Structure and function in bacteriorhodopsin: the effect of the interhelical loops on the protein folding kinetics.
The loops connecting the seven transmembrane helices of bacteriorhodopsin have each been replaced in turn by structureless linkers of Gly-Gly-Ser repeat sequences, and the effect on the protein folding kinetics has been determined. An SDS-denatured state of each loop mutant bacterio-opsin was folded in l-alpha-1,2-dihexanoylphosphatidylcholine/l-alpha-1,2-dimyristoylphosphatidylcholine micelles, containing retinal, to give functional bacteriorhodopsin. Stopped-flow mixing was used to initiate the folding reaction, giving a time resolution of milliseconds, and changes in protein fluorescence were used to monitor folding. All loop mutant proteins folded according to the same reaction scheme as wild-type protein. The folding kinetics of the AB, BC and DE loop mutants were the same as wild-type protein, despite the blue-shifted chromophore band of the BC loop mutant bR state. A partially folded apoprotein intermediate state of the AB loop mutant did however appear to decay in the absence of retinal. The most significant effects on the folding kinetics were seen for mutant protein with structureless linkers in place of the CD, EF and FG loops. The rate-limiting apoprotein folding step of the CD loop mutant was about ten times slower than wild-type, whilst that of the EF loop mutant was almost four times slower than wild-type. Wild-type behaviour was observed for the other folding and retinal binding events of the CD and EF loop mutant proteins. These effects of the CD and EF loop mutations on apoprotein folding correlate with the fact that these two loop mutants also have the least stable, partially folded apoprotein intermediate of all the loop mutants, and are the most affected by a decrease in lipid lateral pressure. In contrast, the FG loop mutant exhibited wild-type apoprotein folding, but altered covalent binding of retinal and final folding to bacteriorhodopsin. This correlates with the fact that the FG loop mutant bacteriorhodopsin is the most susceptible to denaturation by SDS of all the loop mutants, but its partially folded apoprotein intermediate is more stable than that of the CD and EF mutants. Thus the CD and EF loops may contribute to the transition state for the rate-limiting apoprotein folding step and the FG loop to that for final folding and covalent binding of retinal.
Allen
S J
SJ
Department of Biochemistry, Imperial College of Science, Technology and Medicine, London, SW7 2AY, UK.
Kim
J M
JM
Khorana
H G
HG
Lu
H
H
Booth
P J
PJ
D001059:Apoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002793:Cholic Acids / Q000378:metabolism; D004134:Dimyristoylphosphatidylcholine / Q000378:metabolism; D005453:Fluorescence; D019617:Halobacterium salinarum / Q000737:chemistry* / Q000235:genetics; D007700:Kinetics; D008823:Micelles; D009154:Mutation / Q000235:genetics; D010742:Phospholipid Ethers / Q000378:metabolism; D017510:Protein Folding; D020673:Protein Renaturation; D012172:Retinaldehyde / Q000378:metabolism; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship; D013816:Thermodynamics
C052298:1,2-dihexadecyl-sn-glycero-3-phosphocholine; D001059:Apoproteins; D002793:Cholic Acids; D008823:Micelles; D010742:Phospholipid Ethers; D012967:Sodium Dodecyl Sulfate; D001436:Bacteriorhodopsins; C028213:3-((3-cholamidopropyl)dimethylammonium)-1-propanesulfonate; D012172:Retinaldehyde; D004134:Dimyristoylphosphatidylcholine