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At least 235 records · Page 13Linked to original sources
PubMed2004

[Proteins structure changes of COL4A5 gene point mutations and structure-phenotype relations in Alport syndrome].

OBJECTIVE: Alport syndrome (AS) is a progressive hereditary nephritis presented with hematuria and renal failure, frequently associated with sensorineural deafness and ocular lesions. So far, more than 300 gene mutations in AS have been identified which provides a better way to analyze the association between genotype and phenotype. It is hard to understand all the phenotype according to the gene mutations, because the structure and function changes of the relevant protein, alpha5(IV) chain, encoded by mutated COL4A5 gene are rare to know. This study aimed to detect the proteins structure encoded by COL4A5 gene with different missense mutations and to analyze the effect of gene mutations on the secondary structure of alpha5(IV) chain and structure-phenotype relations. METHODS: Two X-linked AS patients with different missense mutations (g.3246G > T resulting in p.G1015V and g.3290G > A resulting in p.G1030S, respectively) diagnosed by clinical manifestations, family history and skin or renal biopsy examinations, as well as a control were included in this study. The fragments of cDNA with the two mutations, respectively, and that of corresponding cDNA from the control were expressed in E. coli. The secondary structure of the recombinant polypeptides were analyzed by using circular dichroism (CD) spectroscopy. RESULTS: CD spectra of the control exhibited a negative peak near 200 nm whereas that of the patient 1 exhibited a negative peak near 220 nm. Furthermore, the magnitude of the negative peak of patient 1 decreased from -9000 deg x cm2 x dmol(-1) to -4000 deg x cm2 x dmol(-1) as compared with that of the control. CD spectra of the patient 2 were slightly changed with the negative peak remaining near 220 nm but the magnitude increasing from -9000 deg x cm2 x dmol(-1) to -11000 deg x cm2 x dmol(-1) as compared with that of the control. In addition, the secondary structure of the control polypeptide was mainly composed of beta-sheet and random coil without alpha-helix, whereas that of the patient 1 presented 12.9% alpha-helix. Although the secondary structure of polypeptide of the patient 2 was also mainly composed of beta-sheet and random coil, the composition of beta-sheet reduced and random coil increased. CONCLUSION: Although the glycine substitutions existed in the same domain of alpha5(IV) chain, the patient 1 with the severe AS phenotype and g.3246G > T mutation, and patient 2 with the mild AS phenotype and g.3290G > A mutation were revealed with different secondary structures of alpha5(IV) chain. Moreover, the secondary structure changes of alpha5(IV) chain were consistent with their corresponding phenotype severity.

Collagen Type IV↗
PubMed1993

The three-dimensional structure of the ligand-binding domain of a wild-type bacterial chemotaxis receptor. Structural comparison to the cross-linked mutant forms and conformational changes upon ligand binding.

The three-dimensional structures of the ligand-binding domain of the wild-type Salmonella typhimurium aspartate receptor have been determined in the absence (apo) and presence of bound aspartate (complex) and compared to a cross-linked mutant containing a cysteine at position 36 which does not change signaling behavior of the intact receptor. The structures of the wild-type forms were determined in order to assess the effects of cross-linking on the structure and its influence on conformational changes upon ligand binding. As in the case of the cross-linked mutant receptor, the non-cross-linked ligand-binding domain is dimeric and is composed of 4-alpha-helical bundle monomer subunits related by a crystallographic 2-fold axis in the unbound form and by a non-crystallographic axis in the aspartate-bound form. A comparative study between the non-cross-linked and cross-linked structures has led to the following observations: 1) The long N-terminal helices of the individual subunits in the cross-linked structures are bent toward each other to accommodate the disulfide bond. 2) The rest of the subunit conformation is very similar to that of the wild-type. 3) The intersubunit angle of the cross-linked apo structure is larger by about 13 degrees when compared to the wild-type apo structure. 4) The nature and magnitude of the aspartate-induced conformational changes in the non-cross-linked wild-type structures are very similar to those of the cross-linked structures.

