A hierarchy of functionally important relaxations within myoglobin based on solvent effects, mutations and kinetic model

Geminate CO rebinding in myoglobin is studied for two viscous solvents, trehalose and sol-gel (bathed in 100% glycerol) at several temperatures. Mutations in key distal hemepocket residues are used to eliminate or enhance specific relaxation modes. The time-resolved data are analyzed with a modified Agmon-Hopfield model which is capable of providing excellent fits in cases where a single relaxation mode is dominant. Using this approach, we determine the relaxation rate constants of specific functionally important modes, obtaining also their Arrhenius activation energies. We find a hierarchy of distal pocket modes controlling the rebinding kinetics. The "heme access mode" (HAM) is responsible for the major slow-down in rebinding. It is a solvent-coupled cooperative mode which restricts ligand return from the xenon cavities. Bulky side-chains, like those His64 and Trp29 (in the L29W mutant), operate like overdamped pendulums which move over and block the binding site. They may be either unslaved (His64) or moderately slaved (Trp29) to the solvent. Small side-chain relaxations, most notably of leucines, are revealed in some mutants (V68L, V68A). They are conjectured to facilitate inter-cavity ligand motion. When all relaxations are arrested (H64L in trehalose), we observe pure inhomogeneous kinetics with no temperature dependence, suggesting that proximal relaxation is not a factor on the investigated timescale.     

Factors affecting the dominance hierarchy dynamics in a hummingbird assemblage

Intra and in terspecific competiti on for n ectar play an imports nt role in hummingbird communities. Larger sized species usually exclude smaller species from the rich floral resources. However, it has been recently postulated that the competitive advantages of a large body size decline as the evolutionary distance between the contending species in creases. In this study, we analyzed dominance hierarchy dynamics in a hummingbird assemblage in central Mexico. By monitoring hummingbird territories established in three plant species through 1 year, we assessed the effects of energy within territories and the territory owners identity in the frequency of inter and intraspecific encounters. We also evaluated if these factors affect the dominance of larger species when they compete against smaller distantly related contenders. Our results show that their frequency of intraspecific encounters was related with the identity of the territory's owner. On the contrary, the frequency of interspecific encounters was related with both the territory and the identity of the territory's owner. We did not find a significant difference between the number of encounters dominated by larger and smaller species and their conte nders. However, the in crease in genetic dista nee between contenders was positively associated with a higher frequency of encounters dominated by small hummingbirds.Our results showed that the ecological factors and evolutionary relationships among contenders play important roles in the dominance hierarchy dynamics.     

Glutathione reductase: solvent equilibrium and kinetic isotope effects

Glutathione reductase catalyzes the NADPH-dependent reduction of oxidized glutathione (GSSG). The kinetic mechanism is ping-pong, and we have investigated the rate-limiting nature of proton-transfer steps in the reactions catalyzed by the spinach, yeast, and human erythrocyte glutathione reductases using a combination of alternate substrate and solvent kinetic isotope effects. With NADPH or GSSG as the variable substrate, at a fixed, saturating concentration of the other substrate, solvent kinetic isotope effects were observed on V but not V/K. Plots of Vm vs mole fraction of D2O (proton inventories) were linear in both cases for the yeast, spinach, and human erythrocyte enzymes. When solvent kinetic isotope effect studies were performed with DTNB instead of GSSG as an alternate substrate, a solvent kinetic isotope effect of 1.0 was observed. Solvent kinetic isotope effect measurements were also performed on the asymmetric disulfides GSSNB and GSSNP by using human erythrocyte glutathione reductase. The Km values for GSSNB and GSSNP were 70 microM and 13 microM, respectively, and V values were 62 and 57% of the one calculated for GSSG, respectively. Both of these substrates yield solvent kinetic isotope effects greater than 1.0 on both V and V/K and linear proton inventories, indicating that a single proton-transfer step is still rate limiting. These data are discussed in relationship to the chemical mechanism of GSSG reduction and the identity of the proton-transfer step whose rate is sensitive to solvent isotopic composition. Finally, the solvent equilibrium isotope effect measured with yeast glutathione reductase is 4.98, which allows us to calculate a fractionation factor for the thiol moiety of GSH of 0.456.     

Nondisjunction of chromosome 15: origin and recombination.

