Direct ATP photolabeling of Escherichia coli recA proteins: identification of regions required for ATP binding
When the Escherichia coli RecA protein is UV irradiated in the presence of (alpha-/sup 32/P)ATP, a labeled protein--ATP adduct is formed. All the experimental evidence indicates that, in forming such an adduct, the ATP becomes specifically immobilized in the catalytically relevant ATP binding site. The adduct can also be identified after irradiation of E. coli cell lysates in a similar manner. This direct ATP photolabeling of RecA proteins has been used to identify regions of the polypeptide chain involved in the binding of ATP. The photolabeling of a RecA protein that lacks wild-type carboxy-terminal amino acids is not detectable. A RecA protein in which the amino-terminal sequence NH2-Ala-Ile-Asp-Glu-Asn- is replaced by NH2-Thr-Met-Ile-Thr-Asn-Ser-Ser-Ser- is only about 5% as efficiently photolabeled as the wild-type protein. Both of these RecA protein constructions, however, contain all the elements previously implicated, directly or indirectly, in the binding of ATP. ATP-photolabeled RecA protein has also been chemically cleaved at specific amino acids in order to identify regions of the polypeptide chain to which the nucleotide becomes covalently photolinked. The evidence is consistent with a region comprising amino acids 116-170. Thus, this work and that of others suggest that several disparate regions of the unfolded polypeptide chain may combine to form the ATP binding site upon protein folding or may influence binding through long-range effects.
- Research Organization:
- National Institute for Medical Research, London, England
- OSTI ID:
- 6790584
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 20; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
560120* -- Radiation Effects on Biochemicals
Cells
& Tissue Culture
59 BASIC BIOLOGICAL SCIENCES
63 RADIATION, THERMAL, AND OTHER ENVIRON. POLLUTANT EFFECTS ON LIVING ORGS. AND BIOL. MAT.
AMINO ACID SEQUENCE
ATP
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
CELL CONSTITUENTS
CHEMISTRY
COVALENCE
DAYS LIVING RADIOISOTOPES
ELECTROMAGNETIC RADIATION
ESCHERICHIA COLI
IRRADIATION
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
LABELLING
LIGHT NUCLEI
MEMBRANE PROTEINS
MICROORGANISMS
MOLECULAR STRUCTURE
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
PEPTIDES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
PHOTOCHEMISTRY
PLASMIDS
POLYPEPTIDES
PROTEINS
RADIATIONS
RADIOISOTOPES
REACTION KINETICS
RECEPTORS
TRACER TECHNIQUES
ULTRAVIOLET RADIATION