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At least 829 records · Page 46Linked to original sources
PubMed2005

Electron cryomicroscopy of biological machines at subnanometer resolution.

Advances in electron cryomicroscopy (cryo-EM) have made possible the structural determination of large biological machines in the resolution range of 6-9 angstroms. Rice dwarf virus and the acrosomal bundle represent two distinct types of machines amenable to cryo-EM investigations at subnanometer resolutions. However, calculating the density map is only the first step, and much analysis remains to extract structural insights and the mechanism of action in these machines. This paper will review the computational and visualization methodologies necessary for analysis (structure mining) of the computed cryo-EM maps of these machines. These steps include component segmentation, averaging based on local symmetry among components, density connectivity trace, incorporation of bioinformatics analysis, and fitting of high-resolution component data, if available. The consequences of these analyses can not only identify accurately some of the secondary structure elements of the molecular components in machines but also suggest structural mechanisms related to their biological functions.

Capsid Proteins↗
PubMed2006

Stealth and mimicry by deadly bacterial toxins.

Diphtheria toxin and exotoxin A are well-characterized members of the ADP-ribosyltransferase toxin family that function as virulence factors in the pathogenic bacteria Corynebacterium diphtheriae and Pseudomonas aeruginosa. Recent high-resolution structural data of the Michaelis (enzyme-substrate) complex of the P. aeruginosa toxin with an NAD(+) analog and eukaryotic elongation factor 2 (eEF2) have provided insights into the mechanism of inactivation of protein synthesis caused by these protein factors. In addition, rigorous steady-state and stopped-flow kinetic analyses of the toxin-catalyzed reaction, in combination with inhibitor studies, have resulted in a quantum leap in our understanding of the mechanistic details of this deadly enzyme mechanism. It is now apparent that these toxins use stealth and molecular mimicry in unleashing their toxic strategy in the infected host eukaryotic cell.

ADP Ribose Transferases↗
PubMed2005

2-Amino-14,16-dimethyloctadecan-3-ol, a new sphingosine analogue toxin in the fungal genus Fusarium.

2-Amino-14,16-dimethyloctadecan-3-ol (2-AOD-3-ol) was isolated from the cytotoxic rice culture extract of a strain of Fusarium avenaceum, which had previously been isolated from Norwegian grain. The structural information was obtained from LC-MS/MS, GC-MS, NMR spectroscopy and high-resolution MS data. The metabolite has a striking similarity to sphinganine, an intermediate in the biosynthesis of the sphingolipids. This similarity is a major feature of the so-called sphingosine analogue toxins; the most studied being the AAL toxins and the fumonisins. 2-AOD-3-ol was found to be cytotoxic to the rat hepatoma cell line H4IIE-W and to the porcine epithelial kidney cell line PK(15) at concentrations (EC(50)) of 16 and 24 microM, respectively. The metabolite has been found in F. avenaceum inoculated wheat that was treated to support ideal conditions for Fusarium growth, demonstrating that the fungus has the potential to produce the metabolite under field conditions, which may occur in Northern Europe.

Animals↗
PubMed2005

Understanding the cytochrome bc complexes by what they don't do. The Q-cycle at 30.

The cytochrome (cyt) bc(1), b(6)f and related complexes are central components of the respiratory and photosynthetic electron transport chains. These complexes carry out an extraordinary sequence of electron and proton transfer reactions that conserve redox energy in the form of a trans-membrane proton motive force for use in synthesizing ATP and other processes. Thirty years ago, Peter Mitchell proposed a general turnover mechanism for these complexes, which he called the Q-cycle. Since that time, many opposing schemes have challenged the Q-cycle but, with the accumulation of large amounts of biochemical, kinetic, thermodynamic and high-resolution structural data, the Q-cycle has triumphed as the accepted model, although some of the intermediate steps are poorly understood and still controversial. One of the major research questions concerning the cyt bc(1) and b(6)f complexes is how these enzymes suppress deleterious and dissipative side reactions. In particular, most Q-cycle models involve reactive semiquinone radical intermediates that can reduce O(2) to superoxide and lead to cellular oxidative stress. Current models to explain the avoidance of side reactions involve unprecedented or unusual enzyme mechanisms, the testing of which will involve new theoretical and experimental approaches.