Thirty-two cases of uniparental disomy (UPD), ascertained from Prader-Willi syndrome patients (N = 27) and Angelman syndrome patients (N = 5), are used to investigate the pattern of recombination associated with nondisjunction of chromosome 15. In addition, the meiotic stage of nondisjunction is inferred by using markers mapping near the centromere. Two basic approaches to the analysis of recombination are utilized. Standard methods of centromere mapping are employed to determine the level of recombination in specific pairwise intervals along the chromosome. This method shows a significant reduction in recombination for two of five intervals examined. Second, the observed frequency of each recombinant class (i.e., zero, one, two, three, or more observable crossovers) is compared with expected values. This is useful for testing whether the reduction in recombination can be attributed solely to a proportion of cases with no recombination at all (because of asynapsis), with the remaining groups showing normal recombination (or even excess recombination), or whether recombination is uniformly reduced. Analysis of maternal UPD(15) data shows a slight reduction in the multiple-recombinant classes, with a corresponding increase in both the zero- and one-recombinant classes over expected values. The majority, more than 82%, of the extra chromosomes in maternal UPD(15) cases are due to meiotic I nondisjunction events. In contrast, most paternal UPD(15) cases so far examined appear to have a postzygotic origin of the extra paternal chromosome.     

Ligand binding and protein relaxation in heme proteins: a room temperature analysis of NO geminate recombination

Ultrafast absorption spectroscopy is used to study heme-NO recombination at room temperature in aqueous buffer on time scales where the ligand cannot leave its cage environment. While a single barrier is observed for the cage recombination of NO with heme in the absence of globin, recombination in hemoglobin and myoglobin is nonexponential. Examination of hemoglobin with and without inositol hexaphosphate points to proximal constraints as important determinants of the geminate rebinding kinetics. Molecular dynamics simulations of myoglobin and heme-imidazole subsequent to ligand dissociation were used to investigate the transient behavior of the Fe-proximal histidine coordinate and its possible involvement in geminate recombination. The calculations, in the context of the absorption measurements, are used to formulate a distinction between nonexponential rebinding that results from multiple protein conformations (substates) present at equilibrium or from nonequilibrium relaxation of the protein triggered by a perturbation such as ligand dissociation. The importance of these two processes is expected to depend on the time scale of rebinding relative to equilibrium fluctuations and nonequilibrium relaxation. Since NO rebinding occurs on the picosecond time scale of the calculated myoglobin relaxation, a time-dependent barrier is likely to be an important factor in the observed nonexponential kinetics. The general implications of the present results for ligand binding in heme proteins and its time and temperature dependence are discussed. It appears likely that, at low temperatures, inhomogeneous protein populations play an important role and that as the temperature is raised, relaxation effects become significant as well.     

Ligand recombination to the alpha and beta subunits of human hemoglobin

The rebinding of CO, O2, NO, methyl, ethyl, n-propyl, and n-butyl isocyanide to isolated alpha and beta chains and intact hemoglobin at pH 7, 20 degrees C was examined both during and after a 30-ns dye laser pulse. The resultant absorbance changes were analyzed in terms of a linear three-step reaction scheme: Hb + X in equilibrium with C in equilibrium with B in equilibrium with A or HbX, where A is the final bound state, and C and B are geminate states. Rate constants were assigned for each of the transitions in this mechanism using fitting procedures described previously for analyzing ligand rebinding to sperm whale myoglobin at room temperature (Gibson, Q. H., Olson, J. S., McKinnie, R. E., and Rohlfs, R. J. (1986) J. Biol. Chem. 261, 10228-10239). Five major conclusions were obtained. First, initial geminate recombination phases for the NO and O2 complexes of hemoglobin and its isolated subunits exhibit half-times equal to approximately 12 and approximately 440 ps, respectively. These values are in excellent agreement with more direct, picosecond measurements of the geminate recombination of HbNO (Cornelius, P. A., Hochstrasser, R. M., and Steele, A. W. (1983) J. Mol. Biol. 163, 119-128) and HbO2 (Friedman, J. M., Scott, T. W., Fisanick, G. J., Simon, S. R., Findsen, E. W., Ondrias, M. R., and MacDonald, V. W. (1985) Science 229, 187-229) following extremely short laser pulses. Second, the correspondence between our nanosecond measurements and the published picosecond data suggests strongly that the intrinsic photochemical yield of all ferrous, hexacoordinate heme complexes approaches one. Third, the major differences between the isolated alpha and beta chains involve the rate of ligand migration to the solvent, kC----X and the extent of recombination from the second geminate state, C, as measured by the ratio kC----B/kC----X. Fourth, for both isolated chains and intact hemoglobin, the rate and equilibrium constants for the formation of the initial O2 geminate state starting from ligand in the solvent (i.e. kX----B and KX----B) are 5-10 times greater than the corresponding parameters for the formation of the first CO geminate state. Fifth, the rate-limiting step for NO, O2, and isonitrile binding to hemoglobin and its isolated subunits is ligand migration up to the initial geminate state (i.e. kX----B). In the case of CO binding, both migration to state B and iron-ligand bond formation (kB----A) affect the overall, bimolecular association rate constant.     

