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At least 865 records · Page 48Linked to original sources
PubMed2006

Molecular characterization of a disease associated conformational epitope on GAD65 recognised by a human monoclonal antibody b96.11.

Autoantibodies to the 65kDa isoform of glutamate decarboxylase (GAD65) are associated with type I diabetes and recognise highly conformational epitope(s) that remain to be defined. The human recombinant Fab from mAb b96.11 inhibits binding of most GAD65 antibody positive sera from patients and its epitope has previously been localized to the middle region of GAD65. Recent studies indicate that b96.11 antibody specificity predicts the risk of developing type 1 diabetes in prediabetic individuals. We describe the use homology modelling, protein-protein docking simulations and biopanning of random peptide phage displayed libraries with b96.11 to predict contact amino acids on the interface of GAD65/Fab b96.11 complex. Further analysis by in vitro mutagenesis of GAD65 followed by radioimmunoprecipitation refined the amino acids contributing to the b96.11 epitope. Our studies show an interface characterized by a protruding antibody-combining site centered on the long heavy chain CDR3 loop of Fab b96.11 establishing interactions with the critical residue Phe(344) in the core of the epitope on GAD65, surrounded by charged sites within (375)RK(376) and (305)DER(307). The epitope requires residues from both middle and the C-terminal domains, and is the first precise definition of an epitope on GAD65. The nature of the b96.11 epitope leads to considerations of potential structural variations for differences in antigenicity between the isoforms GAD65 and GAD67. The study shows the utility of using a combination of in silico techniques and experimental data for molecular characterization and localization of conformational epitopes for which crystal structures are lacking.

Amino Acid Sequence↗
PubMedDate not supplied

Molecular modeling of averaged rigor crossbridges from tomograms of insect flight muscle.

Electron tomography, correspondence analysis, molecular model building, and real-space refinement provide detailed 3-D structures for in situ myosin crossbridges in the nucleotide-free state (rigor), thought to represent the end of the power stroke. Unaveraged tomograms from a 25-nm longitudinal section of insect flight muscle preserved native structural variation. Recurring crossbridge motifs that repeat every 38.7 nm along the actin filament were extracted from the tomogram and classified by correspondence analysis into 25 class averages, which improved the signal to noise ratio. Models based on the atomic structures of actin and of myosin subfragment 1 were rebuilt to fit 11 class averages. A real-space refinement procedure was applied to quantitatively fit the reconstructions and to minimize steric clashes between domains introduced during the fitting. These combined procedures show that no single myosin head structure can fit all the in situ crossbridges. The validity of the approach is supported by agreement of these atomic models with fluorescent probe data from vertebrate muscle as well as with data from regulatory light chain crosslinking between heads of smooth muscle heavy meromyosin when bound to actin.

Actomyosin↗
PubMed2004

Conformational changes in human Arf1 on nucleotide exchange and deletion of membrane-binding elements.

Conformational changes associated with nucleotide exchange or truncation of the N-terminal alpha-helix of human Arf1 have been investigated by using forms of easily acquired NMR data, including residual dipolar couplings and amide proton exchange rates. ADP-ribosylation factors (Arfs) are 21-kDa GTPases that regulate aspects of membrane traffic in all eukaryotic cells. An essential component of the biological actions of Arfs is their ability to reversibly bind to membranes, a process that involves exposure of the myristoylated N-terminal amphipathic alpha-helix upon activation and GTP binding. Deletion of this helix results in a protein, termed Delta17Arf1, that has a reduced affinity for GDP and the ability to bind GTP in the absence of lipids or detergents. Previous studies, comparing crystal structures for Arf1.GDP and Delta17Arf1.GTP, identified several regions of structural variation and suggested that these be associated with nucleotide exchange rather than removal of the N-terminal helix. However, separation of conformational changes because of nucleotide binding and N-terminal truncation cannot be addressed in comparing these structures, because both the bound nucleotide and the N terminus differ. Resolving the two effects is important as any structural changes involving the N terminus may represent membrane-mediated conformational adjustments that precede GTP binding. Results from NMR experiments presented here on Arf1.GDP and Delta17Arf1.GDP in solution reveal substantial structural differences that can only be associated with N-terminal truncation.