Animals↗
PubMed2005

Experimental and theoretical improvements on understanding of the O K-edge of TeO2.

Using an electron monochromator attached to an electron microscope, high energy-resolution electron energy-loss spectra collected from TeO2 have revealed new features in the Oxygen K-edge. Using density-functional theory in the local density and the generalized gradient approximation, we find that core-hole strength of 1.3 gives an excellent fit to our high-resolution experimental data. This indicates that screening is not weak in this oxide, as normally assumed, and that neither the ground state nor a full core-hole model is adequate in quantitative reproduction of the O K-edge in the TeO2 system.

Journal Article↗
PubMed2002

Transient binding of CO to Cu(B) in cytochrome c oxidase is dynamically linked to structural changes around a carboxyl group: a time-resolved step-scan Fourier transform infrared investigation.

The redox-driven proton pump cytochrome c oxidase is that enzymatic machinery of the respiratory chain that transfers electrons from cytochrome c to molecular oxygen and thereby splits molecular oxygen to form water. To investigate the reaction mechanism of cytochrome c oxidase on the single vibrational level, we used time-resolved step-scan Fourier transform infrared spectroscopy and studied the dynamics of the reduced enzyme after photodissociation of bound carbon monoxide across the mid-infrared range (2300-950 cm(-1)). Difference spectra of the bovine complex were obtained at -20 degrees C with 5 micros time resolution. The data demonstrate a dynamic link between the transient binding of CO to Cu(B) and changes in hydrogen bonding at the functionally important residue E(I-286). Variation of the pH revealed that the pK(a) of E(I-286) is >9.3 in the fully reduced CO-bound oxidase. Difference spectra of cytochrome c oxidase from beef heart are compared with those of the oxidase isolated from Rhodobacter sphaeroides. The bacterial enzyme does not show the environmental change in the vicinity of E(I-286) upon CO dissociation. The characteristic band shape appears, however, in redox-induced difference spectra of the bacterial enzyme but is absent in redox-induced difference spectra of mammalian enzyme. In conclusion, it is demonstrated that the dynamics of a large protein complex such as cytochrome c oxidase can be resolved on the single vibrational level with microsecond Fourier transform infrared spectroscopy. The applied methodology provides the basis for future investigations of the physiological reaction steps of this important enzyme.

Animals↗
PubMed1994

Conformational dynamics of bovine Cu, Zn superoxide dismutase revealed by time-resolved fluorescence spectroscopy of the single tyrosine residue.

The structural dynamics of bovine erythrocyte Cu, Zn superoxide dismutase (BSOD) was studied by time-resolved fluorescence spectroscopy. BSOD is a homodimer containing a single tyrosine residue (and no tryptophan) per subunit. Frequency-domain fluorometry revealed a heterogeneous fluorescence decay that could be described with a Lorentzian distribution of lifetimes. The lifetime distribution parameters (center and width) were markedly dependent on temperature. The distribution center (average lifetime) displayed Arrhenius behavior with an Ea of 4.2 kcal/mol, in contrast with an Ea of 7.4 kcal/mol for the single-exponential decay of L-tyrosine. This indicated that thermal quenching of tyrosine emission was not solely responsible for the effect of temperature on the lifetimes of BSOD. The distribution width was broad (1 ns at 8 degrees C) and decreased significantly at higher temperatures. Furthermore, the width of the lifetime distribution increased in parallel to increasing viscosity of the medium. The combined effects of temperature and viscosity on the fluorescence decay suggest the existence of multiple conformational substrates in BSOD that interconvert during the excited-state lifetime. Denaturation of BSOD by guanidine hydrochloride produced an increase in the lifetime distribution width, indicating a larger number of conformations probed by the tyrosine residue in the denatured state. The rotational mobility of the tyrosine in BSOD was also investigated. Analysis of fluorescence anisotropy decay data enabled resolution of two rotational correlation times. One correlation time corresponded to a fast (picosecond) rotation that contributed 62% of the anisotropy decay and likely reported local mobility of the tyrosine ring. The longer correlation time was 50% of the expected value for rotation of the whole (dimeric) BSOD molecule and appeared to reflect segmental motions in the protein in addition to overall tumbling. Comparison between rotational correlation times and fluorescence lifetimes of BSOD indicates that the heterogeneity in lifetimes does not arise from mobility of the tyrosine per se, but rather from dynamics of the protein matrix surrounding this residue which affect its fluorescence decay.

