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B Kobe

Publications and source records attributed to B Kobe.

At least 37 records · Page 2Linked to original sources
PubMed1999

Crystallization of importin alpha, the nuclear-import receptor.

Crystals of recombinant importin alpha, the nuclear-import receptor, have been obtained at two different pH conditions by vapour diffusion using sodium citrate as precipitant and dithiothreitol as an additive. At pH 4-5, the crystals have the symmetry of the trigonal space group P3121 or P3221 (a = b = 78.0, c = 255.8 A, gamma = 120 degrees ); at pH 6-7, the crystals have the symmetry of the orthorhombic space group P212121 (a = 78.5, b = 89.7, c = 100.5 A). In both cases, there is probably one molecule of importin alpha in the asymmetric unit. At least one of the crystal forms diffracts to a resolution higher than 3 A using the laboratory X-ray source; the crystals are suitable for crystal structure determination.

Amino Acid Sequence↗
PubMed1998

Crystallization and preliminary X-ray analysis of ferric enterobactin receptor FepA, an integral membrane protein from Escherichia coli.

Diffraction-quality crystals have been obtained of the integral membrane protein ferric enterobactin receptor (FepA) from the outer membrane of Escherichia coli. Crystals were grown using the zwitterionic detergent lauryldimethylamine oxide (LDAO), the precipitants polyethylene glycol (PEG) 1000 and sodium chloride, and the additive heptane-1,2,3-triol; they have the symmetry of the orthorhomic space group C2221 with a = 112.2, b = 137.2 and c = 135. 4 A and diffract to 2.5 A resolution. The crystals were flash-cooled and a preliminary data set was collected at 103 K. The crystals are suitable for three-dimensional structure analysis.

Bacterial Outer Membrane Proteins↗
PubMed1998

Molecular typing of Salmonella enterica subsp. enterica serovar Hadar: evaluation and application of different typing methods.

A highly discriminatory molecular typing system for isolates of Salmonella enterica subsp. enterica (S.) serovar Hadar was developed based on the data obtained from 34 epidemiologically unrelated S. Hadar isolates from Germany and Brazil. Plasmid profile analysis and macrorestriction analysis with the enzymes XbaI, SpeI and BlnI represented the most discriminative typing methods. In contrast, ribotyping as performed with a gene probe that recognized the entire rrn operon did not reveal any differences between the S. Hadar isolates. IS200 typing was not applicable due to the lack of insertion elements of this type in all S. Hadar isolates included in this study. The combination of the results of plasmid analysis and macrorestriction analysis subdivided the 34 S. Hadar isolates into 32 different genomic groups. The high discriminatory power of these methods confirmed their suitability as effective typing methods for epidemiological studies on S. Hadar isolates. The practical application of this molecular typing system to S. Hadar isolates of the same flock obtained during a 15 month period strongly suggested recurring introduction of genomically different S. Hadar isolates instead of a single introduction of S. Hadar followed by subsequent spread within the flock.

Animals↗
PubMed1998

Crystallization of a trimeric human T cell leukemia virus type 1 gp21 ectodomain fragment as a chimera with maltose-binding protein.

We present a novel protein crystallization strategy, applied to the crystallization of human T cell leukemia virus type 1 (HTLV-1) transmembrane protein gp21 lacking the fusion peptide and the transmembrane domain, as a chimera with the Escherichia coli maltose binding protein (MBP). Crystals could not be obtained with a MBP/gp21 fusion protein in which fusion partners were separated by a flexible linker, but were obtained after connecting the MBP C-terminal alpha-helix to the predicted N-terminal alpha-helical sequence of gp21 via three alanine residues. The gp21 sequences conferred a trimeric structure to the soluble fusion proteins as assessed by sedimentation equilibrium and X-ray diffraction, consistent with the trimeric structures of other retroviral transmembrane proteins. The envelope protein precursor, gp62, is likewise trimeric when expressed in mammalian cells. Our results suggest that MBP may have a general application for the crystallization of proteins containing N-terminal alpha-helical sequences.

ATP-Binding Cassette Transporters↗
PubMed1998

Inter- and intra-phage type differentiation of Salmonella enterica subsp. enterica serovar enteritidis isolates using molecular typing methods.

