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Titolo:
Quantitative measurement of water diffusion lifetimes at a protein/DNA interface by NMR
Autore:
Gruschus, JM; Ferretti, JA;
Indirizzi:
NHLBI, Biophys Chem Lab, NIH, Bethesda, MD 20892 USA NHLBI Bethesda MD USA 20892 Biophys Chem Lab, NIH, Bethesda, MD 20892 USA
Titolo Testata:
JOURNAL OF BIOMOLECULAR NMR
fascicolo: 2, volume: 20, anno: 2001,
pagine: 111 - 126
SICI:
0925-2738(2001)20:2<111:QMOWDL>2.0.ZU;2-I
Fonte:
ISI
Lingua:
ENG
Soggetto:
HOMEODOMAIN-DNA COMPLEX; VND/NK-2 HOMEODOMAIN; CRYSTAL-STRUCTURE; ANGSTROM RESOLUTION; HYDRATION WATER; SPECTROSCOPY; MOLECULES; DYNAMICS; HELIX; MACROMOLECULES;
Keywords:
B-factors; binding specificity and affinity; conserved water; cross-relaxation; homeodomain; hydrogen bond; relay magnetization transfer; ROE and NOE; temperature factors; water residence time; X-ray;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
36
Recensione:
Indirizzi per estratti:
Indirizzo: Ferretti, JA NHLBI, Biophys Chem Lab, NIH, Bethesda, MD 20892 USA NHLBI Bethesda MD USA 20892 Lab, NIH, Bethesda, MD 20892 USA
Citazione:
J.M. Gruschus e J.A. Ferretti, "Quantitative measurement of water diffusion lifetimes at a protein/DNA interface by NMR", J BIOM NMR, 20(2), 2001, pp. 111-126

Abstract

Hydration site lifetimes of slowly diffusing water molecules at the protein/DNA interface of the vnd/NK-2 homeodomain DNA complex were determined using novel three-dimensional NMR techniques. The lifetimes were calculated using the ratios of ROE and NOE cross-relaxation rates between the water and the protein backbone and side chain amides. This calculation of the lifetimes is based on a model of the spectral density function of the water-protein interaction consisting of three timescales of motion: fast vibrational/rotational motion, diffusion into/out of the hydration site, and overall macromolecular tumbling. The lifetimes measured ranged from approximately 400 ps to more than 5 ns, and nearly all the slowly diffusing water molecules detected lie at the protein/DNA interface. A quantitative analysis of relayedwater cross-relaxation indicated that even at very short mixing times, 5 ms for ROESY and 12 ms for NOESY, relay of magnetization can make a small but detectable contribution to the measured rates. The temperature dependences of the NOE rates were measured to help discriminate direct dipolar cross-relaxation from chemical exchange. Comparison with several X-ray structuresof homeodomain/DNA complexes reveals a strong correspondence between watermolecules in conserved locations and the slowly diffusing water molecules detected by NMR. A homology model based on the X-ray structures was createdto visualize the conserved water molecules detected at the vnd/NK-2 homeodomain DNA interface. Two chains of water molecules are seen at the right and left sides of the major groove, adjacent to the third helix of the homeodomain. Two water-mediated hydrogen bond bridges spanning the protein/DNA interface are present in the model, one between the backbone of Phe8 and a DNA phosphate, and one between the side chain of Asn51 and a DNA phosphate. The hydrogen bond bridge between Asn51 and the DNA might be especially important since the DNA contact made by the invariant Asn51 residue, seen in allknown homeodomain/DNA structures, is critical for binding affinity and specificity.

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Documento generato il 05/12/20 alle ore 13:06:53