113 resultados para Folding coadjuvant
Resumo:
The finding that peptides containing -amino acid residues give rise to folding patterns hitherto unobserved in -amino acid peptides[1] has stimulated considerable interest in the conformational properties of peptides built from , and residues,[2] as the introduction of additional methylene (CH2) units into peptide chains provides further degrees of conformational freedom.
Resumo:
The structure of bovine prothrombin fragment 1 has been refined at 2.25 Å resolution using high resolution measurements made with the synchrotron beam at CHESS. The synchrotron data were collected photographically by oscillation methods (R-merge = 0.08). These were combined with lower order diffractometer data for refinement purposes. The structure was refined using restrained least-squares methods with the program PROLSQ to a crystallographic R-value of 0.175. The structure includes 105 water molecules with occupancies of >0·6. The first 35 residues (Ala1-Leu35) of the N-terminal ?-carboxy glutamic acid-domain (Ala1-Cys48) of fragment 1 are disordered as are two carbohydrate chains of Mr ? 5000; the latter two combine to render 40% of the structure disordered. The folding of the kringle of fragment 1 is related to the close intramolecular contact between the inner loop disulfide groups. Half of the conserved sequence of the kringle forms an inner core surrounding these disulfide groups. The remainder of the sequence conservation is associated with the many turns of the main chain. The Pro95 residue of the kringle has a cis conformation and Tyr74 is ordered in fragment 1, although nuclear magnetic resonance studies indicate that the comparable residue of plasminogen kringle 4 has two positions. Surface accessibility calculations indicate that none of the disulfide groups of fragment 1 is accessible to solvent.
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Determining the sequence of amino acid residues in a heteropolymer chain of a protein with a given conformation is a discrete combinatorial problem that is not generally amenable for gradient-based continuous optimization algorithms. In this paper we present a new approach to this problem using continuous models. In this modeling, continuous "state functions" are proposed to designate the type of each residue in the chain. Such a continuous model helps define a continuous sequence space in which a chosen criterion is optimized to find the most appropriate sequence. Searching a continuous sequence space using a deterministic optimization algorithm makes it possible to find the optimal sequences with much less computation than many other approaches. The computational efficiency of this method is further improved by combining it with a graph spectral method, which explicitly takes into account the topology of the desired conformation and also helps make the combined method more robust. The continuous modeling used here appears to have additional advantages in mimicking the folding pathways and in creating the energy landscapes that help find sequences with high stability and kinetic accessibility. To illustrate the new approach, a widely used simplifying assumption is made by considering only two types of residues: hydrophobic (H) and polar (P). Self-avoiding compact lattice models are used to validate the method with known results in the literature and data that can be practically obtained by exhaustive enumeration on a desktop computer. We also present examples of sequence design for the HP models of some real proteins, which are solved in less than five minutes on a single-processor desktop computer Some open issues and future extensions are noted.
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Sequence repeats constituting the telomeric regions of chromosomes are known to adopt a variety of unusual structures, consisting of a G tetraplex stem and short stretches of thymines or thymines and adenines forming loops over the stem. Detailed model building and molecular mechanics studies have been carried out for these telomeric sequences to elucidate different types of loop orientations and possible conformations of thymines in the loop. The model building studies indicate that a minimum of two thymines have to be interspersed between guanine stretches to form folded-back structures with loops across adjacent strands in a G tetraplex (both over the small as well as large groove), while the minimum number of thymines required to build a loop across the diagonal strands in a G tetraplex is three. For two repeat sequences, these hairpins, resulting from different types of folding, can dimerize in three distinct ways-i.e., with loops across adjacent strands and on same side, with loops across adjacent strands and on opposite sides, and with loops across diagonal strands and on opposite sides-to form hairpin dimer structures. Energy minimization studies indicate that all possible hairpin dimers have very similar total energy values, though different structures are stabilized by different types of interactions. When the two loops are on the same side, in the hairpin dimer structures of d(G(4)T(n)G(4)), the thymines form favorably stacked tetrads in the loop region and there is interloop hydrogen bonding involving two hydrogen bonds for each thymine-thymine pair. Our molecular mechanics calculations on various folded-back as well as parallel tetraplex structures of these telomeric sequences provide a theoretical rationale for the experimentally observed feature that the presence of intervening thymine stretches stabilizes folded-back structures, while isolated stretches of guanines adopt a parallel tetraplex structure
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Beta-Lactamase, which catalyzes beta-lactam antibiotics, is prototypical of large alpha/beta proteins with a scaffolding formed by strong noncovalent interactions. Experimentally, the enzyme is well characterized, and intermediates that are slightly less compact and having nearly the same content of secondary structure have been identified in the folding pathway. In the present study, high temperature molecular dynamics simulations have been carried out on the native enzyme in solution. Analysis of these results in terms of root mean square fluctuations in cartesian and [phi, psi] space, backbone dihedral angles and secondary structural hydrogen bonds forms the basis for an investigation of the topology of partially unfolded states of beta-lactamase. A differential stability has been observed for alpha-helices and beta-sheets upon thermal denaturation to putative unfolding intermediates. These observations contribute to an understanding of the folding/unfolding processes of beta-lactamases in particular, and other alpha/beta proteins in general.
