Kinetics of an individual transmembrane helix during bacteriorhodopsin folding.
The kinetics of an individual helix of bacteriorhodopsin have been monitored during folding of the protein into lipid bilayer vesicles. A fluorescence probe was introduced at individual sites throughout helix D of bacteriorhodopsin and the changes in the fluorescence of the label were time-resolved. Partially denatured, labelled bacteriorhodopsin in SDS was folded directly into phosphatidylcholine lipid vesicles. Stopped-flow mixing of the reactants allowed the folding kinetics to be monitored with millisecond time resolution by time-resolving changes in the label fluorescence, intrinsic protein fluorescence as well as in the absorption of the retinal chromophore. Monitoring specific positions on helix D showed that two kinetic phases were altered compared to those determined by monitoring the average protein behaviour. These two phases, of 6.7 s(-1) and 0.33 s(-1), were previously assigned to formation of a key apoprotein intermediate during bacteriorhodopsin folding. The faster 6.7s(-1) phase was missing when time-resolving fluorescence changes of labels attached to the middle of helix D. The amplitude of the 0.33 s(-1) phase increased along the helix, as single labels were attached in turn from the cytoplasmic to the extracellular side. An interpretation of these results is that the 6.7 s(-1) phase involves partitioning of helix D within the lipid headgroups of the bilayer vesicle, while the 0.33 s(-1) phase could reflect transmembrane insertion of this helix. In addition, a single site on helix G was monitored during folding. The results indicate that, unlike helix D, the insertion of helix G cannot be differentiated from the average protein behaviour. The data show that, while folding of bacteriorhodopsin from SDS into lipids is a co-operative process, it is nevertheless possible to obtain information on specific regions of a membrane protein during folding in vitro.
Compton
Emma L R
EL
Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, UK.
Farmer
Nicola A
NA
Lorch
Mark
M
Mason
Jody M
JM
Moreton
Kathleen M
KM
Booth
Paula J
PJ
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry; D015394:Molecular Structure; D009154:Mutation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011720:Pyrazoles / Q000737:chemistry; D013816:Thermodynamics
D005456:Fluorescent Dyes; D008051:Lipid Bilayers; D011720:Pyrazoles; D001436:Bacteriorhodopsins; C059597:monochlorobimane; D003545:Cysteine