Animals↗
PubMed1998

A topological approach to nucleosome structure and dynamics: the linking number paradox and other issues.

The linking number paradox of DNA in chromatin (two negative crossings around the octamer, associated with a unit linking number reduction), which is 21 years old this year, has come of age. After stirring much debate in the past, the initially hypothetical explanation of the paradox by DNA overtwisting on the nucleosome surface is now presented as a hard fact in recent textbooks. The first part of this article presents a historical perspective of the problem and details the numerous attempts to measure DNA local periodicity, which in one remarkable example sowed the seeds for the discovery of DNA bending. The second part is devoted to the DNA minicircle system, which has been developed in the author's laboratory as an alternative to the local-periodicity-measurement approach. It offers a simple proposal: a unit linking number reduction associated with a single crossing. This conclusion is contrasted with the latest high-resolution crystallographic data of the nucleosome in the third part of the article, and the fourth part examines the available evidence supporting an extension of these results to nucleosomes in chromatin. The last part addresses another basic question pertaining to nucleosome dynamics, the conformational flexibility of the histone tetramer.

Alternative Splicing↗
PubMed2002

Quantum mechanical and NMR spectroscopy studies on the conformations of the hydroxymethyl and methoxymethyl groups in aldohexosides.

The potential energy surfaces of the hydroxymethyl and methoxymethyl groups in methyl hexopyranosides have been extensively studied, employing quantum mechanical calculations and high resolution NMR data. The structure and energy of the C-5-C-6 rotamers were calculated at the B3LYP level of the density functional theory (DFT). For all, geometry optimizations were carried out for 264 conformers of 16 methyl D-gluco- and methyl D-galactopyranoside derivatives 1-16 at the B3LYP/6-31G** level. For all calculated minima, single-point calculations were performed at the B3LYP/6-311++G** level. Solvent effects were considered using a self-consistent reaction field method. Values of the vicinal coupling constants 3J(H-5-H-6R), 3J(H-5-H-6S), 3J(C-4-H-6R), and 3J(C-4-H-6S) for methyl D-glucopyranosides, methyl D-galactopyranosides and their 6-O-methyl derivatives 9-16 were measured in two solvents, methanol and water. The calculated gg, gt, and tg rotamer populations of the hydroxymethyl and methoxymethyl groups in 9-16 agreed well with experimental data. The results clearly showed that the population of gg, gt, and tg rotamers is sensitive to solvent effects. It was concluded that the preference of rotamers in 1-16 is due to the hydrogen bonding and solvent effects.

Carbohydrate Conformation↗
PubMed1979

Identification and quantitation of nucleosides, bases and other UV-absorbing compounds in serum, using reversed-phase high-performance liquid chromatography. I. Chromatographic methodology.

A comprehensive investigation of the high-performance liquid chromatographic separation of nucleosides, their bases and other low-molecular-weight UV-absorbing compounds that might be found in serum is reported. A buffer-methanol gradient was used in conjunction with chemically bonded, microparticulate columns to separate many of the biologically important compounds under study in minimal time with maximal resolution. Retention data, absorbance ratios (280/254 nm) and fluorescence responses are reported for 86 nucleosides, bases, nucleotides and other UV-absorbing compounds commonly encountered in biological studies.

Amino Acids↗
PubMed1994

Thermal expansion of hen egg-white lysozyme. Comparison of the 1.9 A resolution structures of the tetragonal form of the enzyme at 100 K and 298 K.

