Home About us Contact
|
Home About us Contact | ||
|
|
||
Residue Protein (residue + protein)
Selected AbstractsThe histidine-phosphocarrier protein of Streptomyces coelicolor folds by a partially folded species at low pHFEBS JOURNAL, Issue 10 2003Gregorio Fernández-Ballester The folding of a 93-residue protein, the histidine-phosphocarrier protein of Streptomyces coelicolor, HPr, has been studied using several biophysical techniques, namely fluorescence, 8-anilinonaphthalene-1-sulfate binding, circular dichroism, Fourier transform infrared spectroscopy, gel filtration chromatography and differential scanning calorimetry. The chemical-denaturation behaviour of HPr, followed by fluorescence, CD and gel filtration, at pH 7.5 and 25 °C, is described as a two-state process, which does not involve the accumulation of thermodynamically stable intermediates. Its conformational stability under those conditions is ,G = 4.0 ± 0.2 kcal·mol,1 (1 kcal = 4.18 kJ), which makes the HPr from S. coelicolor the most unstable member of the HPr family described so far. The stability of the protein does not change significantly from pH 7,9, as concluded from the differential scanning calorimetry and thermal CD experiments. Conformational studies at low pH (pH 2.5,4) suggest that, in the absence of cosmotropic agents, HPr does not unfold completely; rather, it accumulates partially folded species. The transition from those species to other states with native-like secondary and tertiary structure, occurs with a pKa = 3.3 ± 0.3, as measured by the averaged measurements obtained by CD and fluorescence. However, this transition does not agree either with: (a) that measured by burial of hydrophobic patches (8-anilinonaphthalene-1-sulfate binding experiments); or (b) that measured by acquisition of native-like compactness (gel-filtration studies). It seems that acquisition of native-like features occurs in a wide pH range and it cannot be ascribed to a unique side-chain titration. These series of intermediates have not been reported previously in any member of the HPr family. [source] Cell-free expression and selective isotope labelling in protein NMRMAGNETIC RESONANCE IN CHEMISTRY, Issue S1 2006David Staunton Abstract Isotope labelling is a very powerful tool in NMR studies of proteins and has been employed in various ways for over 40 years. 15N and 13C incorporation, using recombinant expression systems, is now commonplace because heteronuclear experiments assist with the fundamental problems of peak resolution and assignment. The use of selective labelling for peak assignment has been restricted by the scrambling of isotope label through metabolic pathways within the expression host organism. The availability of efficient cell-free expression systems with low levels of metabolic conversion allow the increasing use of selective isotope labelling as a tool in protein NMR. We describe two examples, one where a selective labelling scheme can identify backbone amide peaks from unassigned 1H15N HSQC and HNCO spectra of a 84 residue protein, and another where a specific backbone amide in a 198 residue construct of the ninth and tenth Type III repeats from human fibronectin can be labelled and rapidly identified using a simple HSQC experiment. Copyright © 2006 John Wiley & Sons, Ltd. [source] Solution structure of HI1506, a novel two-domain protein from Haemophilus influenzaePROTEIN SCIENCE, Issue 5 2007Nese Sari Abstract HI1506 is a 128-residue hypothetical protein of unknown function from Haemophilus influenzae. It was originally annotated as a shorter 85-residue protein, but a more detailed sequence analysis conducted in our laboratory revealed that the full-length protein has an additional 43 residues on the C terminus, corresponding with a region initially ascribed to HI1507. As part of a larger effort to understand the functions of hypothetical proteins from Gram-negative bacteria, and H. influenzae in particular, we report here the three-dimensional solution NMR structure for the corrected full-length HI1506 protein. The structure consists of two well-defined domains, an ,/, 50-residue N-domain and a 3-, 32-residue C-domain, separated by an unstructured 30-residue linker. Both domains have positively charged surface patches and weak structural homology with folds that are associated with RNA binding, suggesting a possible functional role in binding distal nucleic acid sites. [source] Structural studies of a baboon (Papio sp.) plasma protein inhibitor of cholesteryl ester transferasePROTEIN SCIENCE, Issue 8 2000Garry W. Buchko Abstract A 38-residue protein associated with cholesteryl ester transfer inhibition has been identified in baboons (Papio sp.). The cholesteryl ester transfer inhibitor protein (CETIP) corresponds to the N-terminus of baboon apoC-I. Relative to CETIP, baboon apoC-I is a weak inhibitor of baboon cholesteryl ester transferase (CET). To study the structural features responsible for CET inhibition, CETIP