Evaluation of lipid exposure of tryptophan residues in membrane peptides and proteins.
Fluorescence quenching is used to gain information on the exposure of tryptophan residues to lipid in membrane-bound proteins and peptides. A protocol is developed to calculate this exposure, based on a comparison of quenching efficiency and of a fluorescence lifetime (or quantum yield) measured for a protein and for a model tryptophan-containing compound. Various methods of analysis of depth-dependent quenching are compared and three universal measures of quenching profile are derived. One of the measures, related to the area under profile, is used to estimate quenching efficiency. The method is applied to single tryptophan mutants of a membrane-anchoring nonpolar peptide of cytochrome b(5) and of an outer membrane protein A. Analysis of quenching of the cytochrome's nonpolar peptide by a set of four brominated lipids reveals a temperature-controlled reversible conformational change, resulting in increased exposure of tryptophan to lipid and delocalization of its transverse position. Kinetic quenching profiles and fluorescence binding kinetics reported by Kleinschmidt et al. (Biochemistry (1999) 38, 5006-5016) were analyzed to extract information on the relative exposure of tryptophan residues during folding of an outer membrane protein A. Trp-102, which translocates across the bilayer, was found to be noticeably shielded from the lipid environment throughout the folding event compared to Trp-7, which remains on the cis side. The approach described here provides a new tool for studies of low-resolution structure and conformational transitions in membrane proteins and peptides.
Ladokhin
A S
AS
Departments of Physiology and Biophysics and Program in Macromolecular Structure, University of California, Irvine, California, 92697-4560, USA.
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry; D015786:Cytochromes b5 / Q000737:chemistry; D005456:Fluorescent Dyes; D066298:In Vitro Techniques; D007700:Kinetics; D008563:Membrane Lipids / Q000737:chemistry*; D008565:Membrane Proteins / Q000737:chemistry*; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D010713:Phosphatidylcholines / Q000737:chemistry; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000031:analogs & derivatives / Q000737:chemistry*
D001425:Bacterial Outer Membrane Proteins; D005456:Fluorescent Dyes; D008563:Membrane Lipids; D008565:Membrane Proteins; D010455:Peptides; D010713:Phosphatidylcholines; C097263:tryptophan octyl ester; C078167:OMPA outer membrane proteins; D014364:Tryptophan; D015786:Cytochromes b5; C028694:1-palmitoyl-2-oleoylphosphatidylcholine