Seventy-six Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) isolates which represented 14 phage types and RDNC isolates were investigated by several independent molecular methods. Eleven different plasmid profiles were detected. Plasmid-encoded virulence genes of the spv gene cluster were shown by hybridization with a spvB/C gene probe to be located on plasmids of either 55 kbp or 95 kbp. Ribotyping as performed with gene probes that recognized either the entire rrn operon or 16S rRNA genes allowed no differentiation between the S. Enteritidis isolates. Three different hybridization patterns could be observed by IS200-typing. Macrorestriction analysis of XbaI-, SpeI- and NotI-digested whole cell DNA proved to be the most discriminatory method as confirmed by the calculation of discriminatory indices. Based on the data obtained from plasmid analysis and macrorestriction analyses with three suitable restriction endonucleases, the 76 S. Enteritidis isolates were assigned to 26 different genomic groups. The observation that several genomic groups included isolates of different phage types has suggested that a combination of suitable molecular methods and phage typing is most useful for tracing S. Enteritidis isolates in epidemiological investigations.

Animals↗
PubMed1997

The myosin-I-binding protein Acan125 binds the SH3 domain and belongs to the superfamily of leucine-rich repeat proteins.

The SH3 domains of src and other nonreceptor tyrosine kinases have been shown to associate with the motif PXXP, where P and X stand for proline and an unspecified amino acid, but a motif that binds to the SH3 domain of myosin has thus far not been characterized. We previously showed that the SH3 domain of Acanthamoeba myosin-IC interacts with the protein Acan125. We now report that the Acan125 protein sequence contains two tandem consensus PXXP motifs near the C terminus. To test for binding, we expressed a polypeptide, AD3p, which includes 344 residues of native C-terminal sequence and a mutant polypeptide, AD3delta977-994p, which lacks the sequence RPKPVPPPRGAKPAPPPR containing both PXXP motifs. The SH3 domain of Acanthamoeba myosin-IC bound AD3p and not AD3delta977-994p, showing that the PXXP motifs are required for SH3 binding. The sequence of Acan125 is related overall to a protein of unknown function coded by Caenorhabditis elegans gene K07G5.1. The K07G5.1 gene product contains a proline-rich segment similar to the SH3 binding motif found in Acan125. The aligned sequences show considerable conservation of leucines and other hydrophobic residues, including the spacing of these residues, which matches a motif for leucine-rich repeats (LRRs). LRR domains have been demonstrated to be sites for ligand binding. Having an LRR domain and an SH3-binding domain, Acan125 and the C. elegans homologue define a novel family of bifunctional binding proteins.

Acanthamoeba↗
PubMed1997

Regulation and crystallization of phosphorylated and dephosphorylated forms of truncated dimeric phenylalanine hydroxylase.

Phenylalanine hydroxylase is regulated in a complex manner, including activation by phosphorylation. It is normally found as an equilibrium of dimeric and tetrameric species, with the tetramer thought to be the active form. We converted the protein to the dimeric form by deleting the C-terminal 24 residues and show that the truncated protein remains active and regulated by phosphorylation. This indicates that changes in the tetrameric quaternary structure of phenylalanine hydroxylase are not required for enzyme activation. Truncation also facilitates crystallization of both phosphorylated and dephosphorylated forms of the enzyme.

Animals↗
PubMed1996

Mechanism of ribonuclease inhibition by ribonuclease inhibitor protein based on the crystal structure of its complex with ribonuclease A.

We describe the mechanism of ribonuclease inhibition by ribonuclease inhibitor, a protein built of leucine-rich repeats, based on the crystal structure of the complex between the inhibitor and ribonuclease A. The structure was determined by molecular replacement and refined to an Rcryst of 19.4% at 2.5 A resolution. Ribonuclease A binds to the concave region of the inhibitor protein comprising its parallel beta-sheet and loops. The inhibitor covers the ribonuclease active site and directly contacts several active-site residues. The inhibitor only partially mimics the RNase-nucleotide interaction and does not utilize the p1 phosphate-binding pocket of ribonuclease A, where a sulfate ion remains bound. The 2550 A2 of accessible surface area buried upon complex formation may be one of the major contributors to the extremely tight association (Ki = 5.9 x 10(-14) M). The interaction is predominantly electrostatic; there is a high chemical complementarity with 18 putative hydrogen bonds and salt links, but the shape complementarity is lower than in most other protein-protein complexes. Ribonuclease inhibitor changes its conformation upon complex formation; the conformational change is unusual in that it is a plastic reorganization of the entire structure without any obvious hinge and reflects the conformational flexibility of the structure of the inhibitor. There is a good agreement between the crystal structure and other biochemical studies of the interaction. The structure suggests that the conformational flexibility of RI and an unusually large contact area that compensates for a lower degree of complementarity may be the principal reasons for the ability of RI to potently inhibit diverse ribonucleases. However, the inhibition is lost with amphibian ribonucleases that have substituted most residues corresponding to inhibitor-binding residues in RNase A, and with bovine seminal ribonuclease that prevents inhibitor binding by forming a dimer.