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The design of folded structures in peptides containing the higher homologues of alpha-amino acid residues requires the restriction of the range of local conformational choices In alpha-amino acids stereochemically constrained residues like alpha,alpha-dialkylated residue, aminoisobutyric acid (Aib), and D-Proline ((D)Pro) have proved extremely useful in the design of helices and hairpins in short peptides Extending this approach, backbone substitution and cyclization are anticipated to bc useful in generating conformationally constrained beta- and gamma-residues This brief review provides a survey of work on hybrid peptide sequences concerning the conformationally constrained gamma-amino acid residue 1-aminomethyl cyclohexane acetic acid, gabapentin (Gpn) This achiral, beta,beta-disubstituted, gamma-residue strongly favors gauche-gauche conformations about the C-alpha-C-beta (0(2)) and C-alpha-C-gamma (0(1)) bonds, facilitating local folding The Gpn residue can adopt both C-7 (NH1 -> CO1) and C-9 (CO1 (I)<- NH1+I) hydrogen bonds which are analogous to the C-5 and C7 (gamma-turn) conformations at alpha-residues In conjunction with adjacent residues, Gpn may be used in ay and gamma alpha segments to generate C-12 hydrogen bonded conformations which may be considered as expanded analogs of conventional beta-turns The structural characterization of C-12 helices, C-12/C-10 helices with mixed hydrogen bond directionalities and beta-hairpins incorporating Gpn residues at the turn segment is illustrated (C) 2010 Wiley Periodicals, Inc Biopolymers (Pept Sci) 94 733-741 2010
Resumo:
A strategy for the modular construction of synthetic protein mimics based on the ability non-protein amino acids to act as stereochemical directors of polypeptide chain folding, is described. The use of alpha-aminoisobutyric acid (Aib) to construct stereochemically rigid helices has been exemplified by crystallographic and spectroscopic studies of several apolar peptides, ranging in length from seven to sixteen residues. The problem of linker design in elaborating alpha,alpha motifs has been considered. Analysis of protein crystal structure data provides a guide to choosing linking sequences. Attempts at constructing linked helical motifs using linking Gly-Pro segments have been described. The use of flexible linkers, like epsilon-aminocaproic acid has been examined and the crystallographic and solution state analysis of a linked helix motif has been presented. The use of bulky sidechain modifications on a helical scaffold, as a means of generating putative binding sites has been exemplified by a crystal structure of a peptide packed in a parallel zipper arrangement.
Resumo:
Heat shock protein 90 participates in diverse biological processes ranging from protein folding, cell cycle, signal transduction and development to evolution in all eukaryotes. It is also critically involved in regulating growth of protozoa such as Dictyostelium discoideum, Leishmania donovani, Plasmodium falciparum, Trypanosoma cruzi, and Trypanosoma evansi. Selective inhibition of Hsp90 has also been explored as an intervention strategy against important human diseases such as cancer, malaria, or trypanosomiasis. Giardia lamblia, a simple protozoan parasite of humans and animals, is an important cause of diarrheal disease with significant morbidity and some mortality in tropical countries. Here we show that the G. lamblia cytosolic hsp90 ( glhsp90) is split in two similar sized fragments located 777 kb apart on the same scaffold. Intrigued by this unique arrangement, which appears to be specific for the Giardiinae, we have investigated the biosynthesis of GlHsp90. We used genome sequencing to confirm the split nature of the giardial hsp90. However, a specific antibody raised against the peptide detected a product with a mass of about 80 kDa, suggesting a post-transcriptional rescue of the genomic defect. We show evidence for the joining of the two independent Hsp90 transcripts in-trans to one long mature mRNA presumably by RNA splicing. The splicing junction carries hallmarks of classical cis-spliced introns, suggesting that the regular cis-splicing machinery may be sufficient for repair of the open reading frame. A complementary 26-nt sequence in the ``intron'' regions adjacent to the splice sites may assist in positioning the two pre-mRNAs for processing. This is the first example of post-transcriptional rescue of a split gene by trans-splicing.
