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Globular Domain (globular + domain)
Selected AbstractsCharacterization of sequence variations in human histone H1.2 and H1.4 subtypesFEBS JOURNAL, Issue 14 2005Bettina Sarg In humans, eight types of histone H1 exist (H1.1,H1.5, H1°, H1t and H1oo), all consisting of a highly conserved globular domain and less conserved N- and C-terminal tails. Although the precise functions of these isoforms are not yet understood, and H1 subtypes have been found to be dispensable for mammalian development, it is now clear that specific functions may be assigned to certain individual H1 subtypes. Moreover, microsequence variations within the isoforms, such as polymorphisms or mutations, may have biological significance because of the high degree of sequence conservation of these proteins. This study used a hydrophilic interaction liquid chromatographic method to detect sequence variants within the subtypes. Two deviations from wild-type H1 sequences were found. In K562 erythroleukemic cells, alanine at position 17 in H1.2 was replaced by valine, and, in Raji B lymphoblastoid cells, lysine at position 173 in H1.4 was replaced by arginine. We confirmed these findings by DNA sequencing of the corresponding gene segments. In K562 cells, a homozygous GCC,GTC shift was found at codon 18, giving rise to H1.2 Ala17Val because the initial methionine is removed in H1 histones. Raji cells showed a heterozygous AAA,AGA codon change at position 174 in H1.4, corresponding to the Lys173Arg substitution. The allele frequency of these sequence variants in a normal Swedish population was found to be 6.8% for the H1.2 GCC,GTC shift, indicating that this is a relatively frequent polymorphism. The AAA,AGA codon change in H1.4 was detected only in Raji cells and was not present in a normal population or in six other cell lines derived from individuals suffering from Burkitt's lymphoma. The significance of these sequence variants is unclear, but increasing evidence indicates that minor sequence variations in linker histones may change their binding characteristics, influence chromatin remodeling, and specifically affect important cellular functions. [source] Modulation of sarcoplasmic reticulum Ca2+ -ATPase by chronic and acute exposure to peroxynitriteFEBS JOURNAL, Issue 13 2004Yolanda Gutiérrez-Martín The Ca2+ -ATPase of skeletal muscle sarcoplasmic reticulum (SERCA), an integral membrane protein, becomes irreversibly inactivated in vitro by the addition of a single bolus of peroxynitrite with a K0.5 of 200,300 µm, and this results in a large decrease of the ATP-dependent Ca2+ gradient across the sarcoplasmic reticulum (SR) membranes. The inactivation of SERCA is raised by treatment of SR vesicles with repetitive micromolar pulses of peroxynitrite. The inhibition of the SERCA is due to the oxidation of thiol groups and tyrosine nitration. Scavengers that react directly with peroxynitrite, such as cysteine, reduced glutathione, NADH, methionine, ascorbate or Trolox, a water-soluble analog of ,-tocopherol, afforded significant protection. However, dimethyl sulfoxide and mannitol, two hydroxyl radical scavengers, and ,-tocopherol did not protect SERCA from inactivation. Our results showed that the target of peroxynitrite is the cytosolic globular domain of the SERCA and that major skeletal muscle intracellular reductants (ascorbate, NADH and reduced glutathione) protected against inhibition of this ATPase by peroxynitrite. [source] An immunohistochemical study of laminin in basal cell carcinomaJOURNAL OF CUTANEOUS PATHOLOGY, Issue 1 2010Wedad Z. Mostafa Background: Laminins are components of the extracellular matrix that mediate cell adhesion, growth, migration, proliferation and differentiation. Basement membrane (BM) laminins, in particular, may play a role in enhancing carcinoma cell motility. Aim: To evaluate the distribution pattern of laminin in basal cell carcinoma (BCC), as regards the basement membrane, cellular cytoplasm, peritumoral lacunae and surface epithelium and to correlate laminin distribution with different variants of BCC. Patients and Methods: Skin biopsy specimens were obtained from 21 BCC patients for routine histopathological and immunohistochemical study. Laminin was evaluated qualitatively and semiquantitatively using monoclonal mouse antihuman antibody (Dako-Laminin, 4C7. Code No: MO638, which reacts with the terminal globular domain of the ,5 chain) Results: The majority of BCC cases showed patchy cytoplasmic distribution of laminin. The BM expression of laminin, in most cases, was well defined, fine and linear with irregular areas of thickening. Staining intensity was moderate in differentiated and mixed variants, weak in superficial spreading and absent in morpheic types. Conclusion: Cytoplasmic and basement membrane laminin is important in the pathogenesis