Chemical crosslinking and mass spectrometry studies of the structure and dynamics of membrane proteins and receptors.

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Membrane proteins make up a diverse and important subset of proteins for which structural information is limited. In this study, chemical cross-linking and mass spectrometry were used to explore the structure of the G-protein-coupled photoreceptor bovine rhodopsin in the dark-state conformation. All experiments were performed in rod outer segment membranes using amino acid 'handles' in the native protein sequence and thus minimizing perturbations to the native protein structure. Cysteine and lysine residues were covalently cross-linked using commercially available reagents with a range of linker arm lengths. Following chemical digestion of cross-linked protein, cross-linked peptides were identified by accurate mass measurement ... continued below

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60 p.

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Haskins, William E.; Leavell, Michael D.; Lane, Pamela; Jacobsen, Richard B.; Hong, Joohee; Ayson, Marites J. et al. March 1, 2005.

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Membrane proteins make up a diverse and important subset of proteins for which structural information is limited. In this study, chemical cross-linking and mass spectrometry were used to explore the structure of the G-protein-coupled photoreceptor bovine rhodopsin in the dark-state conformation. All experiments were performed in rod outer segment membranes using amino acid 'handles' in the native protein sequence and thus minimizing perturbations to the native protein structure. Cysteine and lysine residues were covalently cross-linked using commercially available reagents with a range of linker arm lengths. Following chemical digestion of cross-linked protein, cross-linked peptides were identified by accurate mass measurement using liquid chromatography-fourier transform mass spectrometry and an automated data analysis pipeline. Assignments were confirmed and, if necessary, resolved, by tandem MS. The relative reactivity of lysine residues participating in cross-links was evaluated by labeling with NHS-esters. A distinct pattern of cross-link formation within the C-terminal domain, and between loop I and the C-terminal domain, emerged. Theoretical distances based on cross-linking were compared to inter-atomic distances determined from the energy-minimized X-ray crystal structure and Monte Carlo conformational search procedures. In general, the observed cross-links can be explained by re-positioning participating side-chains without significantly altering backbone structure. One exception, between C3 16 and K325, requires backbone motion to bring the reactive atoms into sufficient proximity for cross-linking. Evidence from other studies suggests that residues around K325 for a region of high backbone mobility. These findings show that cross-linking studies can provide insight into the structural dynamics of membrane proteins in their native environment.

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60 p.

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  • Report No.: SAND2005-1699
  • Grant Number: AC04-94AL85000
  • DOI: 10.2172/922763 | External Link
  • Office of Scientific & Technical Information Report Number: 922763
  • Archival Resource Key: ark:/67531/metadc900599

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Office of Scientific & Technical Information Technical Reports

Reports, articles and other documents harvested from the Office of Scientific and Technical Information.

Office of Scientific and Technical Information (OSTI) is the Department of Energy (DOE) office that collects, preserves, and disseminates DOE-sponsored research and development (R&D) results that are the outcomes of R&D projects or other funded activities at DOE labs and facilities nationwide and grantees at universities and other institutions.

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  • March 1, 2005

Added to The UNT Digital Library

  • Sept. 27, 2016, 1:39 a.m.

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  • Nov. 22, 2016, 10:21 p.m.

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Haskins, William E.; Leavell, Michael D.; Lane, Pamela; Jacobsen, Richard B.; Hong, Joohee; Ayson, Marites J. et al. Chemical crosslinking and mass spectrometry studies of the structure and dynamics of membrane proteins and receptors., report, March 1, 2005; United States. (digital.library.unt.edu/ark:/67531/metadc900599/: accessed November 16, 2018), University of North Texas Libraries, Digital Library, digital.library.unt.edu; crediting UNT Libraries Government Documents Department.