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PubMed1983

Skeletal muscle myosin light chain kinase. A refined structural model.

A hydrodynamic, enzymatic and CD spectroscopic study of skeletal muscle myosin light chain kinase, three proteolytic fragments and corresponding complexes with calmodulin was performed. A refined shape model was built for the enzyme. It was shown that a head-and-tail structure is formed from two major fragments which are aligned end-to-end. The one fragment (Mr 36000) is compact, of high alpha-helix content and contains the catalytic center with the light chain and the calmodulin binding domains. The other fragment (Mr 33000) with unknown function is asymmetric (a/b greater than 10), of low alpha-helix and of unusually high proline content.

Animals↗

PubMed2002

A novel short neurotoxin, cobrotoxin c, from monocellate cobra (Naja kaouthia) venom: isolation and purification, primary and secondary structure determination, and tertiary structure modeling.

A novel short neurotoxin, cobrotoxin c (CBT C) was isolated from the venom of monocellate cobra (Naja kaouthia) using a combination of ion-exchange chromatography and FPLC. Its primary structure was determined by Edman degradation. CBT C is composed of 61 amino acid residues. It differs from cobrotoxin b (CBT B) by only two amino acid substitutions, Thr/Ala11 and Arg/Thr56, which are not located on the functionally important regions by sequence similarity. However, the LD50 is 0.08 mg/g to mice, i.e. approximately five-fold higher than for CBT B. Strikingly, a structure-function relationship analysis suggests the existence of a functionally important domain on the outside of Loop III of CBT C. The functionally important basic residues on the outside of Loop III might have a pairwise interaction with alpha subunit, instead of gamma or delta subunits of the nicotinic acetylcholine receptor (nAChR).

Amino Acid Sequence↗

PubMed2002

Envelope proteins containing single amino acid substitutions support a structural model of the receptor-binding domain of bovine leukemia virus surface protein.

Functional domains of the strikingly conserved envelope (Env) glycoproteins of bovine leukemia virus (BLV) and its close relative, human T-cell leukemia virus type 1 (HTLV-1), are still being defined. We have used BLV Env protein variants to gain insights into the structure and function of this important determinant of viral infectivity. Each of 23 different single amino acid variants found in cDNA clones of env transcripts present after short-term culture of peripheral blood mononuclear cells from BLV-infected sheep was expressed in COS-1 cells and tested for the ability to mediate cell fusion and to be cleaved to surface (SU) and transmembrane (TM) protein subunits. Of 11 Env variants that failed to induce syncytia or did so poorly, 7 contained changes in amino acids identical or chemically conserved in the HTLV-1 Env protein. These seven included the four variants that showed aberrant proteolytic cleavage and poor cell surface expression, underscoring their importance for Env structure. Ten of 12 variants that retained wild-type syncytium-inducing ability clustered in the N-terminal half of BLV SU, which forms the putative receptor-binding domain (RBD). Several variants in the RBD showed evidence of subtle misfolding, as judged by reduced binding to monoclonal antibodies recognizing conformational epitopes F, G, and H formed by the N terminus of SU. We modeled the BLV RBD by aligning putative structural elements with known elements of the ecotropic Friend murine leukemia virus RBD monomer. All the variant RBD residues but one are exposed on the surface of this BLV model. These variants as well as function-altering, antibody-reactive residues defined by other investigators group on one face of the molecular model. They are strikingly absent from the opposite face, implying that it is likely to face inward in Env complexes. This surface might interact with the C-terminal domain of SU or with an adjacent monomer in the Env oligomer. This location suggests an orientation for the monomer of ecotropic Friend murine leukemia virus RBD.

Amino Acid Sequence↗

PubMed1989

Raison d'être and structural model for the B-Z transition of poly d(G-C).poly d(G-C).

In DNA oligonucleotides crystallized in the A form, the nucleotides adopt standard conformation except for steps 5'-CpG-3' where reduced base-pair twist and a sliding motion of the base pairs along their long axes causes pronounced interstrand guanine-guanine overlap. As a consequence, torsion angles alpha, beta and gamma are consistently trans, trans, trans instead of the common-gauche, trans, +gauche. This conformation significantly increases the intraresidue distance between the guanine base and the 5'-phosphate group. A molecular model of poly d(G-C).poly d(G-C) built with these structural characteristics in the A form, which we call A2-DNA, shows that rotation of the guanosine sugar into the syn orientation is easily achieved and pushes the base pair across the helix axis. If successive guanosines are changed this way, a smooth transformation occurs to the left-handed Z-DNA. We suggest that A- and A2-DNA forms of poly d(G-C).poly d(G-C) are metastable and that the actual transition is B in equilibrium (A in equilibrium A2) in equilibrium Z-DNA.

