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Multidomain Proteins (multidomain + protein)
Selected AbstractsFolding of epidermal growth factor-like repeats from human tenascin studied through a sequence frame-shift approachFEBS JOURNAL, Issue 21 2004Francesco Zanuttin In order to investigate the factors that determine the correct folding of epidermal growth factor-like (EGF) repeats within a multidomain protein, we prepared a series of six peptides that, taken together, span the sequence of two EGF repeats of human tenascin, a large protein from the extracellular matrix. The peptides were selected by sliding a window of the average length of tenascin EGF repeats over the sequence of EGF repeats 13 and 14. We thus obtained six peptides, EGF-f1 to EGF-f6, that are 33 residues long, contain six cysteines each, and bear a partial overlap in the sequence. While EGF-f1 corresponds to the native EGF-14 repeat, the others are frame-shifted EGF repeats. We carried out the oxidative folding of these peptides in vitro, analyzed the reaction mixtures by acid trapping followed by LC-MS, and isolated some of the resulting products. The oxidative folding of the native EGF-14 peptide is fast, produces a single three-disulfide species with an EGF-like disulfide topology and a marked difference in the RP-HPLC retention time compared with the starting product. On the contrary, frame-shifted peptides fold more slowly and give mixtures of three-disulfide species displaying RP-HPLC retention times that are closer to those of the reduced peptides. In contrast to the native EGF-14, the three-disulfide products that could be isolated are mainly unstructured, as determined by CD and NMR spectroscopy. We conclude that both kinetics and thermodynamics drive the correct pairing of cysteines, and speculate about how cysteine mispairing could trigger disulfide reshuffling in vivo. [source] Truncation of the MLL gene in exon 5 by gene targeting leads to early preimplantation lethality of homozygous embryosGENESIS: THE JOURNAL OF GENETICS AND DEVELOPMENT, Issue 4 2001Paul Ayton Abstract Summary: The mixed lineage leukemia gene (MLL) was originally identified through its involvement in reciprocal translocations in leukemias. MLL codes for a large multidomain protein and bears homology to the Drosophila developmental control gene trithorax in two small domains in the amino terminal region, the central zinc finger domain and the carboxy SET domain. Like the Drosophila trx, MLL has also been shown to be a positive regulator of Hox gene expression. We have targeted Mll (the murine homologue of MLL) in exon 5 causing expression of three truncated in-frame Mll transcripts. These transcripts retain all or some of the AT hook motifs and the DMT domain. This mutant allele causes early in vivo preimplantation lethality of homozygous embryos prior to the 2-cell stage. Embryos cultured in vitro progress to the 2-cell stage, but further development is arrested. The heterozygotes exhibit mild skeletal defects as well as defects in some neuroectodermal derivatives. genesis 30:201,212, 2001. © 2001 Wiley-Liss, Inc. [source] Effect of metal cations on the conformation and inactivation of recombinant human factor VIIIJOURNAL OF PHARMACEUTICAL SCIENCES, Issue 10 2004Tiffany S. Derrick Abstract Heavy metals have been implicated in the aggregation of proteins and the pathophysiology of several neurodegenerative diseases. Herein, we describe the interaction of recombinant human factor VIII (rhFVIII) with Al+3, Tb+3, Co+2, and Fe+3 using a combination of intrinsic fluorescence, circular dichroism, and high-resolution fourth-derivative absorbance analysis. rhFVIII in solution was titrated with the metal cations and the properties of the resulting complexes were examined. rhFVIII has a tendency to aggregate and inactivate slowly over time under physiological conditions, but this aggregation process is greatly accelerated in the presence of metals with Al+3 being the most efficient. This leads to a complete loss of activity of the protein. Al+3 -induced conformational changes in the protein were small but detectable with limited changes seen in secondary and tertiary structure. Because rhFVIII is a multidomain protein with subunits linked through divalent metal cations, the small intramolecular changes seen may be attributed to rearrangements of the subunits to an aggregation-competent conformer that is very similar to that of the native form. