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Results for “conformational ensemble”

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PubMed2002

Single-molecule fluorescence of nucleic acids.

Less than a decade old, single-molecule fluorescence of nucleic acids has rapidly become an important tool in the arsenal of biological probes. A variety of novel approaches to investigate conformational dynamics, catalytic mechanisms, folding pathways and protein-nucleic-acid interactions have recently been devised for nucleic acids using this technique. Combined with biomechanical tools and ensemble measurements, single-molecule fluorescence methods extend our ability to observe and understand biomolecules and complex biological processes.

DNA↗

PubMed1996

Simulations of the overshoot in the build-up of orientation of long DNA during gel electrophoresis based on a distribution of oscillation times.

The periodic extension-contraction motion observed for long DNA molecules undergoing agarose gel electrophoresis in a constant field is believed to be important for the separation mechanism in pulsed field gel electrophoresis. These oscillations give rise to an overshoot and an undershoot in the ensemble orientation of DNA in the beginning of a field pulse, when the molecules oscillate coherently. After approximately one oscillation cycle, the coherence between the molecules is lost, and a constant, cycle-averaged orientation is reached. In this paper we simulate this build-up of the ensemble orientation of DNA by using a distribution of oscillation times (the time between two consecutive compressed conformations) determined by fluorescence microscopy for YOYO-stained DNA. Six different orientation profiles, describing the orientation during one oscillation cycle, were used. The simulated orientation responses are compared with an orientation response measured by linear dichroism (LD) under the same experimental conditions as in the microscopy study. We found that the choice of orientation profile during the oscillation is important. Best agreement between the simulated and the experimental orientation response was obtained for an orientation profile based on a theoretical model by Schurr and Smith (Biopolymers 1990, 29, 1161-1165). The influence of the distribution of oscillation times and its standard deviation on the orientation response was also investigated. Furthermore, simulations at different field strengths and DNA sizes were performed and found to agree quite well with the experimentally obtained LD data.

Computer Simulation↗

PubMed2006

Probabilistic approach to determining unbiased random-coil carbon-13 chemical shift values from the protein chemical shift database.

We describe a probabilistic model for deriving, from the database of assigned chemical shifts, a set of random coil chemical shift values that are "unbiased" insofar as contributions from detectable secondary structure have been minimized (RCCSu). We have used this approach to derive a set of RCCSu values for 13Calpha and 13Cbeta for 17 of the 20 standard amino acid residue types by taking advantage of the known opposite conformational dependence of these parameters. We present a second probabilistic approach that utilizes the maximum entropy principle to analyze the database of 13Calpha and 13Cbeta chemical shifts considered separately; this approach yielded a second set of random coil chemical shifts (RCCSmax-ent). Both new approaches analyze the chemical shift database without reference to known structure. Prior approaches have used either the chemical shifts of small peptides assumed to model the random coil state (RCCSpeptide) or statistical analysis of chemical shifts associated with structure not in helical or strand conformation (RCCSstruct-stat). We show that the RCCSmax-ent values are strikingly similar to published RCCSpeptide and RCCSstruct-stat values. By contrast, the RCCSu values differ significantly from both published types of random coil chemical shift values. The differences (RCCSpeptide - RCCSu) for individual residue types show a correlation with known intrinsic conformational propensities. These results suggest that random coil chemical shift values from both prior approaches are biased by conformational preferences. RCCSu values appear to be consistent with the current concept of the "random coil" as the state in which the geometry of the polypeptide ensemble samples the allowed region of (phi, psi)-space in the absence of any dominant stabilizing interactions and thus represent an improved basis for the detection of secondary structure. Coupled with the growing database of chemical shifts, this probabilistic approach makes it possible to refine relationships among chemical shifts, their conformational propensities, and their dependence on pH, temperature, or neighboring residue type.

Carbon Isotopes↗

PubMed1994

Nuclear magnetic resonance and conformational investigations of the pentasaccharide of the Forssman antigen and overlapping di-, tri-, and tetra-saccharide sequences.