Crystallography↗

PubMed2005

Protein-DNA binding specificity predictions with structural models.

Protein-DNA interactions play a central role in transcriptional regulation and other biological processes. Investigating the mechanism of binding affinity and specificity in protein-DNA complexes is thus an important goal. Here we develop a simple physical energy function, which uses electrostatics, solvation, hydrogen bonds and atom-packing terms to model direct readout and sequence-specific DNA conformational energy to model indirect readout of DNA sequence by the bound protein. The predictive capability of the model is tested against another model based only on the knowledge of the consensus sequence and the number of contacts between amino acids and DNA bases. Both models are used to carry out predictions of protein-DNA binding affinities which are then compared with experimental measurements. The nearly additive nature of protein-DNA interaction energies in our model allows us to construct position-specific weight matrices by computing base pair probabilities independently for each position in the binding site. Our approach is less data intensive than knowledge-based models of protein-DNA interactions, and is not limited to any specific family of transcription factors. However, native structures of protein-DNA complexes or their close homologs are required as input to the model. Use of homology modeling can significantly increase the extent of our approach, making it a useful tool for studying regulatory pathways in many organisms and cell types.

Base Sequence↗

PubMed1993

Protein secondary structure modelling with probabilistic networks.

In this paper we study the performance of probabilistic networks in the context of protein sequence analysis in molecular biology. Specifically, we report the results of our initial experiments applying this framework to the problem of protein secondary structure prediction. One of the main advantages of the probabilistic approach we describe here is our ability to perform detailed experiments where we can experiment with different models. We can easily perform local substitutions (mutations) and measure (probabilistically) their effect on the global structure. Window-based methods do not support such experimentation as readily. Our method is efficient both during training and during prediction, which is important in order to be able to perform many experiments with different networks. We believe that probabilistic methods are comparable to other methods in prediction quality. In addition, the predictions generated by our methods have precise quantitative semantics which is not shared by other classification methods. Specifically, all the causal and statistical independence assumptions are made explicit in our networks thereby allowing biologists to study and experiment with different causal models in a convenient manner.

Algorithms↗

PubMed1991

Structural models of ribonuclease H domains in reverse transcriptases from retroviruses.

Tertiary models of ribonuclease H (RNase H) domains in reverse transcriptases (RTs) from Moloney murine leukemia virus (MuLV) and human immunodeficiency virus (HIV-1) were built based upon the X-ray structure of RNase H from Escherichia coli (E. coli RNase H). In two models of RT-RNase H domains, not only active site residues but also residues, which construct a hydrophobic core and hydrogen bonds, are located in the same positions as those of E. coli RNase H. The whole backbone structure and the electrostatic molecular surface of MuLV RT-RNase H model are similar to those of E. coli RNase H. On the contrary, HIV-1 RT-RNase H model lacks the third helix and the following loop, resulting no positive charge clusters around the hybrid recognition site. Referring the complex models of RTs with their substrate hybrid, the interaction between DNA-polymerase and RNase H domains in RTs was discussed.

Amino Acid Sequence↗

PubMed1997

NMR and structural model of dynorphin A (1-17) bound to dodecylphosphocholine micelles.

Dynorphin A (1-17) (dynorphin) acts preferentially and with high affinity at the kappa-opioid receptor, for which it is the natural, endogenous ligand. Interest in designing new ligands to interact at the kappa-opioid receptor is based in part on the desire to circumvent some of the problems associated with mu-opioid ligands such as morphine. The high-resolution structure of dynorphin in an environment which closely resembles its environment in vivo could be considered as an important lead for new drugs. The interactions that occur between dynorphin and a model membrane are potentially important, as peptide hormone activity is thought to be mediated by interactions with the cell membrane. Therefore, we have determined the high-resolution structures of dynorphin in a model membrane. Results from our laboratory have shown the existence of an alpha-helical region in dynorphin from residues Gly3 through Arg9 when bound to perdeuterated dodecylphosphocholine (DPC) micelles. In this report we show that dynorphin is bound to DPC micelles and describe a family of dynorphin structures that is alpha-helical from residues Gly3 through Pro10 and that contains a beta-turn from residues Trp14 through Gln17. A model of interaction with the micelle is also reported and is discussed in the context of hormone action in vivo. The structures were determined with 1D and 2D nuclear magnetic resonance spectroscopy, distance geometry in dihedral angle space, and restrained molecular dynamics simulations.

