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At least 1,261 records · Page 70Linked to original sources
PubMed1994

A detailed comparison of the refined structures of cytochrome c3 molecules from two strains in Desulfovibrio vulgaris: the relationship between the heme structures and their redox properties.

The refined structures of the cytochrome c3 molecules from Desulfovibrio vulgaris Miyazaki and Hildenborough have been compared in detail. Though there are no significant differences of the overall structure and spatial arrangement of four heme groups between two molecules, there are some unique features with regard to the local structures near the heme pockets. Two of the heme groups show significant differences including a hydrogen bonding scheme between the imidazole rings and water molecules in heme pocket. The water molecule near the heme 1 pocket in Miyazaki cytochrome c3 is replaced by the C delta 1 atom of Leu9 in the Hildenborough structure. The rotation around the C alpha-C beta bond of Leu9 results in the increase of the solvent accessibility of the portion of the heme 1 edge. On the other hand, in heme3 one water molecule is additionally included in the hydrogen bonding network with a N delta 1 atom of the imidazole ring and carbonyl oxygen atom of the main chain in Hildenborough cytochrome c3. This additional hydrogen bonding network will contribute to the possible passage between the imidazole ring and the molecular surface. These features have been related to the upfield shift of the C2 proton signals of NMR spectra and the low redox potentials of the cytochrome c3 molecules of Desulfovibrio vulgaris Hildenborough in comparison with those from Miyazaki.

Amino Acid Sequence↗
PubMed1988

Fine structure of the A-band in cryo-sections. Diversity of M-band structure in chicken breast muscle.

Electron micrographs of longitudinal ultrathin cryo-sections and plastic sections of chicken pectoralis muscle together with their average images have been used to study in detail the axial structure of the M-band. It was found that M-band structure could vary markedly in different fibres, even within the white part of the muscle. Strong M-band density ("M-bridges") could be seen at M4 and M4' in all fibres. On the other hand the density at M1 or M6 could vary systematically. Some fibres (probably fast) had M1 strong, M6 weak (a "3-line" M-band), and the Z-band was narrow. Other fibres, especially (but not exclusively) in the red part of the muscle and probably slow, had M6 strong, M1 weak (a "4-line" M-band), and the Z-band was broad. However, the majority of fibres ranged in structure between those two extremes and had a more or less "5-line" M-band with M1 and M6 both strong and a Z-band of intermediate width. Since they were such a constant feature, the M4 lines may be the sites of the primarily structural component of the M-band, whereas the different proteins at M1 and M6 may vary in quantity according to the physiological needs of the fibre. Finally, detailed analysis sometimes revealed substructure within the strong M-bridge lines. This substructure may represent additional unknown M-band proteins or may be an indication of the shape of single proteins at these positions.

Animals↗
PubMed1993

Determination of the NMR solution structure of the Hoechst 33258-d(GTGGAATTCCAC)2 complex and comparison with the X-ray crystal structure.

BACKGROUND: The chromosomal stain, Hoechst 33258, binds to the minor groove of the DNA double helix and specifically recognizes a run of four A-T base pairs. Extensive biochemical and biophysical studies have been aimed at understanding the binding of the dye to DNA at the atomic level. Among these studies there have been several crystal structure determinations and some preliminary structural studies by NMR. RESULTS: On the basis of our own previously reported NMR data, we have now determined the three-dimensional solution structure of the 1:1 complex between Hoechst 33258 and the self-complementary DNA duplex d(GTGGAATTCCAC)2. Two coexisting families of con formers, which exhibit differences in their intermolecular hydrogen bonding pattern, were found and the two terminal rings of the dye displayed greater internal mobility than the rest of the molecule. CONCLUSIONS: The observed multiple ligand-binding modes in the complex between Hoechst 33258 and DNA and differential internal mobility along the bound ligand provide a novel, dynamic picture of the specific inter actions between ligands that bind in the minor groove and DNA. The dynamic state revealed by these studies may account for some of the significant differences previously observed between different crystal structures of Hoechst 33258 complexed with a different DNA duplex, d(CGCGAATTCGCG)2.