Amino Acid Sequence↗
PubMed2005

Recent developments in structural proteomics for protein structure determination.

The major challenges in structural proteomics include identifying all the proteins on the genome-wide scale, determining their structure-function relationships, and outlining the precise three-dimensional structures of the proteins. Protein structures are typically determined by experimental approaches such as X-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. However, the knowledge of three-dimensional space by these techniques is still limited. Thus, computational methods such as comparative and de novo approaches and molecular dynamic simulations are intensively used as alternative tools to predict the three-dimensional structures and dynamic behavior of proteins. This review summarizes recent developments in structural proteomics for protein structure determination; including instrumental methods such as X-ray crystallography and NMR spectroscopy, and computational methods such as comparative and de novo structure prediction and molecular dynamics simulations.

Aspergillus↗
PubMed1997

Solution structure of Lqh-8/6, a toxin-like peptide from a scorpion venom--structural heterogeneity induced by proline cis/trans isomerization.

Lqh-8/6 is a minor fraction isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus. Here we describe the purification, amino acid sequencing and solution structure determination by NMR and molecular modeling of this peptide. Lqh-8/6 is a small polypeptide (38 residues) which contains 8 half-cystines and is highly similar to another venom component, chlorotoxin. Standard homonuclear methods were used to sequentially assign the proton NMR spectra and to collect spatial restraints for structure determination. Two populations, identified early in the assignment step, are in slow interconversion on the NMR timescale. The two conformers were shown to originate from a cis/trans peptidyl-prolyl isomerization. Using a distance geometry program and simulated annealing protocol under the NMR restraints we obtained 10 final structures for the major conformation (trans isomer). None of the structures showed NOE violations larger than 0.05 nm, and the rmsd value relative to the mean structure (considering the main chain atoms in well-defined secondary structure) is 0.07 nm. The three-dimensional structure contains a short alpha-helix strapped on a small antiparallel beta-strand and an N-terminal extended fragment. The sequence/structure and structure/function relationships of the new scorpion toxin-like peptide are discussed in the context of the present structure determination. This toxin shows a stable, highly populated cis conformer of a peptidyl-prolyl peptide bond.

Amino Acid Sequence↗
PubMed2001

At nonzero temperatures, stacked structures of methylated nucleic acid base pairs and microhydrated nonmethylated nucleic acid base pairs are favored over planar hydrogen-bonded structures: a molecular dynamics simulations study.

The dynamic structure of all ten possible nucleic acid (NA) base pairs and methylated NA base pairs hydrated by a small number of water molecules (from 1 to 16) was determined by using molecular dynamics simulations in the NVE microcanonical and NVT canonical ensembles with the Cornell force field (W. D. Cornell, P. Cieplak, C. I. Bayly, I. R. Gould, K. M. Merz, D. M. Ferguson, D. C. Spellmeyer, T. Fox, J. E. Caldwell, P. Kollman, J. Am. Chem. Soc. 1995, 117, 5179). The presence of one water molecule does not affect the structure of any hydrogen-bonded (H-bonded) nonmethylated base pair. An equal population of H-bonded and stacked structures of adenine...adenine, adenine...guanine and adenine... thymine pairs is reached if as few as two water molecules are present, while obtaining equal populations of these structures in the case of adenine...cytosine, cytosine...thymine, guanine... guanine and guanine...thymine required the presence of four water molecules, and in the case of guanine...cytosine, six. A comparable population of planar, H-bonded and stacked structures for cytosine...cytosine and thymine... thymine base pairs was only obtained if at least eight water molecules hydrated a pair. Methylation of bases changed the situation dramatically and stacked structures were favoured over H-bonded ones even in the absence of water molecules in most cases. Only in the case of methyl cytosine...methyl cytosine, methyl guanine...methyl guanine and methyl guanine...methyl cytosine pairs were two, two or six water molecules, respectively, needed in order to obtain a comparable population of planar, H-bonded and stacked structures. We believe that these results give clear evidence that the preferred stacked structure of NA base pairs in the microhydrated environment, and also apparently in a regular solvent, is due to the hydrophilic interaction of a small number of water molecules. In the case of methylated bases, it is also due to the fact that the hydrogen atoms most suitable for the formation of H-bonds have been replaced by a methyl group. A preferred stacked structure is, thus, not due to a hydrophobic interaction between a large bulk of water molecules and the base pair, as believed.

