Mapping membrane protein interactions in cell signaling systems.

Technical Report · Mon Dec 01 04:00:00 EST 2003

DOI:https://doi.org/10.2172/918234· OSTI ID:918234

Light, Yooli Kim; Hadi, Masood Z; Lane, Pamela; Jacobsen, Richard B; Hong, Joohee; Ayson, Marites J; Wood, Nichole L; Schoeniger, Joseph S; Young, Malin M

We proposed to apply a chemical cross-linking, mass spectrometry and modeling method called MS3D to the structure determination of the rhodopsin-transducin membrane protein complex (RTC). Herein we describe experimental progress made to adapt the MS3D approach for characterizing membrane protein systems, and computational progress in experimental design, data analysis and protein structure modeling. Over the past three years, we have developed tailored experimental methods for all steps in the MS3D method for rhodopsin, including protein purification, a functional assay, cross-linking, proteolysis and mass spectrometry. In support of the experimental effort. we have out a data analysis pipeline in place that automatically selects the monoisotopic peaks in a mass spectrometric spectrum, assigns them and stores the results in a database. Theoretical calculations using 24 experimentally-derived distance constraints have resulted in a backbone-level model of the activated form of rhodopsin, which is a critical first step towards building a model of the RTC. Cross-linked rhodopsin-transducin complexes have been isolated via gel electrophoresis and further mass spectrometric characterization of the cross-links is underway.

• Research Organization: Sandia National Laboratories
• Sponsoring Organization: USDOE
• DOE Contract Number: AC04-94AL85000
• OSTI ID: 918234
• Report Number(s): SAND2003-8795
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
Biological systems-Computer simulation.
DATA ANALYSIS
DESIGN
ELECTROPHORESIS
FUNCTIONALS
MASS SPECTROSCOPY
MEMBRANE PROTEINS
Mass spectrometry.
PIPELINES
PROTEIN STRUCTURE
PROTEINS
PROTEOLYSIS
PURIFICATION
Protein-analysis.
RHODOPSIN