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The concept of one enzyme-one activity had influenced biochemistry for over half a century. Over 1000 enzymes are now described. Many of them are highly 'specific'. Some of them are crystallized and their three-dimensional structures determined. They range from 12 to 1000 kDa in molecular weight and possess 124 to several hundreds of amino acids. They occur as single polypeptides or multiple-subunit proteins. The active sites are assembled on these by appropriate tertiary folding of the polypeptide chain, or by interaction of the constituent subunits. The substrate is held by the side-chains of a few amino acids at the active site on the surface, occupying a tiny fraction of the total area. What is the bulk of the protein behind the active site doing? Do all proteins have only one function each? Why not a protein have more than one active site on its large surface? Will we discover more than one activity for some proteins? These newer possibilities are emerging and are finding experimental support. Some proteins purified to homogeneity using assay methods for different activities are now recognized to have the same molecular weight and a high degree of homology of amino acid sequence. Obviously they are identical. They represent the phenomenon of one protein-many functions.
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Telomeric DNA of a variety of vertebrates including humans contains the tandem repeat d(TTAGGG)(n). We have investigated the structural properties of the human telomeric repeat oligonucleotide models d(T(2)AG(3))(4), d(G(3)T(2)A)(3)G(3), and d(G(3)T(2)AG(3)) using CD, gel electrophoresis, and chemical probing techniques. The sequences d(G(3)T(2)A)(3)G(3) and d(T(2)AG(3))(4) assume an antiparallel G quartet structure by intramolecular folding, while the sequence d(G(3)T(2)AG(3)) also adopts an antiparallel G quartet structure but by dimerization of hairpins. In all the above cases, adenines are in the loop. The TTA loops are oriented at the same end of the G tetrad stem in the case of hairpin dimer. Further, the oligonucleotide D(G(3)T(2)AG(3)) forms a higher order structure by the association of two hairpin dimers via stacking of G tetrad planes. Here we show that N-7 of adenine in the hairpin dimer is Hoogsteen hydrogen-bonded. The partial reactivity of loop adenines with DEPC in d(T(2)AG(3))(4) suggests that the intramolecular G quartet structure is highly polymorphic and structures with different loop orientations and topologies are formed in solution. Intra- and interloop hydrogen bonding schemes for the TTA loops are proposed to account for the observed diethyl pyrocarbonate reactivities of adenines. Sodium-induced G quartet structures differ from their potassium-induced counterparts not only in stability but also in loop conformation and interactions. Thus, the overall structure and stability of telomeric sequences are modulated by the cation present, loop sequence, and the number of G tracts, which might be important for the telomere function.
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A new class of nets, called S-nets, is introduced for the performance analysis of scheduling algorithms used in real-time systems Deterministic timed Petri nets do not adequately model the scheduling of resources encountered in real-time systems, and need to be augmented with resource places and signal places, and a scheduler block, to facilitate the modeling of scheduling algorithms. The tokens are colored, and the transition firing rules are suitably modified. Further, the concept of transition folding is used, to get intuitively simple models of multiframe real-time systems. Two generic performance measures, called �load index� and �balance index,� which characterize the resource utilization and the uniformity of workload distribution, respectively, are defined. The utility of S-nets for evaluating heuristic-based scheduling schemes is illustrated by considering three heuristics for real-time scheduling. S-nets are useful in tuning the hardware configuration and the underlying scheduling policy, so that the system utilization is maximized, and the workload distribution among the computing resources is balanced.