and invasion of BCC. Most laminin was in basement membrane zone (BMZ), and the more differentiated the tumor, the more cytoplasmic and BM staining it expressed. [source] Structural characterization of a neurotoxic threonine-rich peptide corresponding to the human prion protein ,2-helical 180,195 segment, and comparison with full-length ,2-helix-derived peptides,JOURNAL OF PEPTIDE SCIENCE, Issue 10 2008Luisa Ronga Abstract The 173,195 segment corresponding to the helix 2 of the globular PrP domain is a good candidate to be one of the several ,spots' of intrinsic structural flexibility, which might induce local destabilization and concur to protein transformation, leading to aggregation-prone conformations. Here, we report CD and NMR studies on the ,2-helix-derived peptide of maximal length (hPrP[180,195]) that is able to exhibit a regular structure different from the prevalently random arrangement of other ,2-helix-derived peptides. This peptide, which has previously been shown to be affected by buffer composition via the ion charge density dependence typical of Hofmeister effects, corresponds to the C -terminal sequence of the PrPC full-length ,2-helix and includes the highly conserved threonine-rich 188,195 segment. At neutral pH, its conformation is dominated by ,-type contributions, which only very strong environmental modifications are able to modify. On TFE addition, an increase of ,-helical content can be observed, but a fully helical conformation is only obtained in neat TFE. However, linking of the 173,179 segment, as occurring in wild-type and mutant peptides corresponding to the full-length ,2-helix, perturbs these intrinsic structural propensities in a manner that depends on whether the environment is water or TFE. Overall, these results confirm that the 180,195 parental region in hPrPC makes a strong contribution to the chameleon conformational behavior of the segment corresponding to the full-length ,2-helix, and could play a role in determining structural rearrangements of the entire globular domain. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd. [source] Atomic-resolution crystal structure of Borrelia burgdorferi outer surface protein A via surface engineeringPROTEIN SCIENCE, Issue 8 2006Koki Makabe Abstract Outer surface protein A (OspA) from Borrelia burgdorferi has an unusual dumbbell-shaped structure in which two globular domains are connected with a "single-layer" ,-sheet (SLB). The protein is highly soluble, and it has been recalcitrant to crystallization. Only OspA complexes with Fab fragments have been successfully crystallized. OspA contains a large number of Lys and Glu residues, and these "high entropy" residues may disfavor crystal packing because some of them would need to be immobilized in forming a crystal lattice. We rationally designed a total of 13 surface mutations in which Lys and Glu residues were replaced with Ala or Ser. We successfully crystallized the mutant OspA without a bound Fab fragment and extended structure analysis to a 1.15 Å resolution. The new high-resolution structure revealed a unique backbone hydration pattern of the SLB segment in which water molecules fill the "weak spots" on both faces of the antiparallel ,-sheet. These well-defined water molecules provide additional structural links between adjacent ,-strands, and thus they may be important for maintaining the rigidity of the SLB that inherently lacks tight packing afforded by a hydrophobic core. The structure also revealed new information on the side-chain dynamics and on a solvent-accessible cavity in the core of the C-terminal globular domain. This work demonstrates the utility of extensive surface mutation in crystallizing recalcitrant proteins and dramatically improving the resolution of crystal structures, and provides new insights into the stabilization mechanism of OspA. [source] A helix-turn motif in the C-terminal domain of histone H1PROTEIN SCIENCE, Issue 4 2000Roger Vila Abstract The structural study of peptides belonging to the terminal domains of histone H1 can be considered as a step toward the understanding of the function of H1 in chromatin. The conformational properties of the peptide Ac-EPKRSVAFKKT KKEVKKVATPKK (CH-1), which belongs to the C-terminal domain of histone Hl° (residues 99,121) and is adjacent to the central globular domain of the protein, were examined by means of 1H-NMR and circular dichroism. In aqueous solution, CH-1 behaved as a mainly unstructured peptide, although turn-like conformations in rapid equilibrium with the unfolded state could be present. Addition of trifluoroethanol resulted in a substantial increase of the helical content. The helical limits, as indicated by (i, i + 3) nuclear Overhauser effect (NOE) cross correlations and significant up-field conformational shifts of the C, protons, span from Pro100 to Val 116, with Glu99 and Ala117 as