Base Pairing↗
PubMed2005

New York-Structural GenomiX Research Consortium (NYSGXRC): a large scale center for the protein structure initiative.

Structural GenomiX, Inc. (SGX), four New York area institutions, and two University of California schools have formed the New York Structural GenomiX Research Consortium (NYSGXRC), an industrial/academic Research Consortium that exploits individual core competencies to support all aspects of the NIH-NIGMS funded Protein Structure Initiative (PSI), including protein family classification and target selection, generation of protein for biophysical analyses, sample preparation for structural studies, structure determination and analyses, and dissemination of results. At the end of the PSI Pilot Study Phase (PSI-1), the NYSGXRC will be capable of producing 100-200 experimentally determined protein structures annually. All Consortium activities can be scaled to increase production capacity significantly during the Production Phase of the PSI (PSI-2). The Consortium utilizes both centralized and de-centralized production teams with clearly defined deliverables and hand-off procedures that are supported by a web-based target/sample tracking system (SGX Laboratory Information Data Management System, LIMS, and NYSGXRC Internal Consortium Experimental Database, ICE-DB). Consortium management is provided by an Executive Committee, which is composed of the PI and all Co-PIs. Progress to date is tracked on a publicly available Consortium web site (http://www.nysgxrc.org) and all DNA/protein reagents and experimental protocols are distributed freely from the New York City Area institutions. In addition to meeting the requirements of the Pilot Study Phase and preparing for the Production Phase of the PSI, the NYSGXRC aims to develop modular technologies that are transferable to structural biology laboratories in both academe and industry. The NYSGXRC PI and Co-PIs intend the PSI to have a transforming effect on the disciplines of X-ray crystallography and NMR spectroscopy of biological macromolecules. Working with other PSI-funded Centers, the NYSGXRC seeks to create the structural biology laboratory of the future. Herein, we present an overview of the organization of the NYSGXRC and describe progress toward development of a high-throughput Gene-->Structure platform. An analysis of current and projected consortium metrics reflects progress to date and delineates opportunities for further technology development.

Cloning, Molecular↗
PubMed2005

X-ray structural studies of Mycobacterium tuberculosis RRF and a comparative study of RRFs of known structure. Molecular plasticity and biological implications.

The crystal structure of Mycobacterium tuberculosis ribosome recycling factor has been determined and refined against three X-ray diffraction data sets, two collected at room temperature and the other at 100K. The two room-temperature data sets differ in the radiation damage suffered by the crystals before the data used for processing were collected. A comparison between the structures refined against the two data sets indicates the possibility of radiation-induced conformational change. The L-shaped molecule is composed of a long three-helix bundle domain (domain I) and a globular domain (domain II) connected by a linker region. The main difference between the room-temperature structure and the low temperature structure is in the rotation of domain II about an axis close to its libration axis. This observation and a detailed comparative study of ribosome recycling factors (RRFs) of known structures led to an elaboration of the present understanding of the structural variability of RRF. The variability involves a change in the angle between the two arms of the molecule, a rotation of domain II in a plane nearly perpendicular to the axis of the helix bundle and an internal rotation of domain II. Furthermore, the domains and the linker could be delineated into fixed and variable regions in a physically meaningful manner. The relative mobility of the domains of the molecule in the crystal structure appears to be similar to that in the ribosome--RRF complex. That permits a meaningful discussion of the structural features of RRF in terms of ribosome--RRF interactions. The structure also provides insights into the results of inter-species complementation studies.

