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Helical Domain (helical + domain)
Selected AbstractsA novel mutation (p.Thr198Ser) in the 1A helix of keratin 5 causes the localized variant of Epidermolysis Bullosa SimplexEXPERIMENTAL DERMATOLOGY, Issue 7 2009Paul E. Bowden Abstract:, A novel missense mutation (p.Thr198Ser) in the 1A helix of keratin 5 (K5) has been identified in a four-generation family with a history of the localized variant of epidermolysis bullosa simplex (EBS-loc), a genetic skin fragility disorder caused by K5 or K14 mutations. Genomic DNA was isolated from blood samples of patients and their healthy relatives, and all exons of the genes encoding K5 and K14 (KRT5 and KRT14) were amplified by PCR and directly sequenced. The identified mutation was confirmed by mismatch allele-specific (MM-AS)-PCR and restriction enzyme digestion with RsaI. K5 p.Thr198Ser lies at the C-terminal end of the 1A helical domain and is considered to be outside of the main mutation hotspot region. This is the first reported mutation to affect position 30 of the 1A helix (1A:T30S) in any of the 54 known keratins. [source] The genetic background of Streptococcus pneumoniae affects protection in mice immunized with PspAFEMS MICROBIOLOGY LETTERS, Issue 2 2007Xiangyun He Abstract Anti-PspA antibodies are less efficient at protecting mice against certain pneumococcal strains. Immunization with PspA from EF5668 provided better protection against WU2 (a different capsular serotype and PspA family) than against EF5668. To understand the role of the pneumococcal genetic background in anti-PspA-mediated protection, we constructed a mutant of WU2 expressing pspA from EF5668. Both passive and active immunization demonstrated that the genetic background impacted the protection mediated by anti-PspA antibodies. We localized the protection-eliciting region to the first 122 amino acid residues of the N-termius of the ,-helical domain of PspA/EF5668. [source] COL5A1 signal peptide mutations interfere with protein secretion and cause classic Ehlers-Danlos syndrome,HUMAN MUTATION, Issue 2 2009Sofie Symoens Abstract Classic Ehlers-Danlos syndrome (EDS) is a heritable connective tissue disease characterized by skin hyperextensibility, atrophic scarring, joint hypermobility and generalized tissue fragility. Mutations in COL5A1 and COL5A2, encoding the type V collagen pro,1- and pro,2-chain, are found in ,50% of patients with classic EDS. The majority of mutations lead to a non-functional COL5A1 allele, as a result of the introduction of a premature stopcodon in one COL5A1 transcript. A minority of mutations affect the structure of the type V collagen central helical domain. We show that mutations in the signal peptide (SP) domain of the preproá1(V)-collagen chain cause classic EDS. The missense mutations (p.L25R and p.L25P) are located in the crucial hydrophobic SP core, which is indispensible for preprotein translocation into the endoplasmic reticulum. As a result, mutant type V procollagen is retained within the cell, leading to a decreased amount of type V collagen in the extracellular matrix and disturbed collagen fibrillogenesis. Our findings further support the observation that decreased availability of type V (pro)collagen is a key factor and a shared mechanism in the pathogenesis of classic EDS. © 2008 Wiley-Liss, Inc. [source] Consortium for osteogenesis imperfecta mutations in the helical domain of type I collagen: regions rich in lethal mutations align with collagen binding sites for integrins and proteoglycans,,HUMAN MUTATION, Issue 3 2007Joan C. Marini Abstract Osteogenesis imperfecta (OI) is a generalized disorder of connective tissue characterized by fragile bones and easy susceptibility to fracture. Most cases of OI are caused by mutations in type I collagen. We have identified and assembled structural mutations in type I collagen genes (COL1A1 and COL1A2, encoding the pro,1(I) and pro,2(I) chains, respectively) that result in OI. Quantitative defects causing type I OI were not included. Of these 832 independent mutations, 682 result in substitution for glycine residues in the triple helical domain of the encoded protein and 150 alter splice sites. Distinct genotype,phenotype relationships emerge for each chain. One-third of the mutations that result in glycine substitutions in ,1(I) are lethal, especially when the substituting residues are charged or have a branched side chain. Substitutions in the first 200 residues are nonlethal and have variable outcome thereafter, unrelated to folding or helix stability domains. Two exclusively lethal regions (helix positions 691,823 and 910,964) align with major ligand binding regions (MLBRs), suggesting crucial interactions of collagen monomers or fibrils with integrins, matrix metalloproteinases (MMPs), fibronectin, and cartilage oligomeric matrix protein (COMP). Mutations in COL1A2 are predominantly nonlethal (80%). Lethal substitutions are located in eight regularly spaced clusters along the chain, supporting a regional model. The lethal regions