ADP-Ribosylation Factor 1↗
PubMed1997

Prediction of Critical Micelle Concentration Using a Quantitative Structure-Property Relationship Approach

Relationships between the molecular structure and the critical micelle concentration (cmc) of anionic surfactants were investigated using a quantitative structure-property relationship approach. Measured cmc values for 119 anionic structures, representing sodium alkyl sulfates and sodium sulfonates with a wide variety of hydrophobic and hydrophilic structures, were considered. The best multiple linear regression model involved three terms (descriptors) and had a correlation coefficient of R2 = 0.940. Very good correlations (R2 = 0.988) were obtained using three descriptors for a subset of 68 structures, with structural variation only in the hydrophobic domain. From the descriptors used in these regressions, one can conclude that the cmc is primarily dependent on the size (volume or surface area) of the hydrophobic domain and to a lesser extent on the structural complexity of the surfactant molecule.

Journal Article↗
PubMed1998

A pseudoknotted tRNA variant is a substrate for tRNA (cytosine-5)-methyltransferase from Xenopus laevis.

tRNA post-transcriptional modification enzymes of Xenopus laevis were proposed previously to belong to two major groups according to their sensitivity to structural perturbations in their substrates. To further investigate the structural variations tolerated by these enzymes, the tRNA-like domain of turnip yellow mosaic virus RNA (88 nucleotides in length) has been microinjected into the oocytes of Xenopus laevis. This RNA possesses 12 potential target nucleotides for modification within a structure including a pseudoknotted folding, an extended anticodon stem, and unusual D-loop/T-loop interactions. Results indicate that only cytosine-42, a position equivalent to C-49 in canonical tRNAs, was quantitatively modified into m5C in the microinjected RNA. Modification was detected to high levels, indicating that at least one enzyme tolerates non-canonical structural features.

Animals↗
PubMed2004

Backbone-rigidified oligo(m-phenylene ethynylenes).

Oligo(m-phenylene ethynylenes) (oligo(m-PE)) with backbones rigidified by intramolecular hydrogen bonds were found to fold into well-defined conformations. The localized intramolecular hydrogen bond involves a donor and an acceptor from two adjacent benzene rings, respectively, which enforces globally folded conformations on these oligomers. Oligomers with two to seven residues have been synthesized and characterized. The persistence of the intramolecular hydrogen bonds and the corresponding curved conformations were established by ab initio and molecular mechanics calculations, 1D and 2D (1)H NMR spectroscopy, and UV spectroscopy. Pentamer 5, hexamer 6, and heptamer 7 adopt well-defined helical conformations. Such a backbone-based conformational programming should lead to molecules whose conformations are resilient toward structural variation of the side groups. These m-PE oligomers have provided a new approach for achieving folded unnatural oligomers under conditions that are otherwise unfavorable for previously described, solvent-driven folding of m-PE foldamers. Stably folded structures based on the design principle described here can be developed and may find important applications.

Hydrogen Bonding↗
PubMed1988

Structure refined to 2A of a nicked DNA octanucleotide complex with DNase I.

The cutting rates of bovine pancreatic deoxyribonuclease I (DNase I) vary along a given DNA sequence, indicating that the enzyme recognizes sequence-dependent structural variations of the DNA double-helix. In an attempt to define the helical parameters determining this sequence-dependence, we have co-crystallized a complex of DNase I with a self-complementary octanucleotide and refined the crystal structure at 2 A resolution. This structure confirms the basic features of an early model, namely that an exposed loop of DNase I binds in the minor groove of B-type DNA and that interactions do occur with the backbone of both strands. Nicked octamer duplexes that have lost a dinucleotide from the 3'-end of one strand are hydrogen-bonded across a two-fold axis in the crystal to form a quasi-continuous double helix of 14 base pairs. The DNA 14-mer has a B-type conformation and shows substantial distortion of both local and overall helix parameters, induced mainly by the tight interaction of Y73 and R38 in the unusually wide minor groove. Directly coupled to the widening of the groove by approximately 3A is a 21.5 degree bend of the DNA away from the bound enzyme towards the major groove, suggesting that both DNA stiffness and groove width are important in determining the sequence-dependence of the enzyme cutting rate. A second cut of the DNA which is induced by diffusion of Mn2+ into the co-crystals suggests that there are two active sites in DNase I separated by more than 15A.

Deoxyribonuclease I↗
PubMed2003

The sucrose transporter gene family in rice.