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:2549,2557, 2004 [source] Design of Peptide Hydroxamate-Based Photoreactive Activity-Based Probes of Zinc-Dependent MetalloproteasesEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 11 2010Paul P. Geurink Abstract Metalloproteases (ADAMs, MMPs) are multidomain proteins that play key roles in extracellular matrix remodelling and degradation, in cell,cell and cell,matrix interactions and in the proteolytic liberation of membrane-anchored proforms of cytokines and growth factors, the so-called ectodomainshedding. In this work we describe the development ofphotoactivatable activity-based probes with which active metalloproteases can be visualised. Our probes are based on the succinyl hydroxamate motif and differ in the positioning of the trifluoromethylphenyldiazirine photoreactive group. We demonstrate that directing the photoactivatable group towards the S1, pocket yields activity-based probes more effective than the corresponding probe with the photoactivatable group directed towards the S2, pocket. [source] Relation between domain evolution, specificity, and taxonomy of the ,-amylase family members containing a C-terminal starch-binding domainFEBS JOURNAL, Issue 4 2003tefan Jane The ,-amylase family (glycoside hydrolase family 13; GH 13) contains enzymes with approximately 30 specificities. Six types of enzyme from the family can possess a C-terminal starch-binding domain (SBD): ,-amylase, maltotetraohydrolase, maltopentaohydrolase, maltogenic ,-amylase, acarviose transferase, and cyclodextrin glucanotransferase (CGTase). Such enzymes are multidomain proteins and those that contain an SBD consist of four or five domains, the former enzymes being mainly hydrolases and the latter mainly transglycosidases. The individual domains are labelled A [the catalytic (,/,)8 -barrel], B, C, D and E (SBD), but D is lacking from the four-domain enzymes. Evolutionary trees were constructed for domains A, B, C and E and compared with the ,complete-sequence tree'. The trees for domains A and B and the complete-sequence tree were very similar and contain two main groups of enzymes, an amylase group and a CGTase group. The tree for domain C changed substantially, the separation between the amylase and CGTase groups being shortened, and a new border line being suggested to include the Klebsiella and Nostoc CGTases (both four-domain proteins) with the four-domain amylases. In the ,SBD tree' the border between hydrolases (mainly,-amylases) and transglycosidases (principally CGTases) was not readily defined, because maltogenic ,-amylase, acarviose transferase, and the archaeal CGTase clustered together at a distance from the main CGTase cluster. Moreover the four-domain CGTases were rooted in the amylase group, reflecting sequence relationships for the SBD. It appears that with respect to the SBD, evolution in GH 13 shows a transition in the segment of the proteins C-terminal to the catalytic (,/,)8 -barrel(domain A). [source] PDK1 and PKB/Akt: Ideal Targets for Development of New Strategies to Structure-Based Drug DesignIUBMB LIFE, Issue 3 2003Thomas Harris Abstract Growth factor binding events to receptor tyrosine kinases result in activation of phosphatidylinositol 3-kinase (PI3K), and activated PI3K generates the membrane-bound second messengers phosphatidylinositol 3,4-diphosphate [PI(3,4)P2] and PI(3,4,5)P3, which mediate membrane translocation of the phosphoinositide-dependent kinase-1 (PDK1) and protein kinase B (PKB, also known as Akt). In addition to the kinase domain, PDK1 and PKB contain a pleckstrin homology (PH) domain that binds to the second messenger, resulting in the phosphorylation and activation of PKB by PDK1. Recent evidence indicates that constitutive activation of PKB contributes to cancer progression by promoting proliferation and increased cell survival. The indicating of PDK1 and PKB as primary targets for discovery of anticancer drugs, together with the observations that both PDK1 and PKB contain small-molecule regulatory binding sites that may be in proximity to the kinase active site, make PDK1 and PKB ideal targets for the development of new strategies to structure-based drug design. While X-ray structures have been reported for the kinase domains of PDK1 and PKB, no suitable crystals have been obtained for either PDK1 or PKB with their PH domains intact. In this regard, a novel structure-based strategy is proposed, which utilizes segmental isotopic labeling of the PH domain in combination with site-directed spin labeling of the kinase active site. Then, long-range distance restraints between the 15N-labeled backbone amide groups of the PH domain and the unpaired electron of the active site spin label can be determined from magnetic resonance studies of the enhancement effect that the paramagnetic spin label has on the nuclear relaxation rates of the amide protons. The determination of the structure and position of the PH domain with respect to the known X-ray structure of the kinase active site could be useful in the rational design of potent and selective inhibitors of PDK1 and PKB by 'linking' the free energies of binding of substrate (ATP) analogs with analogs of the inositol polar head group of the phospholipid second messenger. The combined use of X-ray crystallography, segmental isotopic and spin labeling, and magnetic resonance studies can be further extended to the study of other dynamic multidomain proteins and targets for structure-based drug design. IUBMB Life, 55: 117-126, 2003 [source] Applications of time-resolved resonance energy transfer