The 1H and 13C NMR parameters, i.e., chemical shifts and coupling constants, for the pentasaccharide of the Forssman antigen and overlapping di-, tri-, and tetra-saccharide sequences thereof have been measured and assigned completely using 1D and 2D techniques, and the oligosaccharide structures have thereby been confirmed. Nuclear Overhauser effect (NOE) experiments have been carried out at three different temperatures to assess the preferred conformations of the pentasaccharide and the component oligosaccharides. The conformational preferences of the compounds mentioned above have subsequently been investigated by theoretical calculations. The flexibility and dynamics of the molecules have been studied by Metropolis Monte Carlo simulations using a modified HSEA force field, and ensemble average data have been generated and compared to data obtained experimentally.

Calorimetry↗

PubMed2000

Binding of retinol induces changes in rat cellular retinol-binding protein II conformation and backbone dynamics.

The structure and backbone dynamics of rat holo cellular retinol-binding protein II (holo-CRBP II) in solution has been determined by multidimensional NMR. The final structure ensemble was based on 3980 distance and 30 dihedral angle restraints, and was calculated using metric matrix distance geometry with pairwise Gaussian metrization followed by simulated annealing. The average RMS deviation of the backbone atoms for the final 25 structures relative to their mean coordinates is 0.85(+/-0.09) A. Comparison of the solution structure of holo-CRBP II with apo-CRBP II indicates that the protein undergoes conformational changes not previously observed in crystalline CRBP II, affecting residues 28-35 of the helix-turn-helix, residues 37-38 of the subsequent linker, as well as the beta-hairpin C-D, E-F and G-H loops. The bound retinol is completely buried inside the binding cavity and oriented as in the crystal structure. The order parameters derived from the (15)N T(1), T(2) and steady-state NOE parameters show that the backbone dynamics of holo-CRBP II is restricted throughout the polypeptide. The T(2) derived apparent backbone exchange rate and amide (1)H exchange rate both indicate that the microsecond to second timescale conformational exchange occurring in the portal region of the apo form has been suppressed in the holo form.

Amides↗

PubMed2004

Generalized simulated tempering realized on expanded ensembles of non-Boltzmann weights.

A generalized version of the simulated tempering operated in the expanded ensembles of non-Boltzmann weights has been proposed to mitigate a quasiergodicity problem occurring in simulations of rough energy landscapes. In contrast to conventional simulated tempering employing the Boltzmann weight, our method utilizes a parametrized, generalized distribution as a workhorse for stochastic exchanges of configurations and subensembles transitions, which allows a considerable enhancement for the rate of convergence of Monte Carlo and molecular dynamics simulations using delocalized weights. A feature of our method is that the exploration of the parameter space encouraging subensembles transitions is greatly accelerated using the dynamic update scheme for the weight via the average guide specific to the energy distribution. The performance and characteristic feature of our method have been validated in the liquid-solid transition of Lennard-Jones clusters and the conformational sampling of alanine dipeptide by taking two types of Tsallis [C. Tsallis, J. Stat. Phys. 52, 479 (1988)] expanded ensembles associated with different parametrization schemes.

Computer Simulation↗

PubMed2005

An efficient and accurate algorithm for assigning nuclear overhauser effect restraints using a rotamer library ensemble and residual dipolar couplings.

Nuclear Overhauser effect (NOE) distance restraints are the main experimental data from protein nuclear magnetic resonance (NMR) spectroscopy for computing a complete three dimensional solution structure including sidechain conformations. In general, NOE restraints must be assigned before they can be used in a structure determination program. NOE assignment is very time-consuming to do manually, challenging to fully automate, and has become a key bottleneck for high-throughput NMR structure determination. The difficulty in automated NOE assignment is ambiguity: there can be tens of possible different assignments for an NOE peak based solely on its chemical shifts. Previous automated NOE assignment approaches rely on an ensemble of structures, computed from a subset of all the NOEs, to iteratively filter ambiguous assignments. These algorithms are heuristic in nature, provide no guarantees on solution quality or running time, and are slow in practice. In this paper we present an accurate, efficient NOE assignment algorithm. The algorithm first invokes the algorithm in [30, 29] to compute an accurate backbone structure using only two backbone residual dipolar couplings (RDCs) per residue. The algorithm then filters ambiguous NOE assignments by merging an ensemble of intra-residue vectors from a protein rotamer database, together with internuclear vectors from the computed backbone structure. The protein rotamer database was built from ultra-high resolution structures (<1.0 A) in the Protein Data Bank (PDB). The algorithm has been successfully applied to assign more than 1,700 NOE distance restraints with better than 90% accuracy on the protein human ubiquitin using real experimentally-recorded NMR data. The algorithm assigns these NOE restraints in less than one second on a single-processor workstation.