In this paper we report the identification, cloning and expression analysis of four putative sucrose transporter (SUT) genes from rice, designated OsSUT2, 3, 4 and 5. Three of the four genes were identified through extensive searches of the recently published draft sequence of the rice genome. Along with the previously reported OsSUT1 we propose that these five genes comprise the rice SUT gene family. Complementary DNA clones were isolated for the four newly identified genes. The deduced proteins of all five SUT genes were predicted to contain 12 membrane-spanning helices and a domain highly conserved throughout all known plant SUTs, suggesting the four additional OsSUT genes encode functional SUTs. Reverse transcription-PCR analysis was performed in order to investigate the expression pattern of each member of the SUT family in rice. A differing but overlapping expression pattern was observed for each member of the SUT family at different stages through plant development. These results, together with the structural variations apparent from the deduced protein sequences, suggest that the five SUTs possess diverse roles in both sink and source tissues. We also discuss the classification and evolution of the rice SUT gene family, using a comparison of the gene structures and deduced amino acid sequences with other known plant SUT genes.

Amino Acid Sequence↗
PubMed1996

Tensile properties of nondegenerate human lumbar anulus fibrosus.

STUDY DESIGN: The in vitro tensile behavior of multiple-layer samples of anulus fibrosus were investigated from nondegenerate intervertebral discs. OBJECTIVES: To quantify the intrinsic tensile behavior of nondegenerate anulus fibrosus and the variations with position and age in the intervertebral disc. SUMMARY OF BACKGROUND DATA: Tension is an important loading mode in the anulus fibrosus. The tensile behavior of single- and multiple-layer samples of anulus fibrosus has been shown to vary with specimen orientation, position in the disc, and environmental conditions. Little is known of the changes in these site-specific tensile properties of the anulus with aging or degeneration of the intervertebral disc. METHODS: Multiple-layer specimens of anulus fibrosus were harvested with an orientation parallel to the circumference of the disc. Constant strain rate and uniaxial tensile tests were performed in 0.15 mol/l NaCl at slow strain rates to measure the intrinsic properties of the collagen-proteoglycan matrix of the anulus fibrosus. The tensile modulus, failure stress, failure strain, and strain energy density were determined. Statistical analyses were done to evaluate regional and age-related differences in these properties. RESULTS: Significant radial and circumferential variations in the intrinsic tensile properties of anular samples were detected. The anterior anulus fibrosus had larger values for tensile moduli and failure stresses than the posterolateral anulus. Also, the outer regions of the anulus had greater moduli and failure stresses and lower failure strains than the inner regions. Strain energy density did not vary significantly with region. Significant, but very weak, correlations were detected between tensile properties and age of the intervertebral disc. CONCLUSIONS: The observed variations in tensile behavior of multiple-layer anulus samples indicate that larger variations in tensile modulus and failure properties occur with radial position in the disc than from anterior to posterolateral regions. This pattern is likely related to site-specific variations in the tensile properties of the single-layer samples of anulus fibrosus lamellae and the organization of successive lamellae and their interactions. The results of the present study suggest that factors other than age, such as compositional and structural variations in the disc, are the most important determinants of tensile behavior of the anulus fibrosus.

Adult↗
PubMed1992

Experimental analysis by site-directed mutagenesis of somatic mutation effects on affinity and fine specificity in antibodies specific for lysozyme.

To experimentally examine the functional roles of somatically derived structural variation in the lysozyme-binding mAb HyHEL-10, we have introduced three different point mutations and one insertion at two different sites in HyHEL-10 by site-directed mutagenesis and expression of the mutant antibodies. Mutation of Asp----Ala at position 101 of the H chain returns a somatically mutated residue to its germline sequence for HyHEL-10, and reduces affinity for chicken lysozyme by approximately 9000-fold. Lengthening the third H chain hypervariable region by two amino acids reduces affinity by about 2000-fold. Two mutations, Asp----Thr at position 101 in the H chain and Lys----Thr at position 49 in the L chain, model somatic differences found in another structurally related but functionally distinguishable mAb and minimally decrease affinity for chicken lysozyme. The H chain mutation Asp101VVH----Thr has little effect on affinity for other avian lysozymes but does alter relative fine specificity for these lysozymes. The L chain mutation Lys49VK----Thr increases affinity for duck lysozyme by approximately fivefold. Neither of the positions mutated, 101 in the H chain nor 49 in the L chain, nor the residues near the insertion contact lysozyme in the x-ray structure of the HyHEL-10 F(ab)-HEL complex. The results suggest that these mutations, which model observed somatic mutations, produce functional variation by indirect or long-range effects.

Amino Acid Sequence↗
PubMed1986

Inhibition of acetylcholinesterase from electric eel by (-)-and (+)-physostigmine and related compounds.