N- and C-caps. A structure calculation performed on the basis of distance constraints derived from NOE cross peaks in 90% trifluoroethanol confirmed the helical structure of this region. The helical region has a marked amphipathic character, due to the location of all positively charged residues on one face of the helix and all the hydrophobic residues on the opposite face. The peptide has a TPKK motif at the C-terminus, following the ,-helical region. The observed NOE connectivities suggest that the TPKK sequence adopts a type (I) ,-turn conformation, a ,-turn conformation or a combination of both, in fast equilibrium with unfolded states. Sequences of the kind (S/T)P(K/R)(K/R) have been proposed as DNA binding motifs. The CH-1 peptide, thus, combines a positively charged amphipathic helix and a turn as potential DNA-binding motifs. [source] Proteome analysis of adipocyte lipid rafts reveals that gC1qR plays essential roles in adipogenesis and insulin signal transductionPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 9 2009Ki-Bum Kim Abstract Since insulin receptors and their downstream signaling molecules are organized in lipid rafts, proteomic analysis of adipocyte lipid rafts may provide new insights into the function of lipid rafts in adipogenesis and insulin signaling. To search for proteins involved in adipocyte differentiation and insulin signaling, we analyzed detergent-resistant lipid raft proteins from 3T3-L1 preadipocytes and adipocytes by 2-DE. Eleven raft proteins were identified from adipocytes. One of the adipocyte-specific proteins was globular C1q receptor (gC1qR), an acidic 32,kDa protein known as the receptor for the globular domain of complement C1q. The targeting of gC1qR into lipid rafts was significantly increased during adipogenesis, as determined by immunoblotting and immunofluorescence. Since the silencing of gC1qR by small RNA interference abolished adipogenesis and blocked insulin-induced activation of insulin receptor, insulin receptor substrate-1 (IRS-1), Akt, and Erk1/2, we can conclude that gC1qR is an essential molecule involved in adipogenesis and insulin signaling. [source] Structure at 1.5,Å resolution of cytochrome c552 with its flexible linker segment, a membrane-anchored protein from Paracoccus denitrificansACTA CRYSTALLOGRAPHICA SECTION D, Issue 7 2010Chitra Rajendran Electron transfer (ET) between the large membrane-integral redox complexes in the terminal part of the respiratory chain is mediated either by a soluble c -type cytochrome, as in mitochondria, or by a membrane-anchored cytochrome c, as described for the ET chain of the bacterium Paracoccus denitrificans. Here, the structure of cytochrome c552 from P. denitrificans with the linker segment that attaches the globular domain to the membrane anchor is presented. Cytochrome c552 including the linker segment was crystallized and its structure was determined by molecular replacement. The structural features provide functionally important information. The prediction of the flexibility of the linker region [Berry & Trumpower (1985), J. Biol. Chem.260, 2458,2467] was confirmed by our crystal structure. The N-terminal region from residues 13 to 31 is characterized by poor electron density, which is compatible with high mobility of this region. This result indicates that this region is highly flexible, which is functionally important for this protein to shuttle electrons between complexes III and IV in the respiratory chain. Zinc present in the crystallization buffer played a key role in the successful crystallization of this protein. It provided rigidity to the long negatively charged flexible loop by coordinating negatively charged residues from two different molecules and by enhancing the crystal contacts. [source] Structure of the lamin A/C R482W mutant responsible for dominant familial partial lipodystrophy (FPLD)ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 7 2009Eugenia Magracheva Proteins of the A-type lamin family, which consists of two members, lamin A and lamin C, are the major components of a thin proteinaceous filamentous meshwork, the lamina, that underlies the inner nuclear membrane. A-type lamins have recently become the focus of extensive functional studies as a consequence of the linking of at least eight congenital diseases to mutations in the lamin A/C gene (LMNA). This spectrum of pathologies, which mostly manifest themselves as dominant traits, includes muscle dystrophies, dilated cardiomyopathies, the premature aging syndrome Hutchinson,Guilford progeria and familial partial lipodystrophy (FPLD). The crystal structure of the lamin A/C mutant R482W, a variant that causes FPLD, has been determined at 1.5,Å resolution. A completely novel aggregation state of the C-terminal globular domain and the position of the mutated amino-acid residue suggest means by which the mutation