Retinal binding during folding and assembly of the membrane protein bacteriorhodopsin

Paula J. Booth, Amjad Farooq, Sabine L. Flitsch

Research output: Contribution to journalArticle

69 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)5902-5909
Number of pages8
JournalBiochemistry
Volume35
Issue number18
DOIs
StatePublished - May 7 1996
Externally publishedYes

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Bacteriorhodopsins
Regeneration
Membrane Proteins
Apoproteins
Micelles
Kinetics
Thermodynamics
Chromophores
Sodium Dodecyl Sulfate
Detergents
Free energy
Rate constants
Proteins
Fluorescence
Lipids
bacterio-opsin

ASJC Scopus subject areas

  • Biochemistry

Cite this

Retinal binding during folding and assembly of the membrane protein bacteriorhodopsin. / Booth, Paula J.; Farooq, Amjad; Flitsch, Sabine L.

In: Biochemistry, Vol. 35, No. 18, 07.05.1996, p. 5902-5909.

Research output: Contribution to journalArticle

Booth, Paula J. ; Farooq, Amjad ; Flitsch, Sabine L. / Retinal binding during folding and assembly of the membrane protein bacteriorhodopsin. In: Biochemistry. 1996 ; Vol. 35, No. 18. pp. 5902-5909.
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