Structural studies on transmembrane proteins. 2. Spin labeling of bacteriorhodopsin mutants at unique cysteines

Journal Article · Tue Sep 19 00:00:00 EDT 1989 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00445a042· OSTI ID:7192296

Altenbach, C; Flitsch, S L; Khorana, H G; Hubbell, W L ^[1]

---

1. Univ. of California, Los Angeles (USA)

Site-directed mutagenesis was used to produce mutants of bacteriorhodopsin where either glycine-72, threonine-90, leucine-92, or serine-169 was replaced by a cysteine. Two different spin labels were then covalently attached to these sites. The selection of attachment sites covered two postulated loops (72,169) and a membrane-spanning segment (90,92). It was not possible to properly refold the protein labeled at position 90, presumably due to steric problems, but the EPR spectra of the other mutants that were successfully reconstituted in phospholipid vesicles provided information on the dynamics of protein side chains in the vicinity of the label site. A power saturation approach was used to investigate the spin relaxation times, which in turn can be influenced by collisions with paramagnetic species. The differential effect of oxygen and a water-soluble chromium complex on the power-saturation behavior of the spin-labeled mutants was used to obtain topographical information on the sites in the membrane-bound protein. The results are consistent with residues 72 and 169 being located in structured loops exposed to the aqueous phase and residue 92 being localized in the membrane interior, possibly near a helix-helix contact region.

• OSTI ID: 7192296
• Journal Information: Biochemistry; (USA), Vol. 28:19; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
RHODOPSIN
MOLECULAR STRUCTURE
CELL MEMBRANES
CYSTEINE
ELECTRON SPIN RESONANCE
ESCHERICHIA COLI
MEMBRANE PROTEINS
MUTANTS
PROTEIN STRUCTURE
AMINO ACIDS
BACTERIA
CARBOXYLIC ACIDS
CELL CONSTITUENTS
MAGNETIC RESONANCE
MEMBRANES
MICROORGANISMS
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
PIGMENTS
PROTEINS
RESONANCE
THIOLS
550200* - Biochemistry