title: Retinal binding during folding and assembly of the membrane protein bacteriorhodopsin.
abstract: The factors driving folding and assembly of integral membrane proteins are largely unknown. In order to determine the role that the retinal chromophore plays in assembly of bacteriorhodopsin, we have determined the kinetics and thermodynamics of retinal binding during regeneration of bacteriorhodopsin, from denatured apoprotein, in vitro. Regeneration is initiated by rapid, stopped-flow, mixing of the denatured apoprotein bacterioopsin in sodium dodecyl sulfate micelles with mixed detergent/lipid micelles containing retinal. Regeneration kinetics are measured by time-resolving changes in protein fluorescence. The dependence of each kinetic component on retinal concentration is determined. Only one experimentally observed rate constant is dependent on retinal concentration, leading to identification of only one second-order reaction involving retinal and bacterioopsin. This reaction occurs after a rate-limiting step in bacterioopsin folding, and results in formation of a noncovalent retinal/protein complex. The free energy change of this retinal binding step is determined, showing that thermodynamic information can be obtained on transient intermediates involved in membrane protein regeneration.
authors.affiliation: Department of Biochemistry, University of Oxford, UK.
authors.forename: P J
authors.initials: PJ
authors.lastname: Booth
authors.forename: A
authors.initials: A
authors.lastname: Farooq
authors.forename: S L
authors.initials: SL
authors.lastname: Flitsch
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism*; D006217:Halobacterium / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008823:Micelles; D011485:Protein Binding; D017510:Protein Folding; D012172:Retinaldehyde / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D008565:Membrane Proteins; D008823:Micelles; D001436:Bacteriorhodopsins; D012172:Retinaldehyde