Bisbenzimidazole↗
PubMed2006

Crystal structures of acetylcholinesterase in complex with HI-6, Ortho-7 and obidoxime: structural basis for differences in the ability to reactivate tabun conjugates.

Inhibition of acetylcholinesterase (AChE) by organophosphorus compounds (OPs) such as pesticides and nerve agents causes acute toxicity or death of the intoxicated individual. The inhibited AChE may be reactivated by certain oximes as antidotes for clinical treatment of OP-intoxications. Crystal structures of the oximes HI-6, Ortho-7 and obidoxime in complex with Mus musculus acetylcholinesterase (mAChE) reveal different roles of the peripheral anionic site (PAS) in the binding of the oximes. A limited structural change of the side chains of Trp286 and Asp74 facilitates the intercalation of the 4-carboxylamide pyridinium ring of HI-6 between the side chains of Tyr124 and Trp286. The 2-carboxyimino pyridinium ring of HI-6 is accommodated at the entrance of the catalytic site with the oximate forming a hydrogen bond to the main-chain nitrogen atom of Phe295. In contrast to HI-6, the coordination of Ortho-7 and obidoxime within the PAS is facilitated by an extended structural change of Trp286 that allows one of the carboxyimino pyridinium rings to form a cation-pi interaction with the aromatic groups of Tyr72 and Trp286. The central chain of Ortho-7 and obidoxime is loosely coordinated in the active-site gorge, whereas the second carboxyimino pyridinium ring is accommodated in the vicinity of the phenol ring of Tyr337. The structural data clearly show analogous coordination of Ortho-7 and obidoxime within the active-site gorge of AChE. Different ability to reactivate AChE inhibited by tabun is shown in end-point reactivation experiments where HI-6, Ortho-7 and obidoxime showed an efficiency of 1, 45 and 38%, respectively. The low efficiency of HI-6 and the significantly higher efficiency of Ortho-7 and obidoxime may be explained by the differential binding of the oximes in the PAS and active-site gorge of AChE.

Acetylcholinesterase↗
PubMedDate not supplied

The Paris-Sud yeast structural genomics pilot-project: from structure to function.

We present here the outlines and results from our yeast structural genomics (YSG) pilot-project. A lab-scale platform for the systematic production and structure determination is presented. In order to validate this approach, 250 non-membrane proteins of unknown structure were targeted. Strategies and final statistics are evaluated. We finally discuss the opportunity of structural genomics programs to contribute to functional biochemical annotation.

Genomics↗
PubMed2005

Structural, mechanical and in vitro characterization of individually structured Ti-6Al-4V produced by direct laser forming.

Direct laser forming (DLF) is a rapid prototyping technique which enables prompt modelling of metal parts with high bulk density on the base of individual three-dimensional data, including computer tomography models of anatomical structures. In our project, we tested DLF-produced material on the basis of the titanium alloy Ti-6Al-4V for its applicability as hard tissue biomaterial. To this end, we investigated mechanical and structural properties of DLF-Ti-6Al-4V. While the tensile and yield strengths of untreated DLF alloy ranged beyond 1000 MPa, a breaking elongation of 6.5+/-0.6% was determined for this material. After an additional post-DLF annealing treatment, this parameter was increased two-fold to 13.0+/-0.6%, while tensile and yield strengths were reduced by approx. 8%. A Young's modulus of 118.000+/-2.300 MPa was determined for post-DLF annealed Ti-6Al-4V. All data gained from tensile testing of post-DLF annealed Ti-6Al-4V matched American Society of Testing and Materials (ASTM) specifications for the usage of this alloy as medical material. Rotating bending tests revealed that the fatigue profile of post-DLF annealed Ti-6Al-4V was comparable to casted/hot isostatic pressed alloy. We characterized the structure of non-finished DLF-Ti-6Al-4V by scanning electron microscopy and observed a surface-associated layer of particles, which was removable by sandblasting as a finishing step. We manufactured porous specimens with nominal pore diameters of 500, 700 and 1000 microm. The diameters were reduced by the used DLF processing by approx. 300 microm. In an in vitro investigation, we cultured human osteoblasts on non-porous and porous blasted DLF-Ti-6Al-4V specimens to study morphology, vitality, proliferation and differentiation of the cells. The cells spreaded and proliferated on DLF-Ti-6Al-4V over a culture time of 14 days. On porous specimens, osteoblasts grew along the rims of the pores and formed circle-shaped structures, as visualized by live/dead staining as well as scanning electron microscopy. Overall, the DLF-Ti-6Al-4V approach proved to be efficient and could be further advanced in the field of hard tissue biomaterials.