Algorithms↗

PubMed1996

The intrinsic optical signal evoked by chiasm stimulation in the rat suprachiasmatic nuclei exhibits GABAergic day-night variation.

Infrared light transmittance imaging was used in rat hypothalamic slices to record an intrinsic optical signal (IOS) of the cell ensemble in the suprachiasmatic nuclei (SCN), the locus of the endogenous circadian clock. Upon optic chiasm stimulation, a transient IOS was observed in an area conforming to the known retinohypothalamic tract innervation in the ventral SCN. An increase in extracellular Mg2+ concentration to 10 mM reduced th IOS, suggesting that the elicited IOS is dependent on synaptic transmission. D-2-amino-5-phosphonopentanoic acid and muscimol suppressed the elicited IOS, indicating that NMDA and GABAA receptor-mediated mechanisms were involved in cell ensemble activity reflected in the IOS. The extracellularly recorded spiking of SCN neurons located outside the IOS area remained largely unaffected by the afferent stimulus. Neurons located within the IOS area responded with a depressed electrical discharge, manifesting an inverse relationship between single-unit discharge and the optical measure. The influence of the endogenous circadian rhythm on the elicited IOS was assessed by carrying out daytime-dependent concentration-response experiments. NMDA and non-NMDA receptor specific compounds did not exhibit significant day-night differences, whereas GABA-specific ligands showed a significant day-night variation in activity. The competitive GABAA receptor antagonist bicuculline enhanced the IOS exclusively in the daytime SCN. 5alpha-Pregnane-3alpha,21-diol-20-one (allotetrahydrodeoxycorticosterone), a neuroactive steroid that potentiates GABAergic inhibition, suppressed the IOS in the night-time SCN more than in the daytime SCN. This suggests that in the rat the level of extracellular GABA is higher in night-time SCN compared to the daytime SCN.

Afferent Pathways↗

PubMed2004

Conformational heterogeneity of an equilibrium folding intermediate quantified and mapped by scanning mutagenesis.

It is challenging to experimentally define an energy landscape for protein folding that comprises multiple partially unfolded states. Experimental results are often ambiguous as to whether a non-native state is conformationally homogeneous. Here, we tested an approach combining systematic mutagenesis and a Brønsted-like analysis to reveal and quantify conformational heterogeneity of folding intermediate states. Using this method, we resolved an otherwise apparently homogeneous equilibrium folding intermediate of Borrelia burgdorferi OspA into two conformationally distinct species and determined their relative populations. Furthermore, we mapped the structural differences between these intermediate species, which are consistent with the non-native species that we previously proposed based on native-state hydrogen exchange studies. When treated as a single state, the intermediate ensemble exhibited fractional Phi-values for mutations and Hammond-type behaviors that are often observed for folding transition states. We found that a change in relative population of the two species within the intermediate ensemble explains these properties well, suggesting that fractional Phi-values and Hammond-type behaviors exhibited by folding intermediates and transition states may arise more often from conformational heterogeneity than from a single partial structure. Our results are consistent with the presence of multiple minima in a rugged energy landscape predicted from theoretical studies. The method described here provides a promising means to probe a complex folding energy landscape.

Antigens, Surface↗

PubMed1995

Conformational analysis of carbohydrates inferred from NMR spectroscopy and molecular modeling. Applications to the behavior of oligo-galactomannan chains.

