The structure of a mutant Antennapedia homeodomain, Antp(C39---S), from Drosophila melanogaster was determined using a set of new programs introduced in the accompanying paper. An input dataset of ...957 distance constraints and 171 dihedral angle constraints was collected using two-dimensional n.m.r. experiments with the 15N-labeled protein. The resulting high quality structure for Antp(C39---S), with an average root-mean-square deviation of 0.53 A between the backbone atoms of residues 7 to 59 in 20 energy-refined distance geometry structures and the mean structure, is nearly identical to the previously reported structure of the wild-type Antp homeodomain. The only significant difference is in the connection between helices III and IV, which was found to be less kinked than was indicated by the structure determination for Antp. The main emphasis of the presentation in this paper is on a detailed account of the practical use of a novel strategy for the computation of nuclear magnetic resonance structures of proteins with the combined use of the programs DIANA, CALIBA, HABAS and GLOMSA.
Pseudocontact shifts (PCSs) induced by a site-specifically bound paramagnetic lanthanide ion are shown to provide fast access to sequence- specific resonance assignments of methyl groups in proteins ...of known three- dimensional structure. Stereospecific assignments of Val and Leu methyls are obtained as well as resonance assignments of all other methyls, including Met epsilonCH sub(3) groups. No prior assignments of the diamagnetic protein are required nor are experiments that transfer magnetization between the methyl groups and the protein backbone. Methyl C sub(z)-exchange experiments were designed to provide convenient access to PCS measurements in situations where a paramagnetic lanthanide is in exchange with a diamagnetic lanthanide. In the absence of exchange, simultaneous super(13)C-HSQC assignments and PCS measurements are delivered by the newly developed program Possum. The approaches are demonstrated with the complex between the N-terminal domain of the subunit epsilon and the subunit theta of the Escherichia coli DNA polymerase III.
The p75 neurotrophin receptor (p75NTR) contains a conserved death domain module similar to that of the cytotoxic receptors Fas and TNFR-1. Here, we describe the selection of peptide ligands from a ...combinatorial library using a variation of the selectively-infective phage (SIP) method directed to the death domain of p75NTR. The binding sites on the death domain of p75NTRwere identified for a 15 amino acid residue peptide by nuclear magnetic resonance (NMR) spectroscopy. The selected peptides may be useful for probing the function of the p75NTRdeath domain and aid in defining its downstream signalling mechanism.
Peptide segments of multiple glycine and alanine residues prevent the proteolytic degradation of ubiquitinated proteins by the proteasome. The structure of a Gly/Ala-rich insert in IκBα was probed by ...nuclear magnetic resonance (NMR) spectroscopy, comparing IκBα samples with and without Gly/Ala-rich insert. Narrow
1H-NMR resonances at chemical shifts indicative of random coil conformations were observed in the difference spectrum. circular dichroism (CD) measurements further confirm that the mechanism of protection against proteolytic degradation is not based on structural transition or stabilization caused by the Gly/Ala-rich segment. In addition, most of the N- and C-terminal residues outside the ankyrin repeats in wild-type IκBα were found to be flexibly disordered.
Schnell und sauber: Ausgezeichnete Ausbeuten in der zellfreien Proteinherstellung können mithilfe von PCR‐amplifizierter DNA erhalten werden, vorausgesetzt die Template sind zur Cyclisierung ...geeignet. Damit werden zeitraubende Klonierungsschritte umgangen, und man gelangt binnen 24 Stunden von cDNA zu einem Protein‐NMR‐Spektrum.