Alloys↗
PubMed2005

Structure and anticoagulant activity of a sulfated galactan from the red alga, Gelidium crinale. Is there a specific structural requirement for the anticoagulant action?

Marine red algae are an abundant source of sulfated galactans with potent anticoagulant activity. However, the specific structural motifs that confer biological activity remain to be elucidated. We have now isolated and purified a sulfated galactan from the marine red alga, Gellidium crinale. The structure of this polysaccharide was determined using NMR spectroscopy. It is composed of the repeating structure -4-alpha-Galp-(1-->3)-beta-Galp1--> but with a variable sulfation pattern. Clearly 15% of the total alpha-units are 2,3-di-sulfated and another 55% are 2-sulfated. No evidence for the occurrence of 3,6-anhydro alpha-galactose units was observed in the NMR spectra. We also compared the anticoagulant activity of this sulfated galactan with a polysaccharide from the species, Botryocladia occidentalis, with a similar saccharide chain but with higher amounts of 2,3-di-sulfated alpha-units. The sulfated galactan from G. crinale has a lower anticoagulant activity on a clotting assay when compared with the polysaccharide from B. occidentalis. When tested in assays using specific proteases and coagulation inhibitors, these two galactans showed significant differences in their activity. They do not differ in thrombin inhibition mediated by antithrombin, but in assays where heparin cofactor II replaces antithrombin, the sulfated galactan from G. crinale requires a significantly higher concentration to achieve the same inhibitory effect as the polysaccharide from B. occidentalis. In contrast, when factor Xa instead of thrombin is used as the target protease, the sulfated galactan from G. crinale is a more potent anticoagulant. These observations suggest that the proportion and/or the distribution of 2,3-di-sulfated alpha-units along the galactan chain may be a critical structural motif to promote the interaction of the protease with specific protease and coagulation inhibitors.

Anticoagulants↗
PubMed2005

The oligomeric structure of vaccinia viral envelope protein A27L is essential for binding to heparin and heparan sulfates on cell surfaces: a structural and functional approach using site-specific mutagenesis.

The soluble domain of the self-assembly vaccinia virus envelope protein A27L, sA27L-aa, consists of a flexible extended coil at the N terminus and a rigid hydrophobic coiled-coil region at the C terminus. In the former, a basic strip of 12 residues is responsible for binding to cell-surface heparan sulfates. Although the latter is believed to mediate self-assembly, its biological role is unclear. However, an in vitro bioassay showed that peptides comprising the 12 residue basic region alone failed to interact with heparin, suggesting that the C-terminal coiled-coil region might serve an indispensable role in biological function. To explore this structural and functional relationship, we performed site-specific mutagenesis in an attempt to specifically disrupt the hydrophobic core of the coiled coil. Three single mutants, L47A, L51A, and L54A, and one triple mutant, L47,51,54A, were expressed and purified from Escherichia coli. The physical properties of the mutants were carefully examined by gel-filtration chromatography, CD, and NMR spectroscopy, and the biological activities were assessed by an in vitro SPR bioassay and three in vivo bioassays: binding to cells, blocking virus infection and blocking cell fusion. We showed that the L47A mutant, which is similar to the parental sA27L-aa in forming a hexamer, is biologically active. L51A and L54A mutants form tetramers and are less active. Notably, in the triple mutant, the self-assembly hydrophobic core structure is uncoiled; as a consequence, the tetrameric structure is biologically inactive. Thus, we conclude that the leucine residues, in particular Leu51 and Leu54, sustain the hydrophobic core structure that is essential for the biological function of vaccinia virus envelope protein A27L, binding to cell-surface heparan sulfate.