Bacterial Proteins↗
PubMed2005

Classification of spider neurotoxins using structural motifs by primary structure features. Single residue distribution analysis and pattern analysis techniques.

In recent years the data on the novel structures of spider toxins have been greatly increasing. The sequence data should be classified. We introduced two primary structure analysis techniques-single residue distribution analysis (SRDA) and pattern analysis for classifying spider polypeptide toxins with molecular weight less than 10kDa. For multiple sequence alignment, we also introduced three novel sequence representation formats named as a simple record, motif record and a pattern record, which can be useful for large-scale analysis of structures. About 300 sequences of spider toxins were analyzed and nine primary structure motifs were identified. New classification of spider toxins was proposed on the basis of previously described principal structural motif (PSM) and extra structural motif (ESM) [Kozlov, S.A., Malyavka, A.A., McCutchen, B., Lu, A., Schepers, E., Herrmann, R., Grishin, E.V., 2005. A novel strategy for the identification of toxin-like structures in spider venom. Proteins 59 (1), 131-140]. Five main structural classes were revealed, and for putative ion channel inhibitors from the most numerous classes 1, 2, and 3, five-digital personal ID numbers were introduced. A reference table with simple, motif and pattern representation sequence formats was created for all analyzed structures.

Amino Acid Motifs↗
PubMed1997

Predicting RNA structures: the model of the RNA element binding Rev meets the NMR structure.

BACKGROUND: How accurate are the predictions of RNA three-dimensional structures? Assessing this accuracy requires the detailed comparison of the prediction with the experimentally determined structure. Previously, sequence variation in RNA aptamers that bind the Rev protein was used to infer a three-dimensional model of the Rev-binding element (RBE) RNA. Although much of this model has been substantiated by subsequent experimental data, its validity remains to be determined by confronting it with the structure determined by NMR spectroscopy. RESULTS: A series of different criteria such as geometric parameters (root mean square deviation, interproton distances, torsions and puckering), helicoidal parameters (base pairing and base stacking) and stability considerations (conformational energies) have been evaluated to identify common and distinguishing structural characteristics of the model and the NMR structure. CONCLUSIONS: The detailed comparison of the two structures reveals striking structural similarities at both the global and local level that validate the RNA modeling approach that we have used. Analysis of the structural differences and the precision of the model suggest that the limitations of the method are related to the amount of structural information available for modeling.

Binding Sites↗
PubMed1999

Formation of metastable RNA structures by sequential folding during transcription: time-resolved structural analysis of potato spindle tuber viroid (-)-stranded RNA by temperature-gradient gel electrophoresis.

A model of functional elements critical for replication and infectivity of the potato spindle tuber viroid (PSTVd) was proposed earlier: a thermodynamically metastable structure containing a specific hairpin (HP II) in the (-)-strand replication intermediate is essential for template activity during (+)-strand synthesis. We present here a detailed kinetic analysis on how PSTVd (-)-strands fold during synthesis by sequential folding into a variety of metastable structures that rearrange only slowly into the structure distribution of the thermodynamic equilibrium. Synthesis of PSTVd (-)-strands was performed by T7-RNA-polymerase; the rate of synthesis was varied by altering the concentration of nucleoside triphosphates to mimic the in vivo synthesis rate of DNA-dependent RNA polymerase II. With dependence on rate and duration of the synthesis, the structure distributions were analyzed by temperature-gradient gel electrophoresis (TGGE). Metastable structures are generated preferentially at low transcription rates--similar to in vivo rates--or at short transcription times at higher rates. Higher transcription rates or longer transcription times lead to metastable structures in low or undetectable amounts. Instead different structures do gradually appear having a more rod-like shape and higher thermodynamic stability, and the thermodynamically optimal rod-like structure dominates finally. It is concluded that viroids are able to use metastable as well as stable structures for their biological functions.

DNA-Directed RNA Polymerases↗
PubMed1996

Hydrogen bonding and solvent structure in an antigen-antibody interface. Crystal structures and thermodynamic characterization of three Fv mutants complexed with lysozyme.