Molecular dynamics simulation of trp-aporepressor in a solvent.

Molecular dynamics simulations of Escherichia coli trp-aporepressor were carried out in the absence and presence of explicit water molecules. The vacuum simulations resulted in significant deformation of the initial X-ray structure. A solvated simulation with a nonbonded cut-off radius of 9 A gave a better result, and the most satisfactory result was obtained when electrostatic interactions within a cut-off radius of 18 A were considered by a twin-range method. The trajectory from the last simulation was used to analyze the dynamical properties of the aporepressor. The root-mean-square fluctuations of the residues showed the rigidity of the central core and the flexibility of the DNA-binding sites, consistent with the X-ray temperature factors. The dynamical cross-correlation map indicated a significant negative correlation between the central core and the two DNA-binding sites, and thus reproduced the three-domain format (a central core and two DNA-binding heads) from a dynamical point of view. The core region showed weak, but many, intra- and inter-molecular correlations, while the helix-turn-helix DNA-binding motifs were free from correlations with other regions.     

Electron transfer within xanthine oxidase: a solvent kinetic isotope effect study

Solvent kinetic isotope effect studies of electron transfer within xanthine oxidase have been performed, using a stopped-flow pH-jump technique to perturb the distribution of reducing equivalents within partially reduced enzyme and follow the kinetics of reequilibration spectrophotometrically. It is found that the rate constant for electron transfer between the flavin and one of the iron-sulfur centers of the enzyme observed when the pH is jumped from 10 to 6 decreases from 173 to 25 s-1 on going from H2O to D2O, giving an observed solvent kinetic isotope effect of 6.9. An effect of comparable magnitude is observed for the pH jump in the opposite direction, the rate constant decreasing from 395 to 56 s-1. The solvent kinetic isotope effect on kobs is found to be directly proportional to the mole fraction of D2O in the reaction mix for the pH jump in each direction, consistent with the effect arising from a single exchangeable proton. Calculations of the microscopic rate constants for electron transfer between the flavin and the iron-sulfur center indicate that the intrinsic solvent kinetic isotope effect for electron transfer from the neutral flavin semiquinone to the iron-sulfur center designated Fe/S I is substantially greater than for electron transfer in the opposite direction and that the observed solvent kinetic isotope effect is a weighted averaged of the intrinsic isotope effects for the forward and reverse microscopic electron-transfer steps.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sticking coefficient-orchestrated selection in a kinetic model for the first origin

The mathematical formalism of the steady-state Poisson equation is applied to a variant of Freeman Dyson's "Toy Model" for a first origin. Our kinetic approach allows for an examination of the requisite conditions under which metabolism is quantized into discrete eigenstates (e.g. Dyson's disordered, saddle point, and metabolically active toy cell states). The surface reaction machinery additionally allows for more realistic modeling, whence the crucial role of sticking coefficients (catalyst precursors) as prebiotic selectors emerges. In our interior source model, a steady influx of vent nutrients fuels the intracellular synthesis of (impermeable) monomers within a rock-encradled cavity. Random adsorptions and desorptions occur at inactive "cell" wall sites (where the inert monomers remain impermeable until their eventual return to the intracellular metabolite pool). Occasionally, metabolizing reactions also occur due to endogeneous source monomers adsorbing at their "active" sites. Dyson's mean field approach is used to simplify the species-specific sticking coefficients at empty active (substrate) sites to functions of the fraction x of sites occupied by (catalytically) active monomers. In short, our work suggests that disorder-order transition models based on random drift between discrete metabolic eigenstates (Dyson's Toy Model) do not, in general, extend to more realistic metabolisms. From a perspective based on quasi-random feedback kinetics, the contraindication for discretization (spontaneous generation) in non-autocatalytic metabolisms is consistent with the emergence of ordered metabolism under hydrothermal driving forces, a provisio the occurrence of each period of vent dormancy coincides with a discrete zero-source (dormant) metabolic state. Cell drift to higher order is induced by the random reactions which happen to enhance the substrate specificity (chemical selectivity) of the sticking coefficients for active monomers. The result is stronger sink effects for metabolizing species, whence active adsorptions are promoted in favor of inactive adsorptions at substrate sites. Positive feedback plays a crucial role in preserving ("propagating") order in the cell wall reaction kinetics and is held in check by negative feedback inhibition of excessive cell growth. Finally, the eventual desorption of randomly growing dysfunctional proteins is postulated as a deterrent to deterioration catastrophes.     

