Nucleotide binding to the C-terminal nucleotide binding domain of ArsA. Studies with an ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine.

The Journal of Biological Chemistry
S Ramaswamy, P Kaur

Abstract

ArsA protein, the catalytic component of the plasmid-encoded anion-translocating ATPase in Escherichia coli, contains two consensus nucleotide binding domains, A1 and A2, that are connected by a flexible linker. ATP has previously been shown to cross-link to the A1 domain upon activation with UV light but not to the A2 domain. The ATP analogue, 5'-p-fluorosulfonylbenzoyladenosine (FSBA) was used to probe the nucleotide binding domains of ArsA. The covalently labeled protein was subjected to partial trypsin proteolysis, followed by Western blot analysis of the fragments with the anti-FSBA serum. The N-terminal amino acid sequence of the labeled fragment showed that FSBA binds preferentially to the C-terminal domain A2 both in the absence and the presence of antimonite. Occupancy of the two nucleotide binding sites was determined by protection from trypsin proteolysis. Trypsin cleaved the ArsA protein at Arg290 in the linker to generate a 32-kDa N-terminal and a 27-kDa C-terminal fragment. The 32-kDa fragment is compact and largely inaccessible to trypsin; however, the 27-kDa was cleaved further. Incubation with FSBA, which binds to the C-terminal domain, resulted in significant protection of the 27-kDa fragment. This fragment wa...Continue Reading

References

Aug 1, 1989·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·P D Boyer
Jan 1, 1983·Annual Review of Biochemistry·R F Colman
Oct 11, 1996·The Journal of Biological Chemistry·J LiB P Rosen

❮ Previous
Next ❯

Citations

Dec 17, 2009·Biochemistry·Hsueh-Liang FuHiranmoy Bhattacharjee
Jan 29, 2000·Molecular Microbiology·J Li, B P Rosen
Jul 5, 2008·Neuropharmacology·Kelvin C AgbohRichard J Evans
May 29, 1999·The Journal of Biological Chemistry·A R WalmsleyB P Rosen
Aug 28, 1999·The Journal of Biological Chemistry·P Kaur
Jan 8, 2005·The Journal of Biological Chemistry·Yong JiangZuben E Sauna
Jun 17, 2008·Biochemistry·Hiranmoy BhattacharjeeBarry P Rosen

❮ Previous
Next ❯

Related Concepts

Related Feeds

ASBMB Publications

The American Society for Biochemistry and Molecular Biology (ASBMB) includes the Journal of Biological Chemistry, Molecular & Cellular Proteomics, and the Journal of Lipid Research. Discover the latest research from ASBMB here.