7T FTICRMS will enable (labeled) peptide identification and the labeled residue.
Project Details
Project type
Exploratory Research
Start Date
2004-01-12
End Date
2005-01-11
Status
Closed
Released Data Link
Team
Principal Investigator
Related Publications
Olson A, DR Ledee, PhD, K Iwamoto, M Kajimoto, CM O'Kelly-Priddy, NG Isern, and MA Portman. 2012. "C-Myc Induced Compensated Cardiac Hypertrophy Increases Free Fatty Acid Utilization for the Citric Acid Cycle." Journal of Molecular and Cellular Cardiology.
Superior Cardiac Function Via Anaplerotic Pyruvate in the Immature Swine Heart After Cardiopulmonary Bypass and Reperfusion AARON K. OLSON1,5, OUTI M. HYYTI1,2,5, GORDON A. COHEN3,5, XUE-HAN NING1,5, MARTIN SADILEK4, NANCY ISERN6 AND MICHAEL A. PORTMAN1,5 Division of Cardiology, Department of Pediatrics1, Department of Radiology2, Division of Pediatric Cardiovascular Surgery, Department of Surgery3 , Department of Chemistry4, University of Washington, Children’s Hospital and Regional Medical Center5, Seattle WA, 98195; Pacific Northwest National Laboratory, Richland WA, 993926