Display Settings:

Format

Send to:

Choose Destination
See comment in PubMed Commons below
J Mol Biol. 2012 May 18;418(5):367-78. doi: 10.1016/j.jmb.2012.02.024. Epub 2012 Mar 3.

Identifying functionally important conformational changes in proteins: activation of the yeast α-factor receptor Ste2p.

Author information

  • 1Department of Biochemistry and Biophysics, P.O. Box 712, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.

Abstract

We have developed a procedure in which disulfide cross-links are used to identify regions of proteins that undergo functionally important intramolecular motion. The approach was applied to the identification of disulfide bonds that stabilize the active state of the yeast α-mating pheromone receptor Ste2p, a member of the superfamily of G protein-coupled receptors. Cysteine residues were introduced at random positions in targeted regions of a starting allele of Ste2p that completely lacks cysteines. Libraries of mutated receptors were then screened for alleles that exhibit constitutive signaling. Two strongly activated alleles were recovered containing cysteine residues in transmembrane (TM) segments 5 and 6. Constitutive activity of these alleles was dependent on the presence of both introduced cysteines and was sensitive to reducing agent. Cross-linked peptides derived from the mutant receptors were detected by immunoblotting. Additional sites of cross-linking between TM segments 5 and 6 that did not lead to constitutive activation were also identified. These results indicate that relative motion of the TM segments 5 and 6 in the extracellular half of the membrane is sufficient to activate the receptor and that TM segment 6, but not TM segment 5, exhibits rotational mobility that is not associated with receptor activation.

Copyright © 2012 Elsevier Ltd. All rights reserved.

PMID:
22387470
[PubMed - indexed for MEDLINE]
PMCID:
PMC3346263
Free PMC Article
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Icon for Elsevier Science Icon for PubMed Central
    Loading ...
    Write to the Help Desk