Site-directed mutagenesis of two conserved charged amino acids in the N-terminal region of alpha subunit of E. coli-F(0)F(1)

FEBS Letters
B HaseH Strotmann

Abstract

Two conserved charged amino acids of the N-terminal 'crown' region of the alpha subunit of E. coli-F(1), alpha-D36 and alpha-R40 were exchanged for chemically related (alpha-D36-->E, alpha-R40-->K) or unrelated amino acids (alpha D-36-->K, alpha R40-->G), respectively, by employing oligonucleotide-directed mutagenesis. ATP formation and ATP hydrolyzing activity of isolated plasma membrane vesicles was strongly inhibited in mutant HS2 (alpha-D36-->K), but only slightly affected in the other mutants. The inhibition is not due to a lower content of F0F1 in HS2. In this mutant the extent of the proton gradient generated by ATP hydrolysis was more than 80% inhibited; in all other transformants much smaller effects were observed. The proton gradient established by NADH oxidation was 33% decreased in HS2, but was decreased to a lesser extent in all other mutants. After blockage of F0 by DCCD treatment, the same NADH-induced proton gradient was obtained in all transformants including HS2. This and the fact that the activity of NADH oxidation was unchanged indicate increased proton leakiness of F0F1 carrying the alpha-D36-->K mutation. In F1 alpha-D36 is located in a domain contacting the beta subunit in the vicinity of the arginine bet...Continue Reading

References

Jan 1, 1990·Annual Review of Biophysics and Biophysical Chemistry·A E Senior
Apr 1, 1989·Proceedings of the National Academy of Sciences of the United States of America·C T ChungR H Miller
Sep 6, 1984·Biochimica Et Biophysica Acta·J E WalkerN J Gay
Jan 1, 1983·Journal of Bacteriology·R HumbertR D Simoni

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Citations

Apr 28, 2004·Molecular and Biochemical Parasitology·Robert E NelsonLarry Simpson
Dec 1, 1996·Trends in Microbiology·S Romagnani
May 23, 2006·Proceedings of the National Academy of Sciences of the United States of America·Srikanta GoswamiSamit Adhya
May 20, 2008·Advances in Applied Microbiology·R E ParalesJ L Ditty
May 29, 1999·The Journal of Biological Chemistry·N BakhtiariD M Mueller
Feb 12, 2000·Journal of Molecular Biology·B BöttcherP Gräber

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