Ligand binding and protein dynamics in lactate dehydrogenase

Recent experimental studies suggest that lactate dehydrogenase (LDH) binds its substrate via the formation of a LDH/NADH.substrate encounter complex through a select-fit mechanism, whereby only a minority population of LDH/NADH is binding-competent. In this study, we perform molecular dynamics calculations to explore the variations in structure accessible to the binary complex with a focus on identifying structures that seem likely to be binding-competent and which are in accord with the known experimental characterization of forming binding-competent species. We find that LDH/NADH samples quite a range of protein conformations within our 2.148 ns calculations, some of which yield quite facile access of solvent to the active site. The results suggest that the mobile loop of LDH is perhaps just partially open in these conformations and that multiple open conformations, yielding multiple binding pathways, are likely. These open conformations do not require large-scale unfolding/melting of the binary complex. Rather, open versus closed conformations are due to subtle protein and water rearrangements. Nevertheless, the large heat capacity change observed between binding-competent and binding-incompetent can be explained by changes in solvation and an internal rearrangement of hydrogen bonds. We speculate that such a strategy for binding may be necessary to get a ligand efficiently to a binding pocket that is located fairly deep within the protein's interior.     

gamma-Glutamylcyclotransferase: inhibition by D-beta-aminoglutaryl-L-alanine and analysis of the solvent kinetic isotope effect

Spin-echo NMR spectroscopy was used to record the cleavage of a gamma-glutamyl--amino-acid by (5-L-glutamyl)-L-amino-acid 5-glutamyltransferase (cyclizing) (gamma-glutamylcyclotransferase) in human erythrocyte hemolysates. The Michaelis-Menten steady-state kinetic parameters were obtained by fitting the integrated Michaelis-Menten equation to the reaction time curves. The product, L-5-oxoproline, was shown to be an inhibitor of the reaction. The active site of the enzyme was probed by studies of the inhibition by D- and L-beta-aminoglutaryl-L-alanine which are the beta-amino-acid isomers of D- and L-gamma-glutamyl-L-alanine (the latter being a natural substrate of the enzyme); the D-isomer was the more potent inhibitor (Ki = 0.30 +/- 0.02 mmol/l water). When the alanyl alpha-carboxyl of the inhibitor was reduced to a hydroxyl (i.e. to give D-beta-aminoglutaryl-L-alaninol) the potency of inhibition was reduced. The previously reported kinetic isotope effect of solvent 2H2O on the enzyme-catalyzed reaction has been further studied using a proton inventory. We propose that the solvent kinetic isotope effect is due to an intramolecular proton transfer between the glutamyl amino group and the peptide bond nitrogen.     

Molecular mechanism of homologous recombination in meiosis: origin and biological significance

Sexual reproduction prevails among eukaryotic organisms. The problem of advantage of sexual reproduction over asexual reproduction remains a subject of not stopping discussions. According to one of the hypotheses, sexual reproduction and homologous recombination which accompanies gamete formation during meiosis has arisen to increase genetic variability and, as consequence, a fitness of organisms. Many researches show that homologous recombination play an important role in reparation of DNA in various groups of organisms irrespective of the way of their reproduction. Involvement of recombination in meiosis, however, is impossible to explain only by DNA repair functions. The hypothesis, that a recombination in the course of sexual process is a source of variability, also is not capable to explain existence of this process well. There is convincing evidence that the homologous recombination in meiosis is necessary for formation of bivalents. A physical connection between homologous chromosomes that is formed by recombination is required for correct chromosome segregation during meiotic division and formation of gametes of full value.     