The average structural and dynamic properties of tetragonal hen egg-white lysozyme have been compared, in structures refined at 1.9 A resolution, using data collected at 100 K and 298 K. The molecule expands by 1.8% over this temperature range with the expansion occurring primarily in its small sub-atomic-sized spaces in an anisotropic manner. Hen egg-white lysozyme consists of two domains: domain 1 (residues 40 to 88) is composed primarily of beta-sheet and is observed to expand by only 0.3%; domain 2 (residues 1 to 39 and 89 to 129) is chiefly alpha-helix and is observed to expand by 2.2%. This is consistent with previous observations that proteins composed primarily of alpha-helix expand more with temperature than do those composed primarily of beta-sheet. The largest movement in the molecule is undergone by the two domains of the structure that move further apart as the temperature is raised. This motion is not a cleft opening but rather consists of a tilt by 2.3 degrees of domain 1 away from domain 2. Within the individual domains the largest movement is undergone by loop T1 of domain 2, consisting of residues 17 to 23. This loop moves in the opposite direction to the rest of the molecule as the temperature is raised. Average temperature factors for the room-temperature and low-temperature structures are 15.2 A2 and 8.1 A2, respectively, when all protein atoms are considered, while these values are 14.0 A2 and 7.8 A2, when only main-chain atoms (N, C alpha, C) are taken into account. An examination of the main-chain averaged B-factor per residue shows that residues involved in intermolecular protein-protein contacts, with symmetry-related molecules, have somewhat lower B-factors than the average and undergo smaller than average changes in B-factor as the temperature is lowered.

Amino Acid Sequence↗
PubMed2002

RNA as a drug target.

In the antiviral and antibacterial area, increasing drug resistance means that there is an ever growing need for novel approaches towards structures and mechanisms which avoid the current problems. The huge increase in high resolution structural data is set to make a dramatic impact on targeting RNA as a drug target. The examples of the RNA binding antibiotics, particularly, the totally synthetic oxazolidinones, should help persuade the skceptics that clinically useful, selective drugs can be obtained from targeting RNA directly.

Animals↗
PubMed1992

Modeling DNA structures.

For a molecule of biological importance, one expects a strong correlation between the three-dimensional structure and its biological function(s). Molecular simulations allow the prediction of physical properties of macromolecules. Many of these properties are closely related to the molecular structure. Model-building studies may thus supplement structurally low-resolution experimental data with detailed three-dimensional hypothetical atomic models. Such studies may give a consistent integral view of a wealth of experimental data. In most cases, such models will predict the outcome of certain experiments. Their actual results will often either confirm the model, be used for further refinement, or demand a major revision. Empirical force-fields provide a large amount of physicochemical knowledge concerning structural and other physical properties about various classes of molecules. They give reasonable bond distances and angles and prevent short van der Waals contacts. Difficulties arise for the prediction of large-scale structural elements. These are not only determined by short-range interactions, but also result from long-range electrostatic, hydration, and hydrophobic forces. In the case of the ionically driven DNA B-Z transition, the point of transition as a function of ionic strength and size can be correctly predicted (33). More must be done for a better understanding of the hydration forces (6). What type of questions will be reliably answered by a force-field? Relatively safe answers concern the local geometry of the molecules. If a conformation leads to strong distortions of bond distances or angles or to close van der Waals contacts, it can safely be rejected. Optimizing such unfavorable structures energetically may lead to structures showing how to avoid such distortions. More difficult are energetic questions: Which of two conformers is more stable, or what is the free energy of the substrate in the active site (63)? One cannot always be sure that the force-field provides the correct answers. Therefore, one should concentrate on questions that can be checked experimentally. The application of such concepts to model curved DNA (19, 60) and the DNA four-way junction (61) provides promising results. To explore the knowledge contained in the force-fields, several methods have been proposed. Taking advantage of structural symmetries may improve critically the convergence, while refining the target molecule or its building blocks. A numerically stable derivative for the torsion potential has been proposed. The optimization method of conjugated gradients (see Section II,B) is a powerful tool to find the way downhill toward a local minimum. To surmount barriers and escape local minima requires nonlocal optimization procedures.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA↗
PubMed1999

Detecting unitary events without discretization of time.

