RETINAL BINDING DURING FOLDING AND ASSEMBLY OF THE MEMBRANE-PROTEIN BACTERIORHODOPSIN

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 regeneratio n of bacteriorhodopsin, from denatured apoprotein, in vitro. Regenerat ion is initiated by rapid, stopped-flow, mixing of the denatured apopr otein bacterioopsin in sodium dodecyl sulfate micelles with mixed dete rgent/lipid micelles containing retinal. Regeneration kinetics are mea sured by time-resolving changes in protein fluorescence. The dependenc e of each kinetic component on retinal concentration is determined. On ly one experimentally observed rate constant is dependent on retinal c oncentration, leading to identification of only one second-order react ion 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 th is retinal binding step is determined, showing that thermodynamic info rmation can be obtained on transient intermediates involved in membran e protein regeneration.