align with proteoglycan binding sites along the fibril, suggesting a role in fibril,matrix interactions. Recurrences at the same site in ,2(I) are generally concordant for outcome, unlike ,1(I). Splice site mutations comprise 20% of helical mutations identified in OI patients, and may lead to exon skipping, intron inclusion, or the activation of cryptic splice sites. Splice site mutations in COL1A1 are rarely lethal; they often lead to frameshifts and the mild type I phenotype. In ,2(I), lethal exon skipping events are located in the carboxyl half of the chain. Our data on genotype,phenotype relationships indicate that the two collagen chains play very different roles in matrix integrity and that phenotype depends on intracellular and extracellular events. Hum Mutat 28(3), 209,221, 2007. Published 2006 Wiley-Liss, Inc. [source] ALys amyloidosis caused by compound heterozygosity in Exon 2 (Thr70Asn) and Exon 4 (Trp112Arg) of the lysozyme gene,,HUMAN MUTATION, Issue 1 2006Christoph Röcken Abstract Hereditary amyloidoses are caused by germline mutations, which increase the propensity of a protein to form cross-, aggregates and deposit as amyloid. Hereditary amyloidoses are particularly interesting as they help to understand how changes in the primary structure of an otherwise non-amyloidogenic protein contribute to amyloidogenesis. Here we report on a novel form of systemic ALys amyloidosis, caused by compound heterozygosity in exon 2 (p.T70N) and exon 4 (p.W112R) of the lysozyme gene (LYZ), with both mutations being present on the same allele. This type of hereditary ALys amyloidosis is characterized by extended amyloid deposits in the upper gastrointestinal tract, entire colon, and kidney, leading to gastrointestinal bleeding. Both mutations are probably effective in disease manifestation. The novel mutation at position 112 in the mature protein is located within the ,-helical domain of the protein and therefore outside the cluster of residues that has so far been implicated in ALys amyloidosis. Taken together with the p.T70N mutation, this results in a lysozyme species where the correct folding of various protein domains is probably impaired and increases the propensity of amyloid fibril formation. Interestingly, this form of ALys amyloidosis is also characterized by the occurrence of proteolytic fragments of lysozyme in the amyloid deposits. © 2005 Wiley-Liss, Inc. [source] The transmembrane homotrimer of ADAM 1 in model lipid bilayersPROTEIN SCIENCE, Issue 2 2007Siok Wan Gan Abstract Fertilin is a transmembrane protein heterodimer formed by the two subunits fertilin , and fertilin , that plays an important role in sperm,egg fusion. Fertilin , and , are members of the ADAM family, and contain each one transmembrane ,-helix, and are termed ADAM 1 and ADAM 2, respectively. ADAM 1 is the subunit that contains a putative fusion peptide, and we have explored the possibility that the transmembrane ,-helical domain of ADAM 1 forms homotrimers, in common with other viral fusion proteins. Although this peptide was found to form various homooligomers in SDS, the infrared dichroic data obtained with the isotopically labeled peptide at specific positions is consistent with the presence of only one species in DMPC or POPC lipid bilayers. Comparison of the experimental orientational data with molecular dynamics simulations performed with sequence homologues of ADAM 1 show that the species present in lipid bilayers is only consistent with an evolutionarily conserved homotrimeric model for which we provide a backbone structure. These results support a model where ADAM 1 forms homotrimers as a step to create a fusion active intermediate. [source] Expression, purification, and activities of full-length and truncated versions of the integral membrane protein Vpu from HIV-1PROTEIN SCIENCE, Issue 3 2002Che Ma HIV-1, human immunodeficiency virus type 1; AIDS, acquired immune deficiency syndrome; NMR, nuclear magnetic resonance; CNBr, cyanogen bromide; DHPC, dihexanoyl phosphatidylcholine; TROSY, transverse relaxation-optimized spectroscopy Abstract Vpu is an 81-residue accessory protein of HIV-1. Because it is a membrane protein, it presents substantial technical challenges for the characterization of its structure and function, which are of considerable interest because the protein enhances the release of new virus particles from cells infected with HIV-1 and induces the intracellular degradation of the CD4 receptor protein. The Vpu-mediated enhancement of the virus release rate from HIV-1-infected cells is correlated with the expression of an ion channel activity associated with the transmembrane hydrophobic helical domain. Vpu-induced CD4 degradation and, to a lesser extent, enhancement of particle release are both dependent on the phosphorylation of two highly conserved serine residues in the cytoplasmic domain of Vpu. To define the minimal folding units of Vpu and to identify their activities, we prepared three truncated forms of Vpu and compared their structural and functional properties to those of