measurements in studies of the molecular crowding effect,JOURNAL OF MOLECULAR RECOGNITION, Issue 5 2004Varda Ittah Abstract The native structures of many globular proteins are only weakly stabilized and form in solution ensembles of multiple conformers. The energy differences between the conformers are assumed to be small. This is the case of flexible multidomain proteins where domain motions were observed. High concentrations of inert macrosolute, which create a crowded or confined environment, can cause shifts of the distribution of the conformers of such proteins towards the more compact structures. This effect may also promote compact structures in partially folded proteins. Time-resolved dynamic non-radiative excitation energy transfer (tr-RET) is suitable for detection of either subtle or major changes in distributions of intramolecular distances in protein molecules in solutions. Two experiments were performed which demonstrated the applicability of tr-RET for detection of the effect of macrosolutes on the conformational ensembles of flexible states of protein molecules. The distribution of distances between residues 203 and 169 in the CORE domain of E. coli adenylate kinase (AK) in the denatured state was determined in the presence of high concentrations of dextran 40. A significant shift of the mean of the distribution was observed without reduction of its width. This was interpreted as a shift to compact structure without change of the degree of disorder of the chain. In a second experiment the distribution of the distance between residues 55 and 169 in AK, which spans the cleft between the CORE and the AMPbind domains, was monitored. No clear effect of high concentrations of dextran 40 was found. These experiments show the strength of the application of tr-RET in investigation of changes in the sub-states of flexible conformations of globular protein. Networks of pairs of labeled sites can be prepared and tr-RET experiments can be performed in order to search for the segments of the protein molecules, which respond to the presence of inert macromolecules in their environment. Copyright © 2004 John Wiley & Sons, Ltd. [source] Prediction of structures of multidomain proteins from structures of the individual domainsPROTEIN SCIENCE, Issue 2 2007Andrew M. Wollacott Abstract We describe the development of a method for assembling structures of multidomain proteins from structures of isolated domains. The method consists of an initial low-resolution search in which the conformational space of the domain linker is explored using the Rosetta de novo structure prediction method, followed by a high-resolution search in which all atoms are treated explicitly and backbone and side chain degrees of freedom are simultaneously optimized. The method recapitulates, often with very high accuracy, the structures of existing multidomain proteins. [source] The 1.35,Å resolution structure of the phosphatase domain of the suppressor of T-cell receptor signaling protein in complex with sulfateACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2010Jean Jakoncic The suppressor of T-cell signaling (Sts) proteins are multidomain proteins that negatively regulate the signaling of membrane-bound receptors, including the T-cell receptor (TCR) and the epidermal growth-factor receptor (EGFR). They contain at their C-terminus a 2H-phosphatase homology (PGM) domain that is responsible for their protein tyrosine phosphatase activity. Here, the crystal structure of the phosphatase domain of Sts-1, Sts-1PGM, was determined at pH 4.6. The asymmetric unit contains two independent molecules and each active site is occupied by a sulfate ion. Each sulfate is located at the phosphate-binding site and makes similar interactions with the catalytic residues. The structure suggests an explanation for the lower Michaelis,Menten constants at acidic pH. [source] Expression, purification and crystallization of the SARS-CoV macro domainACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2006Hélène Malet Macro domains or X domains are found as modules of multidomain proteins, but can also constitute a protein on their own. Recently, biochemical and structural studies of cellular macro domains have been performed, showing that they are active as ADP-ribose-1,,-phosphatases. Macro domains are also present in a number of positive-stranded RNA viruses, but their precise function in viral replication is still unknown. The major human pathogen severe acute respiratory syndrome coronavirus (SARS-CoV) encodes 16 non-structural proteins (nsps), one of which (nsp3) encompasses a macro domain. The SARS-CoV nsp3 gene region corresponding to amino acids 182,355 has been cloned, expressed in Escherichia coli, purified and crystallized. The crystals belong to space group P21, with unit-cell parameters a = 37.5, b = 55.6, c = 108.9,Å, , = 91.4°, and the asymmetric unit contains either two or three molecules. Both native and selenomethionine-labelled crystals diffract to 1.8,Å. [source] | |||||||||||||