Amino Acid Sequence↗

PubMed2002

Evaluation of a novel structural model to describe the endogenous release of lead from bone.

The aim of this paper was to assess the endogenous release of lead from bone to blood, in 204 exposed subjects. resuming their duties after a 10-month strike in a primary lead smelter in 1991. In vivo 109Cd K X-ray Fluorescence (109Cd K XRF) was used to measure the bone lead concentration in tibia and calcaneus in the smelter, in 1994 and five years later. The 1994 data were used to derive the post-strike bone lead concentrations retrospectively from the significant association between bone lead and the cumulative blood lead index (CBLI). When a linear model was used to predict the current blood lead upon the level of lead in bone, structural analysis of the data produced slopes for tibia (2.0, 95% CI 1.66-2.54) and calcaneus (0.19, 95% CI 0.16-0.23) that were significantly higher than those predicted by the commonly used simple linear regression method, for tibia (0.73, 95%, CI 0.58-0.88) and calcaneus (0.08, 95% CI 0.06-0.09). This suggests that more lead than previously predicted by regression is released from bone to blood. Furthermore, the structural analysis of the data produced an estimation of the contribution of the bone lead stores to the bloodstream that was more consistent with the 1999 epidemiological data than did the regression estimation. Moreover, a non-linear relationship between tibia lead and blood lead was suggested from the assumption checking procedures for regression. When a non-linear regression model was fit to the data, the method produced estimates of important parameters in human lead kinetics, namely the blood lead saturation constant, showing a good agreement with current knowledge of lead metabolism. Finally, the likelihood of a non-linear bone lead release seems to be supported by the recently described dependence of the half-life of lead in bone on age and intensity of occupational exposure.

Adult↗

PubMed2003

Archaeal-type rhodopsins in Chlamydomonas: model structure and intracellular localization.

Phototaxis in the unicellular green alga Chlamydomonas reinhardtii is mediated by rhodopsin-type photoreceptor(s). Recent expressed sequence tag database from the Kazusa DNA Research Institute has provided the basis for unequivocal identification of two archaeal-type rhodopsins in it. Here we demonstrate that one is located near the eyespot, wherein the photoreceptor(s) has long been thought to be enriched, along with the results of bioinformatic analyses. Secondary structure prediction showed that the second putative transmembrane helices (helix B) of these rhodopsins are rich in glutamate residues, and homology modeling suggested that some additional intra- or intermolecular interactions are necessary for opsin-like folding of the N-terminal ca. 300-aa membrane spanning domains of 712 and 737-aa polypeptides. These results complement physiological and electrophysiological experiments combined with the manipulation of their expression [O.A. Sineshchekov, K.H. Jung, J.H. Spudich, Proc. Natl. Sci. USA 99 (2002) 8689; G. Nagel, D. Olig, M. Fuhrmann, S. Kateriya, A.M. Musti, E. Bamberg, P. Hegemann, Science 296 (2002) 2395].

Algal Proteins↗

PubMed2002

Esterolytic antibodies as mechanistic and structural models of hydrolases-a quantitative analysis.

Understanding enzymes quantitatively and mimicking their remarkable catalytic efficiency is a paramount challenge. Here, we applied esterolytic antibodies (the D-Abs) to dissect and quantify individual elements of enzymatic catalysis such as transition state (TS) stabilization, nucleophilic reactivity and conformational changes. Kinetic and mutagenic analysis of the D-Abs were combined with existing structural evidence to show that catalysis by the D-Abs is driven primarily by stabilization of the tetrahedral oxyanionic intermediate of ester hydrolysis formed by the nucleophilic attack of an exogenous (solution) hydroxide anion. The side-chain of TyrH100d is shown to be the main H-bond donor of the D-Abs oxyanion hole. The pH-rate and pH-binding profiles indicate that the strength of this H-bond increases dramatically as the neutral substrate develops into the oxyanionic TS, resulting in TS stabilization of 5-7 kcal/mol, which is comparable to oxyanionic TS stabilization in serine hydrolases. We show that the rate of the exogenous (intermolecular) nucleophilic attack can be enhanced by 2000-fold by replacing the hydroxide nucleophile with peroxide, an alpha-nucleophile that is much more reactive than hydroxide. In the presence of peroxide, the rate saturates (k(cat)(max)) at 6 s(-1). This rate-ceiling appears to be dictated by the rate of the induced-fit conformational rearrangement leading to the active antibody-TS complex. The selective usage of negatively charged exogenous nucleophiles by the D-Abs led to the identification of a positively charged channel. Imprinted by the negatively-charged TS-analogue against which these antibodies were elicited, this channel presumably directs the nucleophile to the antibody-bound substrate. Our findings are discussed in comparison with serine esterases and, in particular, with cocaine esterase (cocE), which possesses a tyrosine based oxyanion hole.