In both the biological functions and industrial applications of carbohydrates, their conformational behavior along with the dynamic fluctuations that their structures experience are of great significance. The elucidation of the three-dimensional characteristics of carbohydrates and carbohydrate-containing molecules is in general performed by a combination of high-resolution nuclear magnetic resonance (NMR) spectroscopy and molecular modeling. Heteronuclear couplings and nuclear Overhauser effect (NOE) data are major tools for structural determination. The angular dependence of carbon-proton coupling constants has been shown to depend also on the pathways involved. Concerning molecular modeling, the goal is to generate the ensemble of structures that are consistent with the experimental data. This approach is illustrated in the galactomannan chain. It consists of the conformational study of two model disaccharides (mannobiose and epimelibiose) of galactomannans through carbon-proton coupling constants and NOE measurements. Experimental data are then compared to results of conformational analysis that predicted the stable conformers in solution. From these results helical conformations of oligomers are generated and are discussed in terms of the influence of the position and number of galactose units on the conformation of the mannan backbone.

Carbohydrates↗

PubMed2006

Local kinetic measures of macromolecular structure reveal partitioning among multiple parallel pathways from the earliest steps in the folding of a large RNA molecule.

At the heart of the RNA folding problem is the number, structures, and relationships among the intermediates that populate the folding pathways of most large RNA molecules. Unique insight into the structural dynamics of these intermediates can be gleaned from the time-dependent changes in local probes of macromolecular conformation (e.g. reports on individual nucleotide solvent accessibility offered by hydroxyl radical (()OH) footprinting). Local measures distributed around a macromolecule individually illuminate the ensemble of separate changes that constitute a folding reaction. Folding pathway reconstruction from a multitude of these individual measures is daunting due to the combinatorial explosion of possible kinetic models as the number of independent local measures increases. Fortunately, clustering of time progress curves sufficiently reduces the dimensionality of the data so as to make reconstruction computationally tractable. The most likely folding topology and intermediates can then be identified by exhaustively enumerating all possible kinetic models on a super-computer grid. The folding pathways and measures of the relative flux through them were determined for Mg(2+) and Na(+)-mediated folding of the Tetrahymena thermophila group I intron using this combined experimental and computational approach. The flux during Mg(2+)-mediated folding is divided among numerous parallel pathways. In contrast, the flux during the Na(+)-mediated reaction is predominantly restricted through three pathways, one of which is without detectable passage through intermediates. Under both conditions, the folding reaction is highly parallel with no single pathway accounting for more than 50% of the molecular flux. This suggests that RNA folding is non-sequential under a variety of different experimental conditions even at the earliest stages of folding. This study provides a template for the systematic analysis of the time-evolution of RNA structure from ensembles of local measures that will illuminate the chemical and physical characteristics of each step in the process. The applicability of this analysis approach to other macromolecules is discussed.

Animals↗

PubMed1997

The crystal structure of vascular endothelial growth factor (VEGF) refined to 1.93 A resolution: multiple copy flexibility and receptor binding.

BACKGROUND: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific angiogenic and vasculogenic mitogen. VEGF also plays a role in pathogenic vascularization which is associated with a number of clinical disorders, including cancer and rheumatoid arthritis. The development of VEGF antagonists, which prevent the interaction of VEGF with its receptor, may be important for the treatment of such disorders. VEGF is a homodimeric member of the cystine knot growth factor superfamily, showing greatest similarity to platelet-derived growth factor (PDGF). VEGF binds to two different tyrosine kinase receptors, kinase domain receptor (KDR) and Fms-like tyrosine kinase 1 (Flt-1), and a number of VEGF homologs are known with distinct patterns of specificity for these same receptors. The structure of VEGF will help define the location of the receptor-binding site, and shed light on the differences in specificity and cross-reactivity among the VEGF homologs. RESULTS: We have determined the crystal structure of the receptor-binding domain of VEGF at 1.93 A resolution in a triclinic space group containing eight monomers in the asymmetric unit. Superposition of the eight copies of VEGF shows that the beta-sheet core regions of the monomers are very similar, with slightly greater differences in most loop regions. For one loop, the different copies represent different snapshots of a concerted motion. Mutagenesis mapping shows that this loop is part of the receptor-binding site of VEGF. CONCLUSIONS: A comparison of the eight independent copies of VEGF in the asymmetric unit indicates the conformational space sampled by the protein in solution; the root mean square differences observed are similar to those seen in ensembles of the highest precision NMR structures. Mapping the receptor-binding determinants on a multiple sequence alignment of VEGF homologs, suggests the differences in specificity towards KDR and Flt-1 may derive from both sequence variation and changes in the flexibility of binding loops. The structure can also be used to predict possible receptor-binding determinants for related cystine knot growth factors, such as PDGF.