full-length Vpu (residues 2,81). Vpu2,37 encompasses the N-terminal transmembrane ,-helix; Vpu2,51 spans the N-terminal transmembrane helix and the first cytoplasmic ,-helix; Vpu28,81 includes the entire cytoplasmic domain containing the two C-terminal amphipathic ,-helices without the transmembrane helix. Uniformly isotopically labeled samples of the polypeptides derived from Vpu were prepared by expression of fusion proteins in E. coli and were studied in the model membrane environments of lipid micelles by solution NMR spectroscopy and oriented lipid bilayers by solid-state NMR spectroscopy. The assignment of backbone resonances enabled the secondary structure of the constructs corresponding to the transmembrane and the cytoplasmic domains of Vpu to be defined in micelle samples by solution NMR spectroscopy. Solid-state NMR spectra of the polypeptides in oriented lipid bilayers demonstrated that the topology of the domains is retained in the truncated polypeptides. The biological activities of the constructs of Vpu were evaluated. The ion channel activity is confined to the transmembrane ,-helix. The C-terminal ,-helices modulate or promote the oligomerization of Vpu in the membrane and stabilize the conductive state of the channel, in addition to their involvement in CD4 degradation. [source] 2.9 Å crystal structure of ligand-free tryptophanyl-tRNA synthetase: Domain movements fragment the adenine nucleotide binding sitePROTEIN SCIENCE, Issue 2 2000Valentin A. Ilyin Abstract The crystal structure of ligand-free tryptophanyl-tRNA synthetase (TrpRS) was solved at 2.9 Å using a combination of molecular replacement and maximum-entropy map/phase improvement. The dimeric structure (R = 23.7, Rfree = 26.2) is asymmetric, unlike that of the TrpRS tryptophanyl-5,AMP complex (TAM; Doublie S, Bricogne G, Gilmore CJ, Carter CW Jr, 1995, Structure 3:17,31). In agreement with small-angle solution X-ray scattering experiments, unliganded TrpRS has a conformation in which both monomers open, leaving only the tryptophan-binding regions of their active sites intact. The amino terminal ,A-helix, TIGN, and KMSKS signature sequences, and the distal helical domain rotate as a single rigid body away from the dinucleotide-binding fold domain, opening the AMP binding site, seen in the TAM complex, into two halves. Comparison of side-chain packing in ligand-free TrpRS and the TAM complex, using identification of nonpolar nuclei (Ilyin VA, 1994, Protein Eng 7:1189,1195), shows that significant repacking occurs between three relatively stable core regions, one of which acts as a bearing between the other two. These domain rearrangements provide a new structural paradigm that is consistent in detail with the"induced-fit" mechanism proposed for TyrRS by Fersht et al. (Fersht AR, Knill-Jones JW, Beduelle H, Winter G, 1988, Biochemistry 27:1581,1587). Coupling of ATP binding determinants associated with the two catalytic signature sequences to the helical domain containing the presumptive anticodon-binding site provides a mechanism to coordinate active-site chemistry with relocation of the major tRNA binding determinants. [source] Crystallization and phasing of focal adhesion protein 52 from Gallus gallusACTA CRYSTALLOGRAPHICA SECTION D, Issue 3 2004Imre Tör Focal adhesion protein 52 (FAP52) is a multidomain adaptor protein of 448 amino acids characterized as an abundant component of focal adhesions. FAP52 binds to filamin via its N-terminal ,-helical domain, suggesting a role in linking focal adhesions to the actin-based cytoskeleton. The recombinant protein was crystallized using the hanging-drop vapour-diffusion method, which yielded two crystal forms. Native data were collected from both crystal forms to 2.8 and 2.1,Å resolution, respectively. For one of the crystal forms, initial MAD phasing was successfully performed using two data sets from xenon-derivatized crystals. The derivative data sets were collected using softer X-rays of 1.5 and 1.9,Å wavelength. Preliminary structural analysis reveals the presence of a dimer in the asymmetric unit. [source] Structure of Mycobacterium tuberculosis Rv2714, a representative of a duplicated gene family in ActinobacteriaACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 10 2009Martin Graña The gene Rv2714 from Mycobacterium tuberculosis, which codes for a hypothetical protein of unknown function, is a representative member of a gene family that is largely confined to the order Actinomycetales of Actinobacteria. Sequence analysis indicates the presence of two paralogous genes in most mycobacterial genomes and suggests that gene duplication was an ancient event in bacterial evolution. The crystal structure of Rv2714 has been determined at 2.6,Å resolution, revealing a trimer in which the topology of the protomer core is similar to that observed in a functionally diverse set of enzymes, including purine nucleoside phosphorylases, some carboxypeptidases, bacterial peptidyl-tRNA hydrolases and even the plastidic form of an intron splicing factor. However, some structural