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Titolo:
ACTIVATION OF THE PHOSPHOSIGNALING PROTEIN CHEY .1. ANALYSIS OF THE PHOSPHORYLATED CONFORMATION BY F-19 NMR AND PROTEIN ENGINEERING
Autore:
DRAKE SK; BOURRET RB; LUCK LA; SIMON MI; FALKE JJ;
Indirizzi:
UNIV COLORADO,DEPT CHEM & BIOCHEM BOULDER CO 80309 UNIV COLORADO,DEPT CHEM & BIOCHEM BOULDER CO 80309 CALTECH,DIV BIOL PASADENA CA 91125
Titolo Testata:
The Journal of biological chemistry
fascicolo: 18, volume: 268, anno: 1993,
pagine: 3081 - 3088
SICI:
0021-9258(1993)268:18<3081:AOTPPC>2.0.ZU;2-8
Fonte:
ISI
Lingua:
ENG
Soggetto:
NUCLEAR-MAGNETIC-RESONANCE; GALACTOSE CHEMOSENSORY RECEPTOR; D-LACTATE DEHYDROGENASE; ESCHERICHIA-COLI; BACTERIAL CHEMOTAXIS; SIGNAL TRANSDUCTION; CRYSTAL-STRUCTURE; RESPONSE REGULATOR; SALMONELLA-TYPHIMURIUM; CONSERVED ASPARTATE;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
62
Recensione:
Indirizzi per estratti:
Citazione:
S.K. Drake et al., "ACTIVATION OF THE PHOSPHOSIGNALING PROTEIN CHEY .1. ANALYSIS OF THE PHOSPHORYLATED CONFORMATION BY F-19 NMR AND PROTEIN ENGINEERING", The Journal of biological chemistry, 268(18), 1993, pp. 3081-3088

Abstract

CheY, the 14-kDa response regulator protein of the Escherichia coli chemotaxis pathway, is activated by phosphorylation of Asp57. In order to probe the structural changes associated with activation, an approach which combines F-19 NMR, protein engineering, and the known crystal structure of one conformer has been utilized. This first of two papersexamines the effects of Mg(II) binding and phosphorylation on the conformation of CheY. The molecule was selectively labeled at its six phenylalanine positions by incorporation of 4-fluorophenylalanine, which yielded no significant effect on activity. One of these F-19 probe positions monitored the vicinity of Lys109, which forms a salt bridge to Asp57 in the apoprotein and has been proposed to act as a structural ''switch'' in activation. F-19 NMR chemical shift studies of the labeled protein revealed that the binding of the cofactor Mg(II) triggered local structural changes in the activation site, but did not perturb the probe of the Lys109 region. The structural changes associated with phosphorylation were then examined, utilizing acetyl phosphate to chemically generate phospho-CheY during NMR acquisition. Phosphorylation triggered a long-range conformational change extending from the activation site to a cluster of 4 phenylalanine residues at the other end of the molecule. However, phosphorylation did not perturb the probe of Lys109. The observed phosphorylated conformer is proposed to be the firststep in the activation of CheY; later steps appear to perturb Lys109,as evidenced in the following paper. Together these results may give insight into the activation of other prokaryotic response regulators.

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Documento generato il 26/11/20 alle ore 20:27:21