Unnatural (+)-physostigmine (2) inhibited acetylcholinesterase (AChE) from electric eel considerably less than natural (-)-physostigmine (1), but 2 may because of its possible lower toxicity still be an interesting anticholinesterase agent. (-)-Eseroline (3), a major metabolite of (-)-physostigmine (1) and a potent narcotic analgetic, and its unnatural (+)-antipode (4), were both poor inhibitors of the enzyme. (-)-Dihydrosecophysostigmine (5), a ring-open analog of (-)-physostigmine was less, but (-)-N-methylphysostigmine (6) much more potent than the natural alkaloid. The availability of compounds related to (-)- and (+)-physostigmine by improved chemical synthesis suggests that further structural variation may well lead to other biologically interesting AChE inhibitors.

Acetylcholinesterase↗
PubMed2002

Titanium isopropoxide as efficient catalyst for the aza-Baylis-Hillman reaction. Selective formation of alpha-methylene-beta-amino acid derivatives.

The direct formation of alpha-methylene-beta-amino acid derivatives is achieved using the aza version of the Baylis-Hillman protocol. The products are readily formed in a three-component one-pot reaction between arylaldehydes, sulfonamides, and alpha,beta-unsaturated carbonyl compounds. The reaction is efficiently catalyzed by titanium isopropoxide and 2-hydroxyquinuclidine in the presence of molecular sieves. The protocol allows for structural variation of the substrates, tolerating electron-poor and electron-rich arylaldehydes and various Michael acceptors.

Amino Acids↗
PubMed1986

Ultrastructural characterization of human granulosa cells in stimulated cycles: correlation with oocyte fertilizability.

The fine structure of human granulosa cells (GC) obtained in the course of an in vitro fertilization procedure was correlated with oocyte fertilizability. The investigation was performed by light-microscopic and electron-microscopic examination of ultrathin sections and replicas of freeze-fractured material. GC associated with nonfertilizable oocytes (group I) had significantly smaller cell areas, tended to be tightly packed, and exhibited abundant intercellular gap junctions and adherence junctions. Multiple structural variations of cytoplasmic organelles were observed in comparison to cells associated with fertilizable oocytes (group II). Cells in group II tended to be widely dispersed, frequently contained interiorized gap junctional elements (annular junctions), and showed morphologic correlates of high steroidogenic activity. Structural dissimilarities between groups I and II were not consistently related to follicle size, as determined by volume of aspirated follicular fluid. In view of the sequential ultrastructural changes known to occur during follicular maturation and ovulation, it is concluded that GCs related to nonfertilizable oocytes were lacking structural correlates of luteinizing hormone/human chorionic gonadotropin responsiveness.

Adult↗
PubMed1995

Response of rubredoxin from Pyrococcus furiosus to environmental changes: implications for the origin of hyperthermostability.

The bases of the hyperthermostability of rubredoxin from Pyrococcus furiosus (RdPf) have been probed by structural perturbations induced by solution pH and ionic strength changes. Comparison of the solution behavior at pH 7 and pH 2, as probed by far- and near-UV circular dichroism, Trp fluorescence emission, 1-anilinonaphthalene-8-sulfonate (ANS) binding, and NMR spectroscopy, reveals the presence of only minimal structural variations at room temperature. At pH 2, the protein displays a surprising nearly native-like behavior at high ionic strength while, at low ionic strength, it is capable of strongly binding the hydrophobic probe ANS. All the secondary and tertiary structural features, including the environment of the hydrophobic core, appear to be intact regardless of pH and ionic strength. The apparent "melting" or denaturation temperature at pH 2, however, is 42 degrees C lower than at pH 7. This is attributed to the perturbation of many electrostatic interactions, including the disruption of all the ion pairs, which is complete at pH 2, as indicated by electrometric pH titrations. The implications of these findings for the origins of the hyperthermostability of rubredoxin are discussed.

Anilino Naphthalenesulfonates↗
PubMed2003

The structure of the periplasmic ligand-binding domain of the sensor kinase CitA reveals the first extracellular PAS domain.

The integral membrane sensor kinase CitA of Klebsiella pneumoniae is part of a two-component signal transduction system that regulates the transport and metabolism of citrate in response to its environmental concentration. Two-component systems are widely used by bacteria for such adaptive processes, but the stereochemistry of periplasmic ligand binding and the mechanism of signal transduction across the membrane remain poorly understood. The crystal structure of the CitAP periplasmic sensor domain in complex with citrate reveals a PAS fold, a versatile ligand-binding structural motif that has not previously been observed outside the cytoplasm or implicated in the transduction of conformational signals across the membrane. Citrate is bound in a pocket that is shared among many PAS domains but that shows structural variation according to the nature of the bound ligand. In CitAP, some of the citrate contact residues are located in the final strand of the central beta-sheet, which is connected to the C-terminal transmembrane helix. These secondary structure elements thus provide a potential conformational link between the periplasmic ligand binding site and the cytoplasmic signaling domains of the receptor.