Using site-directed mutagenesis, X-ray crystallography, and titration calorimetry, we have examined the structural and thermodynamic consequences of removing specific hydrogen bonds in an antigen-antibody interface. Crystal structures of three antibody FvD1.3 mutants, VLTyr50Ser (VLY50S), VHTyr32Ala (VHY32A), and VHTyr101Phe (VHY101F), bound to hen egg white lysozyme (HEL) have been determined at resolutions ranging from 1.85 to 2.10 A. In the wild-type (WT) FvD1.3-HEL complex, the hydroxyl groups of VLTyr50, VHTyr32, and VHTyr101 each form at least one hydrogen bond with the lysozyme antigen. Thermodynamic parameters for antibody-antigen association have been measured using isothermal titration calorimetry, giving equilibrium binding constants Kb (M-1) of 2.6 x 10(7) (VLY50S), 7.0 x 10(7) (VHY32A), and 4.0 x 10(6) (VHY101F). For the WT complex, Kb is 2.7 x 10(8) M-1; thus, the affinities of the mutant Fv fragments for HEL are 10-, 4-, and 70-fold lower than that of the original antibody, respectively. In all three cases entropy compensation results in an affinity loss that would otherwise be larger. Comparison of the three mutant crystal structures with the WT structure demonstrates that the removal of direct antigen-antibody hydrogen bonds results in minimal shifts in the positions of the remaining protein atoms. These observations show that this complex is considerably tolerant, both structurally and thermodynamically, to the truncation of antibody side chains that form hydrogen bonds with the antigen. Alterations in interface solvent structure for two of the mutant complexes (VLY50S and VHY32A) appear to compensate for the unfavorable enthalpy changes when protein-protein interactions are removed. These changes in solvent structure, along with the increased mobility of side chains near the mutation site, probably contribute to the observed entropy compensation. For the VHY101F complex, the nature of the large entropy compensation is not evident from a structural comparison of the WT and mutant complexes. Differences in the local structure and dynamics of the uncomplexed Fv molecules may account for the entropic discrepancy in this case.

Antigen-Antibody Complex↗
PubMedDate not supplied

Enzymatic copolymerization alters the structure of unpolymerized mixtures of the biomimetic monomers: the amphiphilic decyl ester of L-tyrosine and L-tyrosineamide--an AFM investigation of nano- to micrometer-scale structure differences.

Previously, we have shown that the amphiphilic decyl esters of both D- and L-tyrosine (DELT) self-assemble in aqueous solution above their critical micelle concentration values to form long rodlike structures that can be enzymatically polymerized. In the current study, we have examined the self-assembled structures of unpolymerized and enzymatically (horseradish peroxidase) copolymerized 1:1 molar mixtures of DELT with the nonamphiphilic comonomer L-tyrosineamide. The structures were examined following adsorption to gold-coated mica surfaces using optical microscopy and scanning electron microscopy, but primarily noncontact atomic force microscopy. Both unpolymerized and copolymerized 1:1 comonomer mixture aggregates produced amorphous to spherical shaped structures, exhibiting increased flexibility that contrasted with our previous observations of the more highly ordered long rodlike structures seen with the pure DELT. The unpolymerized comonomer aggregates were amorphous and of varying size. Interestingly, they contained occasional novel structures-smooth, sharp, nipplelike features that rose hundreds of nanometers above the smooth aggregate surface. However, upon enzymatic copolymerization, the structures are altered, forming nearly hemispherical aggregates in contact with each other on the surface. These structures possessed diameters of 1.51 +/- 0.24 microm. The copolymerized structures lacked any evidence of the sharp nipplelike features observed in the unpolymerized sample, but they did exhibit nanometer-scale detailed surface features, indicative of a higher degree of internal organization. The measured surface roughness of the copolymerized comonomer mixture was more than 10 times greater than the surface roughness of the unpolymerized comonomer mixture.

Biomimetic Materials↗