In earlier studies we developed the 'Unitary Events' analysis (Grün S. Unitary Joint-Events in Multiple-Neuron Spiking Activity: Detection, Significance and Interpretation. Reihe Physik, Band 60. Thun, Frankfurt/Main: Verlag Harri Deutsch, 1996.) to detect the presence of conspicuous spike coincidences in multiple single unit recordings and to evaluate their statistical significance. The method enabled us to study the relation between spike synchronization and behavioral events (Riehle A, Grün S, Diesmann M, Aertsen A. Spike synchronization and rate modulation differentially involved in motor cortical function. Science 1997;278:1950-1953.). There is recent experimental evidence that the timing accuracy of coincident spiking events, which might be relevant for higher brain function, may be in the range of 1-5 ms. To detect coincidences on that time scale, we sectioned the observation interval into short disjunct time slices ('bins'). Unitary Events analysis of this discretized process demonstrated that coincident events can indeed be reliably detected. However, the method looses sensitivity for higher temporal jitter of the events constituting the coincidences (Grün S. Unitary Joint-Events in Multiple-Neuron Spiking Activity: Detection, Significance and Interpretation. Reihe Physik, Band 60. Thun, Frankfurt/Main: Verlag Harri Deutsch, 1996.). Here we present a new approach, the 'multiple shift' method (MS), which overcomes the need for binning and treats the data in their (original) high time resolution (typically 1 ms, or better). Technically, coincidences are detected by shifting the spike trains against each other over the range of allowed coincidence width and integrating the number of exact coincidences (on the time resolution of the data) over all shifts. We found that the new method enhances the sensitivity for coincidences with temporal jitter. Both methods are outlined and compared on the basis of their analytical description and their application on simulated data. The performance on experimental data is illustrated.

Action Potentials↗
PubMed1997

IL-6 and APPs: anti-inflammatory and immunosuppressive mediators.

Acute inflammation is accompanied by changes in the concentrations of acute phase proteins (APPs). While much is known about the cytokines involved in the initiation of inflammation, less is known about the mediators involved in its resolution. Recent data suggest that interleukin 6 (IL-6) and IL-6-regulated APPs are anti-inflammatory and immuno-suppressive, and may negatively regulate the acute phase response.

Acute-Phase Proteins↗
PubMed2001

The design of antiparallel coiled coils.

Recent structural studies have highlighted the importance of antiparallel coiled coils in nature. In addition, well-behaved, model antiparallel coiled coils have been designed and used for the reassembly of protein fragments and for the study of the energetic contributions of various interactions to helix orientation specificity. Finally, high-resolution structural data are available for designed helical bundles, allowing an evaluation of the success of state-of-the-art protein design efforts.

Algorithms↗
PubMed2002

Machinery of protein folding and unfolding.

During the past two years, a large amount of biochemical, biophysical and low- to high-resolution structural data have provided mechanistic insights into the machinery of protein folding and unfolding. It has emerged that dual functionality in terms of folding and unfolding might exist for some systems. The majority of folding/unfolding machines adopt oligomeric ring structures in a cooperative fashion and utilise the conformational changes induced by ATP binding/hydrolysis for their specific functions.

Adenosine Triphosphate↗
PubMed2002

TSE agent strains and PrP: reconciling structure and function.

The molecular structures of the infectious agents that cause transmissible spongiform encephalopathy (TSE) diseases are still not known despite the current wide acceptance of the prion hypothesis as the basis for their resolution. Here, data supporting and challenging the prion hypothesis in relation to both the biochemical and biological properties of TSE infectious agents are discussed. The need for the independent transmission of TSE agent-specific genetic information is described and the requirements for the molecule to carry this information are proposed. Such a molecule is likely to be a small nucleic acid encoding information to determine the diversity of the pathogenesis of TSE agents.

Animals↗