Amino Acid Sequence↗

PubMedDate not supplied

[A disorder of myocardial contractile function in acute experimental coronary failure: the submolecular mechanisms and the action of cardiac glycosides].

Skinned and hybrid myocardial fibers were studied by methods of tensometry, determination of the ATP hydrolysis intensity, and resonance fluorescent energy transfer between highly selective labels bound to various amino acid residues. It was established that development of the early stage of heart failure in the case of acute myocardial ischemia caused by 15-min coronary artery occlusion (CAO) is related to a reversible damage or adaptive (functional) depression of the contractile protein system. As a result, the system features isolated submolecular post-translational variation in the properties of major proteins in a thin actin filament (myosin is not significantly damaged). This leads to a decrease in the force developed by the hybrid fibers (reconstructed using ghost myocardial fibers taken from ischemic area and normal myosin) and in the ATPase activity of actomyosin (ATP hydrolysis intensity) without any significant change in the Ca-sensitivity, cooperativity of the Ca-response of the actomyosin ensemble, and efficiency of the contractile process. In actin of the ischemic area, CAO results in a serious damage of the Lys61 and Cys374 regions and in a less pronounced damage of the Tyr69 and Cys10 regions. These results suggest that the Lys61 and, probably, Cys374-Lys61 regions are included in the actin monomer as a protomer, without adequate prepolymerization structural-conformational changes necessary to provide for the normal functioning of the filament. In the CAO-induced early stage of heart failure, cardiac glycosides (beta-acetyldigoxin, beta-methyldigoxin, and strophanthin K) produce a direct effect upon the intramolecular structure of myocardial actin, restore the generated force level, and increase the intensity of ATP hydrolysis by actomyosin ensemble. This is achieved by improving or normalizing the structural-conformational state and conformational mobility of the Lys61 and Cys374 region of actin.

Actins↗

PubMed2002

Fluorescence correlation spectroscopy for the detection and study of single molecules in biology.

The recent development of single molecule detection techniques has opened new horizons for the study of individual macromolecules under physiological conditions. Conformational subpopulations, internal dynamics and activity of single biomolecules, parameters that have so far been hidden in large ensemble averages, are now being unveiled. Herein, we review a particular attractive solution-based single molecule technique, fluorescence correlation spectroscopy (FCS). This time-averaging fluctuation analysis which is usually performed in Confocal setups combines maximum sensitivity with high statistical confidence. FCS has proven to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solution, and in living cells. The introduction of dual-color cross-correlation and two-photon excitation in FCS experiments is currently increasing the number of promising applications of FCS to biological research.

Animals↗

PubMed2003

Clinically validated peptides as templates for de novo peptidomimetic drug design at G-protein-coupled receptors.

Recent advances in the development of potent and selective peptide and non-peptide ligands for peptidergic receptors are anticipated to help further unravel the roles of class I and II G-protein-coupled receptors in the pathogenesis of human diseases and to accelerate the clinical utility of small molecule peptidomimetics. Peptidomimetic drug discovery directed towards somatostatin agonists, urotensin II antagonists, gonadotropin-releasing hormone antagonists, neurotensin and complement C5a modulators, melanocortin-4 agonists and vasopressin V(2) agonists has achieved success through integration of conformational-based drug design, site-directed mutagenesis, screening, combinatorial chemistry and classical medicinal chemistry. Acceptance that discreet ensembles of secondary structural motifs underpin the interactions of peptides with their cognate receptors has enabled the development of molecules which mimic or stabilize such pharmacophores.