Cell Fusion↗
PubMed2006

Crystal structure of a highly acidic neurotoxin from scorpion Buthus tamulus at 2.2A resolution reveals novel structural features.

The crystal structure of a highly acidic neurotoxin from the scorpion Buthus tamulus has been determined at 2.2A resolution. The amino acid sequence determination shows that the polypeptide chain has 64 amino acid residues. The pI measurement gave a value of 4.3 which is one of the lowest pI values reported so far for a scorpion toxin. As observed in other alpha-toxins, it contains four disulphide bridges, Cys12-Cys63, Cys16-Cys36, Cys22-Cys46, and Cys26-Cys48. The crystal structure reveals the presence of two crystallographically independent molecules in the asymmetric unit. The conformations of two molecules are identical with an r.m.s. value of 0.3A for their C(alpha) tracings. The overall fold of the toxin is very similar to other scorpion alpha-toxins. It is a betaalphabetabeta protein. The beta-sheet involves residues Glu2-Ile6 (strand beta1), Asp32-Trp39 (strand beta3) and Val45-Val55 (strand beta4). The single alpha-helix formed is by residues Asn19-Asp28 (alpha2). The structure shows a trans peptide bond between residues 9 and 10 in the five-membered reverse turn Asp8-Cys12. This suggests that this toxin belongs to classical alpha-toxin subfamily. The surface features of the present toxin are highly characteristic, the first (A-site) has residues, Phe18, Trp38 and Trp39 that protrude outwardly presumably to interact with its receptor. There is another novel face (N-site) of this neurotoxin that contains several negatively charged residues such as, Glu2, Asp3, Asp32, Glu49 and Asp50 which are clustered in a small region of the toxin structure. On yet another face (P-site) in a triangular arrangement, with respect to the above two faces there are several positively charged residues, Arg58, Lys62 and Arg64 that also protrude outwardly for a potentially potent interaction with other molecules. This toxin with three strong features appears to be one of the most toxic molecules reported so far. In this sense, it may be a new subclass of neurotoxins with the largest number of hot spots.

Amino Acid Sequence↗
PubMed2004

The interplay of fold recognition and experimental structure determination in structural genomics.

Achieving the goals of structural genomics initiatives depends on the outcomes of two groups of factors: the number and distribution of experimentally determined protein structures, and our ability to assign novel proteins to known structures (fold recognition) and use them to build models (modeling). The quality of the tools used for fold recognition defines the scope of experimental effort - the more distant the templates that can be recognized, the smaller the number of proteins that have to be solved. Recent improvements in fold recognition may have suggested that the goals of structural genomics initiatives are getting closer. However, problems that surfaced during the first few years of active work have put many of the early estimates in doubt and new ones are still slow in coming.

Animals↗
PubMed2005

Same structure, different function crystal structure of the Epstein-Barr virus IL-10 bound to the soluble IL-10R1 chain.

Human IL-10 (hIL-10) is a cytokine that modulates diverse immune responses. The Epstein-Barr virus (EBV) genome contains an IL-10 homolog (vIL-10) that shares high sequence and structural similarity with hIL-10. Although vIL-10 suppresses inflammatory responses like hIL-10, it cannot activate many other immunostimulatory functions performed by the cellular cytokine. These functional differences have been correlated with the approximately 1000-fold lower affinity of vIL-10, compared to hIL-10, for the IL-10R1 receptor chain. To define the structural basis for these observations, crystal structures of vIL-10 and a vIL-10 point mutant were determined bound to the soluble IL-10R1 receptor fragment (sIL-10R1) at 2.8 and 2.7 A resolution, respectively. The structures reveal that subtle changes in the conformation and dynamics of the vIL-10 AB and CD loops and an orientation change of vIL-10 on sIL-10R1 are the main factors responsible for vIL-10's reduced affinity for sIL-10R1 and its distinct biological profile.

Amino Acid Sequence↗
PubMed1987

Structural responsiveness of filamentous bacteriophage Pf1: comparison of virion structure in fibers and solution. The effect of temperature and ionic strength.