Animals↗

PubMed2007

Assignment of polar states for protein amino acid residues using an interaction cluster decomposition algorithm and its application to high resolution protein structure modeling.

We have developed a new method (Independent Cluster Decomposition Algorithm, ICDA) for creating all-atom models of proteins given the heavy-atom coordinates, provided by X-ray crystallography, and the pH. In our method the ionization states of titratable residues, the crystallographic mis-assignment of amide orientations in Asn/Gln, and the orientations of OH/SH groups are addressed under the unified framework of polar states assignment. To address the large number of combinatorial possibilities for the polar hydrogen states of the protein, we have devised a novel algorithm to decompose the system into independent interacting clusters, based on the observation of the crucial interdependence between the short range hydrogen bonding network and polar residue states, thus significantly reducing the computational complexity of the problem and making our algorithm tractable using relatively modest computational resources. We utilize an all atom protein force field (OPLS) and a Generalized Born continuum solvation model, in contrast to the various empirical force fields adopted in most previous studies. We have compared our prediction results with a few well-documented methods in the literature (WHATIF, REDUCE). In addition, as a preliminary attempt to couple our polar state assignment method with real structure predictions, we further validate our method using single side chain prediction, which has been demonstrated to be an effective way of validating structure prediction methods without incurring sampling problems. Comparisons of single side chain prediction results after the application of our polar state prediction method with previous results with default polar state assignments indicate a significant improvement in the single side chain predictions for polar residues.

Algorithms↗

PubMed2006

Comparison of different chondrocytes for use in tissue engineering of cartilage model structures.

This study compares bovine chondrocytes harvested from four different animal locations--nasoseptal, articular, costal, and auricular--for tissue-engineered cartilage modeling. While the work serves as a preliminary investigation for fabricating a human ear model, the results are important to tissue- engineered cartilage in general. Chondrocytes were cultured and examined to determine relative cell proliferation rates, type II collagen and aggrecan gene expression, and extracellular matrix production. Respective chondrocytes were then seeded onto biodegradable poly(L-lactide-epsilon-caprolactone) disc-shaped scaffolds. Cell-copolymer constructs were cultured and subsequently implanted in the subcutaneous space of athymic mice for up to 20 weeks. Neocartilage development in harvested constructs was assessed by molecular and histological means. Cell culture followed over periods of up to 4 weeks showed chondrocyte proliferation from the tissue sources varied, as did levels of type II collagen and aggrecan gene expression. For both genes, highest expression was found for costal chondrocytes, followed by nasoseptal, articular, and auricular cells. Retrieval of 20-week discs from mice revealed changes in construct dimensions with different chondrocytes. Greatest disc diameter was found for scaffolds seeded with auricular chondrocytes, followed by those with costal, nasoseptal, and articular cells. Greatest disc thickness was measured for scaffolds containing costal chondrocytes, followed by those with nasoseptal, auricular, and articular cells. Retrieved copolymer alone was smallest in diameter and thickness. Only auricular scaffolds developed elastic fibers after 20 weeks of implantation. Type II collagen and aggrecan were detected with differing expression levels on quantitative RT-PCR of discs implanted for 20 weeks. These data demonstrate that bovine chondrocytes obtained from different cartilaginous sites in an animal may elicit distinct responses during their respective development of a tissue-engineered neocartilage. Thus, each chondrocyte type establishes or maintains its particular developmental characteristics, and this observation is critical in the design and elaboration of any tissue-engineered cartilage model.

Aggrecans↗

PubMed2005

Structural models for the interaction of Cd(II) with DNA: trans-[Cd(9-RGH-N7)2(H2O)4]2+.