may affect lamin A/C,protein and protein,DNA interactions. [source] Protocols for the Sequential Solid-State NMR Spectroscopic Assignment of a Uniformly Labeled 25 kDa Protein: HET-s(1-227)CHEMBIOCHEM, Issue 11 2010Anne Schuetz Abstract The sequence-specific resonance assignment of a protein forms the basis for studies of molecular structure and dynamics, as well as to functional assay studies by NMR spectroscopy. Here we present a protocol for the sequential 13C and 15N resonance assignment of uniformly [15N,13C]-labeled proteins, based on a suite of complementary three-dimensional solid-state NMR spectroscopy experiments. It is directed towards the application to proteins with more than about 100 amino acid residues. The assignments rely on a walk along the backbone by using a combination of three experiments that correlate nitrogen and carbon spins, including the well-dispersed C, resonances. Supplementary spectra that correlate further side-chain resonances can be important for identifying the amino acid type, and greatly assist the assignment process. We demonstrate the application of this assignment protocol for a crystalline preparation of the N-terminal globular domain of the HET-s prion, a 227-residue protein. [source] Global analysis of functional surfaces of core histones with comprehensive point mutantsGENES TO CELLS, Issue 1 2007Kazuko Matsubara The core histones are essential components of the nucleosome that act as global negative regulators of DNA-mediated reactions including transcription, DNA replication and DNA repair. Modified residues in the N-terminal tails are well characterized in transcription, but not in DNA replication and DNA repair. In addition, roles of residues in the core globular domains are not yet well characterized in any DNA-mediated reactions. To comprehensively understand the functional surface(s) of a core histone, we constructed 320 yeast mutant strains, each of which has a point mutation in a core histone, and identified 42 residues responsible for the suppressor of Ty (Spt - ) phenotypes, and 8, 30 and 61 residues for sensitivities to 6-azauracil (6AU), hydroxyurea (HU) and methyl-methanesulfonate (MMS), respectively. In addition to residues that affect one specific assay, residues involved in multiple reactions were found, and surprisingly, about half of them were clustered at either the nucleosome entry site, the surface required for nucleosome,nucleosome interactions in crystal packing or their surroundings. This comprehensive mutation approach was proved to be powerful for identification of the functional surfaces of a core histone in a variety of DNA-mediated reactions and could be an effective strategy for characterizing other evolutionarily conserved hub-like factors for which surface structural information is available. [source] Plectin deficiency leads to both muscular dystrophy and pyloric atresia in epidermolysis bullosa simplex,HUMAN MUTATION, Issue 10 2010Ken Natsuga Abstract Plectin is a cytoskeletal linker protein which has a long central rod and N- and C-terminal globular domains. Mutations in the gene encoding plectin (PLEC) cause two distinct autosomal recessive subtypes of epidermolysis bullosa: EB simplex (EBS) with muscular dystrophy (EBS-MD), and EBS with pyloric atresia (EBS-PA). Previous studies have demonstrated that loss of full-length plectin with residual expression of the rodless isoform leads to EBS-MD, whereas complete loss or marked attenuation of expression of full-length and rodless plectin underlies the more severe EBS-PA phenotype. However, muscular dystrophy has never been identified in EBS-PA, not even in the severe form of the disease. Here, we report the first case of EBS associated with both pyloric atresia and muscular dystrophy. Both of the premature termination codon-causing mutations of the proband are located within exon 32, the last exon of PLEC. Immunofluorescence and immunoblot analysis of skin samples and cultured fibroblasts from the proband revealed truncated plectin protein expression in low amounts. This study demonstrates that plectin deficiency can indeed lead to both muscular dystrophy and pyloric atresia in an individual EBS patient. © 2010 Wiley-Liss, Inc. [source] Atomic-resolution crystal structure of Borrelia burgdorferi outer surface protein A via surface engineeringPROTEIN SCIENCE, Issue 8 2006Koki Makabe Abstract Outer surface protein A (OspA) from Borrelia burgdorferi has an unusual dumbbell-shaped structure in which two globular domains are connected with a "single-layer" ,-sheet (SLB). The protein is highly soluble, and it has been recalcitrant to crystallization. Only OspA complexes with Fab fragments have been successfully crystallized. OspA contains a large number of Lys and Glu residues, and these "high entropy" residues may disfavor crystal packing because some of them would need to