title: Quantitative measurement of water diffusion lifetimes at a protein/DNA interface by NMR.
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 relayed water 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 structures of homeodomain/DNA complexes reveals a strong correspondence between water molecules in conserved locations and the slowly diffusing water molecules detected by NMR. A homology model based on the X-ray structures was created to 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 all known homeodomain/DNA structures, is critical for binding affinity and specificity.
authors.affiliation: Laboratory of Biophysical Chemistry, National Heart Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892-0380, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Gruschus
authors.forename: J A
authors.initials: JA
authors.lastname: Ferretti
mesh_terms: D004247:DNA / Q000737:chemistry*; D004058:Diffusion; D029721:Drosophila Proteins; D018398:Homeodomain Proteins / Q000737:chemistry; D046911:Macromolecular Substances; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D011506:Proteins / Q000737:chemistry*; D014157:Transcription Factors; D014867:Water / Q000737:chemistry; D014961:X-Ray Diffraction
chemical_list: D029721:Drosophila Proteins; D018398:Homeodomain Proteins; D046911:Macromolecular Substances; D011506:Proteins; D014157:Transcription Factors; C094797:vnd protein, Drosophila; D014867:Water; D004247:DNA