was synthesized by solid-phase methods. Using sodium dodecyl sulfate (SDS) to model the lipoprotein environment, the solution structure of CETIP was probed by optical and 1HNMR spectroscopy. Circular dichroism data show that the protein lacks a well-defined structure in water but, upon the addition of SDS, becomes helical (56%). A small blue shift of 8 nm was observed in the intrinsic tryptophan fluorescence of CETIP in the presence of saturating amounts of SDS, suggesting that tryptophan-23 is not buried deeply in the lipid environment. The helical nature of CETIP in the presence of SDS was confirmed by upfield 1H, secondary shifts and an average solution structure determined by distance geometry/simulated annealing calculations using 476 NOE-based distance restraints. The backbone (N , C, , C, = O ) root-mean-square deviation of an ensemble of 17 out of 25 calculated structures superimposed on the average structure was 1.06 ± 0.30 Ĺ using residues V4-P35 and 0.51 ± 0.17 Ĺ using residues A7-S32. Although the side-chain orientations fit the basic description of a class A amphipathic helix, both intramolecular salt bridge formation and "snorkeling" of basic side chains toward the polar face play minor, if any, roles in stabilizing the lipid-bound amphipathic structure. Conformational features of the calculated structures for CETIP are discussed relative to models of CETIP inhibition of cholesteryl ester transferase. [source] The X-ray structure of a chitinase from the pathogenic fungus Coccidioides immitisPROTEIN SCIENCE, Issue 3 2000Thomas Hollis Abstract The X-ray structure of chitinase from the fungal pathogen Coccidioides immitis has been solved to 2.2 Ĺ resolution. Like other members of the class 18 hydrolase family, this 427 residue protein is an eight-stranded ,/,-barrel. Although lacking an N-terminal chitin anchoring domain, the enzyme closely resembles the chitinase from Serratia marcescens. Among the conserved features are three cis peptide bonds, all involving conserved active site residues. The active site is formed from conserved residues such as tryptophans 47, 131, 315, 378, tyrosines 239 and 293, and arginines 52 and 295. Glu171 is the catalytic acid in the hydrolytic mechanism; it was mutated to a Gln, and activity was abolished. Allosamidin is a substrate analog that strongly inhibits the class 18 enzymes. Its binding to the chitinase hevamine has been observed, and we used conserved structural features of the two enzymes to predict the inhibitors binding to the fungal enzyme. [source] Novel cytochrome P450s, CYP6BB1 and CYP6P10, from the salt marsh mosquito Aedes sollicitans (Walker) (Diptera: Culicidae)ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2008Shaoming Huang Abstract Based on the conserved heme-binding region and the charge pair consensus of insect cytochrome P450s, two novel full-length P450 cDNAs, CYP6BB1 and CYP6P10, were cloned from the salt marsh mosquito Aedes sollicitans (Walker). CYP6BB1 and CYP6P10 had open reading frames of 1,518 and 1,521 nucleotides encoding 506 and 507 amino acid residue proteins, respectively. Several alleles with amino acid substitutions were found both in CYP6BB1 and CYP6P10. The deduced proteins are typical microsomal P450s sharing signature sequences with other insect CYP6 P450s. Sequence analysis showed that both CYP6BB1 and CYP6P10 shared highest sequence identities with P450 CYP6P4, 56% and 65%, respectively. Phylogenetic analysis showed both CYP6BB1 and CYP6P10 were grouped into the clade containing several P450s from subfamily CYP6P. Real-time RT-PCR analysis showed CYP6BB1 but not CYP6P10 transcription in females was significantly increased 24 h after a blood meal. Neither CYP6BB1 nor CYP6P10 were life stage or gender specific. Protein expression experiments are needed to determine the functions of these proteins. Arch. Insect Biochem. Physiol. 2007. © 2007 Wiley-Liss, Inc. [source] Identification of the ornithine decarboxylase gene in the putrescine-producer Oenococcus oeni BIFI-83FEMS MICROBIOLOGY LETTERS, Issue 2 2004Angela Marcobal Abstract We report here the identification of an ornithine decarboxylase (ODC) gene in the putrescine-producer Oenococcus oeni BIFI-83 strain. The gene contains a 2,235-nucleotide open reading frame encoding a 745-amino acid residues protein with a deduced molecular mass of 81 kDa. The primary structure of the ODC deduced from the nucleotide sequence has a consensus sequence containing the pyridoxal-5-phosphate (PLP) binding domain, and the critical amino acids residues involved in enzymatic activity are also conserved. As determined by BLAST analysis, the deduced amino acid sequence of the odc gene shares a 67% identity with the ODC protein from Lactobacillus 30a. The odc gene appears to be rarely present in the genome of O. oeni, since in a screening for the presence of this gene in 42 oenococcal strains none of the strains possessed an odc gene copy. [source] | ||||||||||