The final stages of folding of the membrane protein bacteriorhodopsin occur by kinetically indistinguishable parallel folding paths that are mediated by pH

TY - JOUR

T1 - The final stages of folding of the membrane protein bacteriorhodopsin occur by kinetically indistinguishable parallel folding paths that are mediated by pH

AU - Lu, H

AU - Booth, P J

PY - 2000/5/26

Y1 - 2000/5/26

N2 - The folding of the transmembrane protein bacteriorhodopsin that occurs during the binding of its retinal cofactor is investigated in a membrane-like environment. Changes in the retinal absorption band reveal two transient retinal-protein intermediate states, with apparent absorption maxima at 380 nm and 440 nm, respectively. Studies on a bacteriorhodopsin mutant of Lys216, which cannot bind retinal covalently, add to evidence that retinal is non-covalently bound in these intermediate states. The two retinal-protein intermediates are genuine intermediate states that form in parallel, each with an observed rate constant of 1.1 s-1. Meanwhile no formation of the folded state is detected. Folded bacteriorhodopsin, with all trans retinal covalently bound, forms from both retinal-bound intermediates with the same apparent rate constant of 0.0070 s-1 that is independent of retinal concentration. Retinal isomerisation then occurs with a rate constant of 0.00033 s-1 to give bacteriorhodopsin containing all trans and 13 cis-retinal. These results provide experimental evidence for multiple folding routes for a membrane protein that are pH dependent, with pH conditions determining the apparent folding route. These observed parallel folding paths are kinetically indistinguishable, which contrasts with most other observations of parallel folding pathways where only pathways with different kinetics have been reported. Furthermore, together with previous work, this study shows that bacteriorhodopsin has to populate at least two folding intermediates, during folding in the mixed lipid micelles investigated here, before the final fold is attained.

AB - The folding of the transmembrane protein bacteriorhodopsin that occurs during the binding of its retinal cofactor is investigated in a membrane-like environment. Changes in the retinal absorption band reveal two transient retinal-protein intermediate states, with apparent absorption maxima at 380 nm and 440 nm, respectively. Studies on a bacteriorhodopsin mutant of Lys216, which cannot bind retinal covalently, add to evidence that retinal is non-covalently bound in these intermediate states. The two retinal-protein intermediates are genuine intermediate states that form in parallel, each with an observed rate constant of 1.1 s-1. Meanwhile no formation of the folded state is detected. Folded bacteriorhodopsin, with all trans retinal covalently bound, forms from both retinal-bound intermediates with the same apparent rate constant of 0.0070 s-1 that is independent of retinal concentration. Retinal isomerisation then occurs with a rate constant of 0.00033 s-1 to give bacteriorhodopsin containing all trans and 13 cis-retinal. These results provide experimental evidence for multiple folding routes for a membrane protein that are pH dependent, with pH conditions determining the apparent folding route. These observed parallel folding paths are kinetically indistinguishable, which contrasts with most other observations of parallel folding pathways where only pathways with different kinetics have been reported. Furthermore, together with previous work, this study shows that bacteriorhodopsin has to populate at least two folding intermediates, during folding in the mixed lipid micelles investigated here, before the final fold is attained.

KW - Bacteriorhodopsins

KW - Darkness

KW - Hydrogen-Ion Concentration

KW - Isomerism

KW - Kinetics

KW - Light

KW - Membrane Proteins

KW - Membranes, Artificial

KW - Micelles

KW - Phosphatidylcholines

KW - Protein Conformation

KW - Protein Denaturation

KW - Protein Folding

KW - Protein Renaturation

KW - Protons

KW - Retinaldehyde

KW - Sodium Dodecyl Sulfate

KW - Spectrophotometry

KW - Thermodynamics

U2 - 10.1006/jmbi.2000.3735

DO - 10.1006/jmbi.2000.3735

M3 - Article

C2 - 10860735

SN - 0022-2836

VL - 299

SP - 233

EP - 243

JO - Journal of Molecular Biology

JF - Journal of Molecular Biology

IS - 1

ER -