Complement C5a↗

PubMed2003

Solution NMR studies of antiamoebin, a membrane channel-forming polypeptide.

Antiamoebin I is a membrane-active peptaibol produced by fungi of the species Emericellopsis which is capable of forming ion channels in membranes. Previous structure determinations by x-ray crystallography have shown the molecule is mostly helical, with a deep bend in the center of the polypeptide, and that the backbone structure is independent of the solvent used for crystallization. In this study, the solution structure of antiamoebin was determined by NMR spectroscopy in methanol, a solvent from which one of the crystal structures was determined. The ensemble of structures produced exhibit a right-handed helical C terminus and a left-handed helical conformation toward the N-terminus, in contrast to the completely right-handed helices found in the crystal structures. The NMR results also suggest that a "hinge" region exists, which gives flexibility to the polypeptide in the central region, and which could have functional implications for the membrane insertion process. A model for the membrane insertion and assembly process is proposed based on the antiamoebin solution and crystal structures, and is contrasted with the assembly and insertion mechanism proposed for other ion channel-forming polypeptides.

Anti-Bacterial Agents↗

PubMed2005

Observing capillarity in hydrophobic silica nanotubes.

The development of template-synthesized silica nanotubes has created a unique opportunity for studying confined fluids by providing nanometer-scale containers in which the inner diameter (i.d.) and surface chemistry can be systematically and independently varied. An interesting question to be answered is the following: do solvents wet nanometer-scale tubes in the same way they wet ordinary capillaries? To answer this question, we have conducted studies to explore the wettability of the hydrophobic interiors of individual nanotubes. In these studies, single nanotubes with i.d.'s of either 30 or 170 nm were investigated over a range of water/methanol mixtures. These studies provide a direct route for comparing wetting phenomena in nanotubes with conventional macroscopic theories of capillarity. Our observations reveal four important aspects of capillary wetting in the 30-170 nm regime, a size range where the application of the Young-Laplace theory has not been experimentally investigated for hydrophobic pores. They are (i) a sharp transition between wetting and nonwetting conditions induced by addition of a cosolvent, (ii) invariance of this transition between nanotubes of 30 and 170 nm pore diameter, (iii) failure of the Young-Laplace equation to accurately predict the cosolvent's (methanol) mol fraction where the transition occurs, and (iv) reversibility of the observed wetting. The first two aspects conform to conventional capillarity (Young-Laplace), but the latter two do not. These measurements were complemented with ensemble experiments. The difference between theory and experiment is likely due to reliance on macroscopic values of contact angles or to liquid-phase instability within the hydrophobic pore.

Journal Article↗

PubMed2005

MovieMaker: a web server for rapid rendering of protein motions and interactions.

MovieMaker is a web server that allows short ( approximately 10 s), downloadable movies of protein motions to be generated. It accepts PDB files or PDB accession numbers as input and automatically calculates, renders and merges the necessary image files to create colourful animations covering a wide range of protein motions and other dynamic processes. Users have the option of animating (i) simple rotation, (ii) morphing between two end-state conformers, (iii) short-scale, picosecond vibrations, (iv) ligand docking, (v) protein oligomerization, (vi) mid-scale nanosecond (ensemble) motions and (vii) protein folding/unfolding. MovieMaker does not perform molecular dynamics calculations. Instead it is an animation tool that uses a sophisticated superpositioning algorithm in conjunction with Cartesian coordinate interpolation to rapidly and automatically calculate the intermediate structures needed for many of its animations. Users have extensive control over the rendering style, structure colour, animation quality, background and other image features. MovieMaker is intended to be a general-purpose server that allows both experts and non-experts to easily generate useful, informative protein animations for educational and illustrative purposes. MovieMaker is accessible at http://wishart.biology.ualberta.ca/moviemaker.

Computer Graphics↗