Amino Acid Sequence↗
PubMed1996

Giant protein kinases: domain interactions and structural basis of autoregulation.

The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules. We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain. The structure of the longer fragment shows that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues. Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I.

Amino Acid Sequence↗
PubMed1996

Ca2+/S100 regulation of giant protein kinases.

Protein phosphorylation by protein kinases plays a central regulatory role in cellular processes and these kinases are themselves tightly regulated. One common mechanism of regulation involves Ca2+-binding proteins (CaBP) such as calmodulin (CaM). Here we report a Ca2+-effector mechanism for protein kinase activation by demonstrating the specific and >1,000-fold activation of the myosin-associated giant protein kinase twitchin by Ca2+/S100A1(2). S100A1(2) is a member of a large CaBP family that is implicated in various cellular processes, including cell growth, differentiation and motility, but whose molecular actions are largely unknown. The S100A1(2)-binding site is a part of the autoregulatory sequence positioned in the active site that is responsible for intrasteric autoinhibition of twitchin kinase; the mechanism of autoinhibition based on the crystal structures of two twitchin kinase fragments is described elsewhere. Ca2+/S100 represents a likely physiological activator for the entire family of giant protein kinases involved in muscle contractions and cytoskeletal structure.

Amino Acid Sequence↗
PubMed1995

A structural basis of the interactions between leucine-rich repeats and protein ligands.

The leucine-rich repeat is a recently characterized structural motif used in molecular recognition processes as diverse as signal transduction, cell adhesion, cell development, DNA repair and RNA processing. We present here the crystal structure at 2.5 A resolution of the complex between ribonuclease A and ribonuclease inhibitor, a protein built entirely of leucine-rich repeats. The unusual non-globular structure of ribonuclease inhibitor, its solvent-exposed parallel beta-sheet and the conformational flexibility of the structure are used in the interaction; they appear to be the principal reasons for the effectiveness of leucine-rich repeats as protein-binding motifs. The structure can serve as a model for the interactions of other proteins containing leucine-rich repeats with their ligands.

Animals↗
PubMed1995

Proteins with leucine-rich repeats.

Leucine-rich repeats are short sequence motifs present in over sixty proteins, all of which appear to be involved in protein-protein interactions. The crystal structure of ribonuclease inhibitor demonstrated that the repeats correspond to beta-alpha structural units. The recently determined crystal structure of the ribonuclease A-ribonuclease inhibitor complex suggests the basis for the protein-binding function of leucine-rich repeats.

Animals↗
PubMed1994

Complex between bovine ribonuclease A and porcine ribonuclease inhibitor crystallizes in a similar unit cell as free ribonuclease inhibitor.

We obtained three different morphologies of co-crystals of bovine ribonuclease A and porcine ribonuclease inhibitor. X-ray quality crystals were grown in 1.3M ammonium sulfate, 100 mM sodium acetate (pH 5.0) and 20 mM dithiothreitol at 21 degrees C. These crystals have the symmetry of the tetragonal space group I4 with a = 133.3 A and c = 86.7 A and diffract to 2.5 A resolution; they have the same symmetry and only slightly different cell parameters than the crystals of free ribonuclease inhibitor. Polyacrylamide gel electrophoresis and the crystal density indicate that both ribonuclease inhibitor and ribonuclease A are present in the crystals. Although small, crystals are suitable for three-dimensional structural analysis.

Animals↗
PubMed1994

The leucine-rich repeat: a versatile binding motif.

Leucine-rich repeats are short sequence motifs present in a number of proteins with diverse functions and cellular locations. All proteins containing these repeats are thought to be involved in protein-protein interactions. The crystal structure of ribonuclease inhibitor protein has revealed that leucine-rich repeats correspond to beta-alpha structural units. These units are arranged so that they form a parallel beta-sheet with one surface exposed to solvent, so that the protein acquires an unusual, nonglobular shape. These two features may be responsible for the protein-binding functions of proteins containing leucine-rich repeats.

Animals↗