Resumo:
Bacteriorhodopsin (bR) continues to be a proven testing ground for the study of integral membrane proteins (IMPs). It is important to study the stability of the individual helices of bR, as they are postulated to exist as independently stable transmembmne helices (TMHs) and also for their utility as templates for modeling other IMPs with the postulated seven-helix bundle topology. Toward this purpose, the seven helices of bR have been studied by molecular dynamics simulation in this study. The suitability of using the backbone-dependent rotamer library of side-chain conformations arrived at from the data base of globular protein structures in the case TMHs has been tested by another set of ? helix simulations with the side-chain orientations taken from this library. The influence of the residue's net charge oil the helix stability was examined by simulating the helices III, IV, and VI (from both of the above sets of helices) with zero net charge on the side chains. The results of these 20 simulations demonstrate in general the stability of the isolated helices of bR in conformity with the two-stage hypothesis of IMP folding. However, the helices I, II, V, and VII are more stable than the other three helices. The helical nature of certain regions of III, IV, and VI are influenced by factors such as the net charge and orientation of several residues. It is seen that the residues Arg, Lys, Asp, and Glu (charged residues), and Ser, Thr, Gly, and Pro, play a crucial role in the stability of the helices of bR. The backbone-dependent rotamer library for the side chains is found to be suitable for the study of TMHs in IMP. (C) 1996 John Wiley & Sons, Inc.
Resumo:
Cys126 is a completely conserved residue in triosephosphate isomerase that is proximal to the active site but has been ascribed no specific role in catalysis. A previous study of the C126S and C126A mutants of yeast TIM reported substantial catalytic activity for the mutant enzymes, leading to the suggestion that this residue is implicated in folding and stability [Gonzalez-Mondragon E et al. (2004) Biochemistry43, 3255–3263]. We re-examined the role of Cys126 with the Plasmodium falciparum enzyme as a model. Five mutants, C126S, C126A, C126V, C126M, and C126T, were characterized. Crystal structures of the 3-phosphoglycolate-bound C126S mutant and the unliganded forms of the C126S and C126A mutants were determined at a resolution of 1.7–2.1 Å. Kinetic studies revealed an approximately five-fold drop in kcat for the C126S and C126A mutants, whereas an approximately 10-fold drop was observed for the other three mutants. At ambient temperature, the wild-type enzyme and all five mutants showed no concentration dependence of activity. At higher temperatures (> 40 °C), the mutants showed a significant concentration dependence, with a dramatic loss in activity below 15 μm. The mutants also had diminished thermal stability at low concentration, as monitored by far-UV CD. These results suggest that Cys126 contributes to the stability of the dimer interface through a network of interactions involving His95, Glu97, and Arg98, which form direct contacts across the dimer interface.
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Methods for macromolecular structure determination (NMR and crystallography) are now being used to get structural information on partially folded and unfolded states of proteins. These techniques, in combination with proton hydrogen exchange studies are powerful tools to extract information on non-native states of proteins. This review discusses progress In this area of protein folding.
Resumo:
In this article, we present a novel application of a quantum clustering (QC) technique to objectively cluster the conformations, sampled by molecular dynamics simulations performed on different ligand bound structures of the protein. We further portray each conformational population in terms of dynamically stable network parameters which beautifully capture the ligand induced variations in the ensemble in atomistic detail. The conformational populations thus identified by the QC method and verified by network parameters are evaluated for different ligand bound states of the protein pyrrolysyl-tRNA synthetase (DhPylRS) from D. hafniense. The ligand/environment induced re-distribution of protein conformational ensembles forms the basis for understanding several important biological phenomena such as allostery and enzyme catalysis. The atomistic level characterization of each population in the conformational ensemble in terms of the re-orchestrated networks of amino acids is a challenging problem, especially when the changes are minimal at the backbone level. Here we demonstrate that the QC method is sensitive to such subtle changes and is able to cluster MD snapshots which are similar at the side-chain interaction level. Although we have applied these methods on simulation trajectories of a modest time scale (20 ns each), we emphasize that our methodology provides a general approach towards an objective clustering of large-scale MD simulation data and may be applied to probe multistate equilibria at higher time scales, and to problems related to protein folding for any protein or protein-protein/RNA/DNA complex of interest with a known structure.