Structure-function studies of bacteriorhodopsin XV

Effects of deletions in loops B-C and E-F on bacteriorhodopsin chromophore and structure

Marie A. Gilles-Gonzalez, Donald M. Engelman, H. Gobind Khorana

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

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 languageEnglish (US)
Pages (from-to)8545-8550
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number13
StatePublished - May 5 1991

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Bacteriorhodopsins
Chromophores
Mutant Proteins
Hydroxylamine
Denaturation
Protein Stability
Escherichia coli Proteins
Bleaching
Sodium Dodecyl Sulfate
Detergents
Escherichia coli
Protons
Phospholipids
Hot Temperature
Amino Acids
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Structure-function studies of bacteriorhodopsin XV : Effects of deletions in loops B-C and E-F on bacteriorhodopsin chromophore and structure. / Gilles-Gonzalez, Marie A.; Engelman, Donald M.; Khorana, H. Gobind.

In: Journal of Biological Chemistry, Vol. 266, No. 13, 05.05.1991, p. 8545-8550.

Research output: Contribution to journalArticle

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AB - 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.

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