X-ray diffraction from fibers and magnetically oriented solutions has been used to study the effect of changes in environment on the helical symmetry and radial structure of the Pf1 virus particle. Detailed analysis of equatorial scattering to a spacing of 8-10 A was used to identify small radial motions of structural elements in the virus particle. R-factor ratios were used to determine the statistical significance of observed changes. Comparison of the structure of virus particles in fibers with those in solution indicated that the helical symmetry of the virions remains unchanged during fiber formation. In most fibers the virions appear to be slightly distorted by the tight packing of virus particles. This distortion results in an apparent increase in the radius of the virus particle of approximately 0.6 A. A change in the radius of the DNA is also observed. Increase in the concentration of solvent molecules during fiber formation results in penetration of the virus interior by some solvent components. NaCl is also able to enter the virus interior. The change in the helical symmetry of the virions at approximately 8 degrees C appears to be the same whether observed by diffraction from fibers or from solutions. Only subtle changes in radial structure are associated with the temperature transition.

Coliphages↗
PubMed1996

Structural origins of redox potentials in Fe-S proteins: electrostatic potentials of crystal structures.

Redox potentials often differ dramatically for homologous proteins that have identical redox centers. For two types of iron-sulfur proteins, the rubredoxins and the high-potential iron-sulfur proteins (HiPIPs), no structural explanations for these differences have been found. We calculated the classical electrostatic potential at the redox site using static crystal structures of four rubredoxins and four HiPIPs to identify important structural determinants of their redox potentials. The contributions from just the backbone and polar side chains are shown to explain major features of the experimental redox potentials. For instance, in the rubredoxins, the presence of Val 44 versus Ala 44 causes a backbone shift that explains a approximately 50 mV lower redox potential in one of the four rubredoxins. This result is consistent with experimental redox potentials of five additional rubredoxins with known sequence. Also, we attribute the unusually lower redox potentials of two of the HiPIPs studied to a less positive electrostatic environment around their redox sites. Finally, molecular dynamics simulations of solvent around static rubredoxin crystal structures indicate that water alone is a major factor in dampening the contribution of charged side chains, in accord with experiments showing that mutations of surface charges produce relatively little effect on redox potentials.

Alanine↗
PubMed2003

Crystal structures of Tritrichomonasfoetus inosine monophosphate dehydrogenase in complex with substrate, cofactor and analogs: a structural basis for the random-in ordered-out kinetic mechanism.

The enzyme inosine monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide biosynthesis. Because it is up-regulated in rapidly proliferating cells, human type II IMPDH is actively targeted for immunosuppressive, anticancer, and antiviral chemotherapy. The enzyme employs a random-in ordered-out kinetic mechanism where substrate or cofactor can bind first but product is only released after the cofactor leaves. Due to structural and kinetic differences between mammalian and microbial enzymes, most drugs that are successful in the inhibition of mammalian IMPDH are far less effective against the microbial forms of the enzyme. It is possible that with greater knowledge of the structural mechanism of the microbial enzymes, an effective and selective inhibitor of microbial IMPDH will be developed for use as a drug against multi-drug resistant bacteria and protists. The high-resolution crystal structures of four different complexes of IMPDH from the protozoan parasite Tritrichomonas foetus have been solved: with its substrate IMP, IMP and the inhibitor mycophenolic acid (MPA), the product XMP with MPA, and XMP with the cofactor NAD(+). In addition, a potassium ion has been located at the dimer interface. A structural model for the kinetic mechanism is proposed.

Animals↗
PubMed1997

The perception of motion and structure in structure-from-motion: comparisons of affine and Euclidean formulations.

I investigated the discrimination of rigid from nonrigid structure and the perception of affine stretches along the line of sight [Norman & Todd (1993). Perception and Psychophysics, 53, pp. 279-291]. Investigations of performance at discriminating rigid from nonrigid structure showed that performance improved when number of views and amount of simulated three-dimensional nonrigidity increased. Investigations of rotations about the vertical which include affine stretches along the line of sight compared Euclidean interpretations of affine-stretching stimuli to human perception. These Euclidean interpretations were obtained from a simple algorithm which recovered structure and motion from this limited class of stimuli under the assumption that distances to the axis of rotation did not change. The algorithm predicted that stretches along the line of sight would be perceived as nearly rigid and have variable angular velocity. These predictions were supported by subjects' reports of occurrences of nonrigidity and minima of angular velocity. The Euclidean algorithm also provided measures of nonrigidity and motion coherence, and experimental results were consistent with a prediction of when perception of nonrigidity would be independent of perception of coherence. The results are discussed relative to the advantages and shortcomings of both the affine and Euclidean approaches to structure-from-motion.

