title: Time-resolved distance determination by tryptophan fluorescence quenching: probing intermediates in membrane protein folding.
abstract: The mechanism of insertion and folding of an integral membrane protein has been investigated with the beta-barrel forming outer membrane protein A (OmpA) of Escherichia coli. This work describes a new approach to this problem by combining structural information obtained from tryptophan fluorescence quenching at different depths in the lipid bilayer with the kinetics of the refolding process. Experiments carried out over a temperature range between 2 and 40 degrees C allowed us to detect, trap, and characterize previously unidentified folding intermediates on the pathway of OmpA insertion and folding into lipid bilayers. Three membrane-bound intermediates were found in which the average distances of the Trps were 14-16, 10-11, and 0-5 A, respectively, from the bilayer center. The first folding intermediate is stable at 2 degrees C for at least 1 h. A second intermediate has been isolated at temperatures between 7 and 20 degrees C. The Trps move 4-5 A closer to the center of the bilayer at this stage. Subsequently, in an intermediate that is observable at 26-28 degrees C, the Trps move another 5-10 A closer to the center of the bilayer. The final (native) structure is observed at higher temperatures of refolding. In this structure, the Trps are located on average about 9-10 A from the bilayer center. Monitoring the evolution of Trp fluorescence quenching by a set of brominated lipids during refolding at various temperatures therefore allowed us to identify and characterize intermediate states in the folding process of an integral membrane protein.
authors.affiliation: Department of Molecular Physiology and Biological Physics, Center for Structural Biology, University of Virginia Health Sciences Center, Charlottesville 22906-0011, USA.
authors.forename: J H
authors.initials: JH
authors.lastname: Kleinschmidt
authors.forename: L K
authors.initials: LK
authors.lastname: Tamm
mesh_terms: D020106:Acrylamide / Q000737:chemistry; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D001692:Biological Transport; D004926:Escherichia coli; D005454:Fluorescence Polarization; D008051:Lipid Bilayers / Q000737:chemistry / Q000378:metabolism; D016011:Normal Distribution; D017510:Protein Folding; D013050:Spectrometry, Fluorescence / Q000379:methods / Q000706:statistics & numerical data; D013696:Temperature; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001425:Bacterial Outer Membrane Proteins; D008051:Lipid Bilayers; C078167:OMPA outer membrane proteins; D020106:Acrylamide; D014364:Tryptophan