Amino Acid Sequence↗
PubMed2006

Development of bioinformatics resources for display and analysis of copy number and other structural variants in the human genome.

The discovery of an abundance of copy number variants (CNVs; gains and losses of DNA sequences >1 kb) and other structural variants in the human genome is influencing the way research and diagnostic analyses are being designed and interpreted. As such, comprehensive databases with the most relevant information will be critical to fully understand the results and have impact in a diverse range of disciplines ranging from molecular biology to clinical genetics. Here, we describe the development of bioinformatics resources to facilitate these studies. The Database of Genomic Variants (http://projects.tcag.ca/variation/) is a comprehensive catalogue of structural variation in the human genome. The database currently contains 1,267 regions reported to contain copy number variation or inversions in apparently healthy human cases. We describe the current contents of the database and how it can serve as a resource for interpretation of array comparative genomic hybridization (array CGH) and other DNA copy imbalance data. We also present the structure of the database, which was built using a new data modeling methodology termed Cross-Referenced Tables (XRT). This is a generic and easy-to-use platform, which is strong in handling textual data and complex relationships. Web-based presentation tools have been built allowing publication of XRT data to the web immediately along with rapid sharing of files with other databases and genome browsers. We also describe a novel tool named eFISH (electronic fluorescence in situ hybridization) (http://projects.tcag.ca/efish/), a BLAST-based program that was developed to facilitate the choice of appropriate clones for FISH and CGH experiments, as well as interpretation of results in which genomic DNA probes are used in hybridization-based experiments.

Algorithms↗
PubMed2004

N-glycan branching requirement in neuronal and postnatal viability.

The structural variations among extracellular N-glycans reflect the activity of glycosyltransferases and glycosidases that operate in the Golgi apparatus. More than other types of vertebrate glycans, N-glycans are highly branched oligosaccharides with multiple antennae linked to an underlying mannose core structure. The branching patterns of N-glycans consist of three types, termed high-mannose, hybrid, and complex. Though most extracellular mammalian N-glycans are of the complex type, some cells variably express hybrid and high-mannose forms. Nevertheless, a requirement for hybrid and complex N-glycan branching exists in embryonic development and postnatal function among mice and humans inheriting defective Mgat1 or Mgat2 alleles. The resulting defects in formation N-glycan branching patterns cause multiple abnormalities, including neurologic defects, and have inferred the presence of distinct functions for hybrid and complex N-glycan branches among different cell lineages. We have further explored N-glycan structure-function relationships in vivo by using Cre-loxP conditional mutagenesis to abolish hybrid and complex N-glycan branching specifically among neuronal cells. Our findings show that hybrid N-glycan branching is an essential posttranslational modification among neurons. Loss of Mgat1 resulted in a unique pattern of neuronal glycoprotein deficiency concurrent with caspase 3 activation and apoptosis. Such animals exhibited severe locomotor deficits, tremors, paralysis, and early postnatal death. Unexpectedly, neuronal Mgat2 deletion resulting in the loss of complex but not hybrid N-glycan branching was well tolerated without phenotypic markers of neuronal or locomotor dysfunction. Structural features associated with hybrid N-glycan branching comprise a requisite posttranslational modification to neuronal glycoproteins that permits normal cellular function and viability.

Acyltransferases↗
PubMed2005

The lexical approach to the study of personality structure: toward the identification of cross-culturally replicable dimensions of personality variation.

The structure of personality variation is discussed from the perspective of the lexical approach, which is based on the examination of relations among personality-descriptive adjectives that are indigenous to various languages. The results of this approach--which reveal a cross-culturally replicated set of six dimensions--are described. Specifically, the obtained structure corresponds rather closely to the Five-Factor Model, but differs from that model in the nature of the Agreeableness and Emotionality/Neuroticism factors and also in the existence of a sixth factor known as Honesty-Humility. It is suggested that the emergence of this structure provides support for attempts to establish a cross-culturally generalizable structural model that could summarize normal and abnormal personality variation.

Cross-Cultural Comparison↗