Reactions of Cd(NO(3))(2) with the model nucleobases 9-alkylguanine in water at neutral pH, give the compounds trans-[Cd(9-RGH-N7)(2)(H(2)O)(4)](NO(3))(2)(R=Me, Et), with the 9-alkylguanine ligands bound to the metal cation at the N(7) position. The X-ray structures of both compounds are reported. The six-coordinate Cd(II) complexes consist of a highly regular octahedral geometry in which the two 9-alkylguanine ligands are in a trans position to each other and approximately collinear with the metal cation. In addition, the networks of both compounds show interesting features. Thus, intramolecular H-bonds between O(6) and a coordinated water molecule are present, and self-association of guanines via H-bonding of N(3)-H...N(2) take place, leading to a 1D supramolecular polymeric ribbon. Density functional theory calculations have been applied to both compounds in order to study the stability of N(7) metalated guanine-guanine associations by comparing experimental and theoretical results. The potential relevance with regard to possible Cd(II)-DNA cross-links is briefly discussed.

Cadmium Compounds↗

PubMed2005

A structural model of an amyloid protofilament of transthyretin.

A docking-and-alignment protocol was devised in order to build amyloid protofilaments of Transthyretin (TTR), starting from partially disrupted TTR monomeric subunits and based on experimentally available information. The docking approach is driven by a combination of shape complementarity and energetic criteria, and uses constraints derived from experimental data obtained for the fibrillar state. The dimeric structures obtained were then subjected to an alignment scheme followed by clustering analysis, producing a collection of protofilaments with distinct geometric properties. The selected protofilament model presented here does agree with known experimental data and general amyloid properties; it is formed by two extended continuous beta-sheets with the beta-strands perpendicular to the main axis of the protofilament and a helical twist with a period of approximately 48 beta-strands. This TTR proto-filament model may be an important step in the understanding of the molecular mechanisms of TTR aggregation, as well as, a valuable instrument in drug design strategies against amyloid diseases.

Amyloid↗

PubMed2004

Testing sequence effects in visual memory: clues for a structural model.

In order to probe the internal organization of visual temporary memory, systematic experiments were performed in which the subjects had to memorize a series of 2-5 images then pass recognition tests, either in all possible testing permutations, (in the case of 2-4 images) or in 20 selected permutations (in the case of 5 images). Over 300,000 tests were performed, generating more than 40,000 errors. The error-rates were found to follow simple rules. Both the 3 and the 4 images results are compatible with the presence of only four typical accuracy levels. On the other hand, the reaction time (RT) results revealed surprisingly rich patterns. The RT for recognizing image i at testing stage t > 1 depends upon which image (j) was tested just before. The ranking of the RTs for (j, i) couples evolves from one testing stage to the next. It is proposed that these RTs reflect, in part, the time needed to localize the trace in memory of a given image, starting from the position at which the previous test occurred. If this assumption is correct, the results of this study are providing a picture of the configurations formed by memorized items in the visual temporary store. A hypothetical minimal model, involving several rows of slots on a triangular mesh is proposed to account for the structures in both the error-rates and the RT results.

Humans↗

PubMed1999

Building new function into glycine receptors: a structural model for the activation of the glycine-gated chloride channel.

1. The glycine receptor chloride channel mediates inhibitory neurotransmission and is a member of the ligand-gated ion channel superfamily, which includes the nicotinic acetylcholine receptor channel. 2. Activation of these channels involves a movement of the pore-lining second membrane-spanning domain with respect to the remainder of the protein. 3. The present review considers the evidence that the loops that connect this domain with the rest of the protein act as crucial components of the channel activation mechanism.

Chloride Channels↗

PubMed2002

A structural model of the chitin-binding domain of cuticle proteins.

The nature of the interaction of insect cuticular proteins and chitin is unknown even though about half of the cuticular proteins sequenced thus far share a consensus region that has been predicted to be the site of chitin binding. We previously predicted the preponderance of beta-pleated sheet in the consensus region and proposed its responsibility for the formation of helicoidal cuticle (Iconomidou et al., Insect Biochem. Mol. Biol. 29 (1999) 285). Consequently, we have also verified experimentally the abundance of antiparallel beta-pleated sheet in the structure of cuticle proteins (Iconomidou et al., Insect Biochem. Mol. Biol. 31 (2001) 877). In this work, based on sequence and secondary structure similarity of cuticle proteins, and especially that of the consensus motif, to that of bovine plasma retinol binding protein (RBP), we propose by homology modelling an antiparallel beta-sheet half-barrel structure as the basic folding motif of cuticle proteins. This folding motif may provide the template for elucidating cuticle protein-chitin interactions in detail and reveal the precise geometrical formation of cuticle's helicoidal architecture. This predicted motif is another example where nature utilizes an almost flat protein surface covered by aromatic side chains to interact with the polysaccharide chains of chitin.

Amino Acid Sequence↗