be immobilized in forming a crystal lattice. We rationally designed a total of 13 surface mutations in which Lys and Glu residues were replaced with Ala or Ser. We successfully crystallized the mutant OspA without a bound Fab fragment and extended structure analysis to a 1.15 Å resolution. The new high-resolution structure revealed a unique backbone hydration pattern of the SLB segment in which water molecules fill the "weak spots" on both faces of the antiparallel ,-sheet. These well-defined water molecules provide additional structural links between adjacent ,-strands, and thus they may be important for maintaining the rigidity of the SLB that inherently lacks tight packing afforded by a hydrophobic core. The structure also revealed new information on the side-chain dynamics and on a solvent-accessible cavity in the core of the C-terminal globular domain. This work demonstrates the utility of extensive surface mutation in crystallizing recalcitrant proteins and dramatically improving the resolution of crystal structures, and provides new insights into the stabilization mechanism of OspA. [source] Structure of laminin-binding adhesin (Lmb) from Streptococcus agalactiaeACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009Preethi Ragunathan Adhesion/invasion of pathogenic bacteria is a critical step in infection and is mediated by surface-exposed proteins termed adhesins. The crystal structure of recombinant Lmb, a laminin-binding adhesin from Streptococcus agalactiae, has been determined at 2.5,Å resolution. Based on sequence and structural homology, Lmb was placed into the cluster 9 family of the ABC (ATP-binding cassette) transport system. The structural organization of Lmb closely resembles that of ABC-type solute-binding proteins (SBPs), in which two structurally related globular domains interact with each other to form a metal-binding cavity at the interface. The bound zinc in Lmb is tetrahedrally coordinated by three histidines and a glutamate from both domains. A comparison of Lmb with other metal transporters revealed an interesting feature of the dimerization of molecules in the crystallographic asymmetric unit in all zinc-binding transporters. A closer comparison of Lmb with the zinc-binding ZnuA from Escherichia coli and Synechocystis 6803 suggested that Lmb might undergo a unique structural rearrangement upon metal binding and release. The crystal structure of Lmb provides an impetus for further investigations into the molecular basis of laminin binding by human pathogens. Being ubiquitous in all serotypes of group B streptococcus (GBS), the structure of Lmb may direct the development of an efficient vaccine. [source] Structural studies of phosphoglucose isomerase from Mycobacterium tuberculosis H37RvACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 5 2010Kanchan Anand Phosphoglucose isomerase (PGI) plays a key role in both glycolysis and gluconeogenesis inside the cell, whereas outside the cell it exhibits cytokine properties. PGI is also known to act as an autocrine motility factor, a neuroleukin agent and a differentiation and maturation mediator. Here, the first crystal structure of PGI from Mycobacterium tuberculosis H37Rv (Mtb) is reported. The structure was refined at 2.25,Å resolution and revealed the presence of one molecule in the asymmetric unit with two globular domains. As known previously, the active site of Mtb PGI contains conserved residues including Glu356, Glu216 and His387 (where His387 is from the neighbouring molecule). The crystal structure of Mtb PGI was observed to be rather more similar to human PGI than other nonbacterial PGIs, with only a few differences being detected in the loops, arm and hook regions of the human and Mtb PGIs, suggesting that the M. tuberculosis enzyme uses the same enzyme mechanism. [source] Structure of the aliphatic sulfonate-binding protein SsuA from Escherichia coliACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2010John Beale Sulfur is an essential component for the biosynthesis of the sulfur-containing amino acids l -methionine and l -cysteine. Under sulfur-starvation conditions, bacteria are capable of scavenging sulfur from sulfur-containing compounds and transporting it across membranes. Here, the crystal structure of the periplasmic aliphatic sulfonate-binding protein SsuA from Escherichia coli is reported at 1.75,Å resolution in the substrate-free state. The overall structure of SsuA resembles the structures of other periplasmic binding proteins and contains two globular domains that form a cleft. Comparison with other periplasmic binding proteins revealed that one of the domains has been displaced by a rigid movement of 17°. Interestingly, the tight crystal packing appears to be mediated by a 13-amino-acid tail from the cloning that folds within the cleft of the next monomer. [source] | |||||||||||||||||||||||||