elements, such as a ,-hairpin insertion involved in protein oligomerization and a C-terminal ,-helical domain that serves as a lid to the putative substrate-binding (or ligand-binding) site, are only found in Rv2714 bacterial homologues and represent specific signatures of this protein family. [source] Insights into the reaction mechanism of glycosyl hydrolase family 49FEBS JOURNAL, Issue 22 2004Site-directed mutagenesis, substrate preference of isopullulanase Aspergillus niger isopullulanase (IPU) is the only pullulan-hydrolase in glycosyl hydrolase (GH) family 49 and does not hydrolyse dextran at all, while all other GH family 49 enzymes are dextran-hydrolysing enzymes. To investigate the common catalytic mechanism of GH family 49 enzymes, nine mutants were prepared to replace residues conserved among GH family 49 (four Trp, three Asp and two Glu). Homology modelling of IPU was also carried out based on the structure of Penicillium minioluteum dextranase, and the result showed that Asp353, Glu356, Asp372, Asp373 and Trp402, whose substitutions resulted in the reduction of activity for both pullulan and panose, were predicted to be located in the negatively numbered subsites. Three Asp-mutated enzymes, D353N, D372N and D373N, lost their activities, indicating that these residues are candidates for the catalytic residues of IPU. The W402F enzyme significantly reduced IPU activity, and the Km value was sixfold higher and the k0 value was 500-fold lower than those for the wild-type enzyme, suggesting that Trp402 is a residue participating in subsite ,1. Trp31 and Glu273, whose substitutions caused a decrease in the activity for pullulan but not for panose, were predicted to be located in the interface between N-terminal and ,-helical domains. The substrate preference of the negatively numbered subsites of IPU resembles that of GH family 49 dextranases. These findings suggest that IPU and the GH family 49 dextranases have a similar catalytic mechanism in their negatively numbered subsites in spite of the difference of their substrate specificities. [source] Is the Rehydrin TrDr3 from Tortula ruralis Associated with Tolerance to Cold, Salinity, and Reduced pH?PLANT BIOLOGY, Issue 3 2005HdeD from Escherichia coli in Response to Abiotic Stress, Physiological Evaluation of the TrDr3 -Orthologue Abstract: We have employed EST analysis in the resurrection moss Tortula ruralis to discover genes that control vegetative desiccation tolerance and describe the characterization of the EST-derived cDNA TrDr3 (Tortula ruralis desiccation-stress related). The deduced polypeptide TRDR3 has a predicted molecular mass of 25.5 kDa, predicted pI of 6.7, and six transmembrane helical domains. Preliminary expression analyses demonstrate that the TrDr3 transcript ratio increases in response to slow desiccation relative to the hydrated control in both total and polysomal mRNA (mRNP fraction), which classifies TrDr3 as a rehydrin. Bioinformatic searches of the electronic databases reveal that Tortula TRDR3 shares significant similarities to the hdeD gene product (HNS-dependent expression) from Escherichia coli. The function of the HdeD protein in E. coli is unknown, but it is postulated to be involved in a mechanism of acid stress defence. To establish the role of E. coli HdeD in abiotic stress tolerance, we determined the log survival percentage from shaking cultures of wild-type bacteria and the isogenic hdeD deletion strain (,hdeD) in the presence of low temperature (28 °C), elevated NaCl (5 % (w/v)), or decreased pH (4.5), or all treatments simultaneously. The ,hdeD deletion strain was less sensitive, as compared to wild-type E. coli, in response to decreased pH (p > 0.009), and the combination of all three stresses (p > 0.0001). [source] NMR studies of dynamics in RNA and DNA by 13C relaxationBIOPOLYMERS, Issue 5-6 2007Zahra Shajani Abstract RNA and DNA molecules experience motions on a wide range of time scales, ranging from rapid localized motions to much slower collective motions of entire helical domains. The many functions of RNA in biology very often require this molecule to change its conformation in response to biological signals in the form of small molecules, proteins or other nucleic acids, whereas local motions in DNA may facilitate protein recognition and allow enzymes acting on DNA to access functional groups on the bases that would otherwise be buried in Watson-Crick base pairs. Although these statements make a compelling case to study the sequence dependent dynamics in nucleic acids, there are few residue-specific studies of nucleic acid dynamics. Fortunately, NMR studies of dynamics of nucleic acids and nucleic acids-protein complexes are gaining increased attention. The aim of this review is to provide an update of the recent progress in studies of nucleic acid dynamics by NMR based on the application of solution relaxation techniques. © 2006 Wiley Periodicals, Inc. Biopolymers 86: 348,359, 2007. This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com [source] | ||||||||||||||||||||||