Bacteriorhodopsin mutants containing deletions in loop B-C, ΔThr67-Glu74 or ΔGly65-Gln75, or a deletion in the loop E-F, ΔGlu161-Ala168, were prepared. Following their expression in Escherichia coli, the mutant proteins were purified to homogeneity and refolded with retinal in detergent-phospholipid mixtures. The mutants containing deletions in the loop B-C were normal at 4°C but showed the following changes at 20°C. 1) The λmax shifted from 540 to below 510 nm; 2) the rates of bleaching by hydroxylamine in the dark increased; and 3) the rate and steady state of proton pumping decreased. Deletion of the eight amino acids in loop E-F did not affect wild-type behavior. However, all the mutant proteins were more prone to thermal and sodium dodecyl sulfate denaturation than the wild-type bacteriorhodopsin. These observations show that the structures of the B-C and E-F loops are not essential for correct folding of bacteriorhodopsin, but they contribute to the stability of the folded protein.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - May 5 1991|
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