Melting of the solvent structure around a RNA duplex: a molecular dynamics simulation study

From three 2.4-ns molecular dynamics simulations of the r(CpG)(12) duplex conducted at 5, 25 and 37 degrees C, a strong temperature dependence of the dynamics of the water molecules and ions located in the first nucleic acid coordination shell is observed. At 5 degrees C, the highest residence times of bound water molecules exceed 1 ns while, at 37 degrees C, they decrease to 0.5 ns in agreement with available NMR data. Similar temperature dependencies are observed for the potassium ions bound to the duplex. In this temperature range, the structure of the RNA helix remains essentially unchanged. Thus, the observed alterations correspond to a 'premelting' of the solvent structure around the duplex. It is proposed that, before the nucleic acid structure melts, the entropy of the solvent increases to a point where it is no longer compensated by the enthalpic contribution of solute-solute and solute-solvent interactions. At this stage, the weakest structural elements start to melt. In other terms, the experimentally observed melting processes are preceded by a melting of the more labile solvent structure.     

Distribution of recombination crossovers and the origin of haplotype blocks: the interplay of population history,recombination, and mutation

Recent studies suggest that haplotypes are arranged into discrete blocklike structures throughout the human genome. Here, we present an alternative haplotype block definition that assumes no recombination within each block but allows for recombination between blocks, and we use it to study the combined effects of demographic history and various population genetic parameters on haplotype block characteristics. Through extensive coalescent simulations and analysis of published haplotype data on chromosome 21, we find that (1) the combined effects of population demographic history, recombination, and mutation dictate haplotype block characteristics and (2) haplotype blocks can arise in the absence of recombination hot spots. Finally, we provide practical guidelines for designing and interpreting studies investigating haplotype block structure.     

DNA recombination: holliday junctions dynamics and branch migration

Holliday junctions are critical intermediates for homologous, site-specific recombination, DNA repair, and replication. A wealth of structural information is available for immobile four-way junctions, but the controversy on the mechanism of branch migration of Holliday junctions remains unsolved. Two models for the mechanism of branch migration were suggested. According to the early model of Alberts-Meselson-Sigal (Sigal, N., and Alberts, B. (1972) J. Mol. Biol. 71, 789-793 and Meselson, M. (1972) J. Mol. Biol. 71, 795-798), exchanging DNA strands around the junction remain parallel during branch migration. Kinetic studies of branch migration (Panyutin, I. G., and Hsieh, P. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2021-2025) suggest an alternative model in which the junction adopts an extended conformation. We tested these models using a Holliday junction undergoing branch migration and time-lapse atomic force microscopy, an imaging technique capable of imaging DNA dynamics. The single molecule atomic force microscopy experiments performed in the presence and in the absence of divalent cations show that mobile Holliday junctions adopt an unfolded conformation during branch migration that is retained despite a broad range of motion in the arms of the junction. This conformation of the junction remains unchanged until strand separation. The data obtained support the model for branch migration having the extended conformation of the Holliday junction.     

How a vicinal layer of solvent modulates the dynamics of proteins

The dynamics of a folded protein is studied in water and glycerol at a series of temperatures below and above their respective dynamical transition. The system is modeled in two distinct states whereby the protein is decoupled from the bulk solvent at low temperatures, and communicates with it through a vicinal layer at physiological temperatures. A linear viscoelastic model elucidates the less-than-expected increase in the relaxation times observed in the backbone dynamics of the protein. The model further explains the increase in the flexibility of the protein once the transition takes place and the differences in the flexibility under the different solvent environments. Coupling between the vicinal layer and the protein fluctuations is necessary to interpret these observations. The vicinal layer is postulated to form once a threshold for the volumetric fluctuations in the protein to accommodate solvents of different sizes is reached. Compensation of entropic-energetic contributions from the protein-coupled vicinal layer quantifies the scaling of the dynamical transition temperatures in various solvents. The protein adapts different conformational routes for organizing the required coupling to a specific solvent, which is achieved by adjusting the amount of conformational jumps in the surface-group dihedrals.     

Pairing dynamics and the origin of species

Whether sexual selection alone can drive the evolution of assortative mating in the presence of gene flow is a long-standing question in evolutionary biology. Here, we report a role for pairing dynamics of individuals when mate choice is mutual, which is sufficient for the evolution of assortative mating by sexual selection alone in the presence of gene flow. Through behavioural observation, individual-based simulation and population genetic analysis, we evaluate the pairing dynamics of coral reef fish in the genus Hypoplectrus (Serranidae), and the role these dynamics can play for the evolution of assortative mating. When mate choice is mutual and the stability of mating pairs is critical for reproductive success, the evolution of assortative mating in the presence of gene flow is not only possible, but is also a robust evolutionary outcome.