Algorithms↗
PubMed2003

Crystal structure of Plasmodium berghei lactate dehydrogenase indicates the unique structural differences of these enzymes are shared across the Plasmodium genus.

As Plasmodium rely extensively on homolactic fermentation for energy production, Plasmodium falciparum lactate dehydrogenase (PfLDH)--the key enzyme in this process--has previously been suggested as a novel target for antimalarials. This enzyme has distinctive kinetic and structural properties that distinguish it from its human homologues. In this study, we now describe the expression, kinetic characterisation and crystal structure determination of the LDH from Plasmodium berghei. This enzyme is seen to have a similar kinetic profile to its P. falciparum counterpart, exhibiting the characteristic lack of substrate inhibition that distinguishes plasmodial from human LDHs. The crystal structure of P. berghei lactate dehydrogenase (PbLDH) shows a very similar active site arrangement to the P. falciparum enzyme. In particular, an insertion of five amino acid residues in the active site loop creates an enlarged volume in the substrate binding site, and characteristic changes in the residues lining the NADH cofactor binding pocket result in displacement of the cofactor relative to its observed position in mammalian and all other LDH structures. These results imply the special features previously described for PfLDH may be shared across the Plasmodium genus, supporting the universal application of therapeutics targeting this enzyme.

Amino Acid Sequence↗
PubMed2002

Structural basis for development of cathepsin B-specific noncovalent-type inhibitor: crystal structure of cathepsin B-E64c complex.

In order to elucidate the substrate specificity of the Sn subsites (n=1-3) of cathepsin B, its crystal structure inhibited by E64c [(+)-(2S,3S)-3-(1-[N-(3-methylbutyl)amino]-leucylcarbonyl)oxirane-2-carboxylic acid] was analyzed by the X-ray diffraction method. Iterative manual rebuilding and convenient conjugate refinement of structure decreased R- and free R-factors to 19.7% and to 23.9%, respectively, where 130 water molecules were included for the refinement using 14,759 independent reflections from 10 to 2.3 A resolution. The epoxy carbonyl carbon of E64c was covalently bonded to the Cys(29) S(gamma) atom and the remaining parts were located at Sn subsites (n=1-3). The substrate specificity of these subsites was characterized based on their interactions with the inhibitor. Base on these structural data, we developed a novel cathepsin B-specific noncovalent-type inhibitor, which may bind to S2'-S3. The molecular design of possessing structural elements of both CA074 and E64c, assisted by energy minimization and molecular dynamics (MD) simulation, may lead to a new lead noncovalent-type inhibitor.

Animals↗
PubMed1998

Probing the structure of the human Ca2+- and Zn2+-binding protein S100A3: spectroscopic investigations of its transition metal ion complexes, and three-dimensional structural model.

A large-scale procedure was developed for the anaerobic purification of the human recombinant Ca2+- and Zn2+-binding protein S100A3 for spectroscopic studies. S100A3 eluted as a non-covalently bound dimer (20.8 kDa). It contained 7.5+/-0.1 free thiol groups/monomer, and bound Ca2+ with a Kd of approximately 4 mM, which corresponds to a tenfold increase in affinity compared to the aerobically purified protein. The transition metal ions Co2+, Zn2+ and Cd2+ were used as spectroscopic probes to investigate the role of the 10 cysteine residues per monomer S100A3 in metal binding. Spectrophotometric titrations suggest the formation of dinuclear thiolate-bridged clusters consisting of a Me2+(S(Cys))4 and a Me2+(S(Cys))3(N(His)) site as described for zinc finger proteins. A three-dimensional structural model of S100A3 was proposed on the basis of the NMR structure of the structurally related rabbit S100A6 protein, and taking into account the structural influence of cysteine residues.

Amino Acid Sequence↗