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Methods used to determine the Nef structure by NMR at pH 8 and 0.6 mM concentration are presented, together with a detailed description of Nef's secondary and tertiary structure. The described techniques have general applicability for the NMR structure determination of proteins that are aggregating and\/or have limited stability at low pH values. Extensive chemical shift assignments are reported for backbone and side chain <jats:sup>1<\/jats:sup>H, <jats:sup>13<\/jats:sup>C, and <jats:sup>15<\/jats:sup>N resonances of the HIV\u20101 Nef deletion mutants NEF<jats:sup>\u03b42\u201039<\/jats:sup>, NEF<jats:sup>\u03b42\u201039,\u03b4159\u2010173<\/jats:sup>, and of NEF<jats:sup>\u03b42\u201039,\u03b4159\u2010173<\/jats:sup> in complex with the SH3 domain of the Hck tyrosine protein kinase. Besides a type II polyproline helix, Nef's structure consists of three \u03b1\u2010helices, a 3<jats:sub>10<\/jats:sub> helix, and a five\u2010stranded anti\u2010parallel \u03b2\u2010sheet. The analysis of <jats:sup>15<\/jats:sup>N relaxation parameters of the backbone amide sites reveals that all the secondary structure elements are non\u2010mobile on the picosecond to nanosecond and on the millisecond time scale. A large number of slowly exchanging amide protons provides evidence for the stability of the Nef core even on the time scale of hours. Significant internal motions on the ps to ns time scale are detected for residues 60 to 71 and for residues 149 to 180, which form solvent\u2010exposed loops. The residues of the HIV\u20101 protease cleavage site (W57\/L58) do not exhibit large amplitude motions on the sub\u2010nanosecond time scale, and their side chains insert themselves into a hydrophobic crevice formed between the C\u2010terminus of helix 1 and the N\u2010terminus of helix 2. A refined structure has been determined based on additional constraints for side\u2010chain and backbone dihedral angles derived from a large number of three\u2010bond J\u2010couplings and ROE data.<\/jats:p>","DOI":"10.1002\/pro.5560060613","type":"journal-article","created":{"date-parts":[[2009,2,10]],"date-time":"2009-02-10T05:41:51Z","timestamp":1234244511000},"page":"1248-1263","source":"Crossref","is-referenced-by-count":141,"title":["Refined solution structure and backbone dynamics of HIV\u20101 Nef"],"prefix":"10.1002","volume":"6","author":[{"given":"Stephan","family":"Grzesiek","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ad","family":"Bax","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jin\u2010Shan","family":"Hu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nico","family":"Tjandra","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Joshua","family":"Kaufman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ira","family":"Palmer","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephen J.","family":"Stahl","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul 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