Energy considerations for kinetic proofreading in biosynthesis

An explicit model for kinetic proofreading in biosynthesis processes is treated in order to clarify the relation between the achieved accuracy, and the required energy which is provided by the hydrolysis of nucleoside triphosphates (atp, gtp). The displacement from equilibrium, which is restrictive for the discrimination, is explicitly taken into account. This means that the process is consistent with the principle of detailed balance. It proceeds in such a way that the molecules to be selected are associated together with nucleoside triphosphates to an enzyme complex. After an initial selection, hydrolysis takes place, whereafter there is a repeated testing in several steps. Our basic idea is that real proofreading systems are designed to use the energy of the triphosphate in the most efficient way to achieve a satisfactory accuracy, and therefore there should exist optimum kinetic properties of the system. A suitable formalism for finding such optimum situations is developed which yields new possibilities to interpret experimental data.     

Identification and analysis of error types in high-throughput genotyping

Although it is clear that errors in genotyping data can lead to severe errors in linkage analysis, there is as yet no consensus strategy for identification of genotyping errors. Strategies include comparison of duplicate samples, independent calling of alleles, and Mendelian-inheritance-error checking. This study aimed to develop a better understanding of error types associated with microsatellite genotyping, as a first step toward development of a rational error-detection strategy. Two microsatellite marker sets (a commercial genomewide set and a custom-designed fine-resolution mapping set) were used to generate 118,420 and 22,500 initial genotypes and 10,088 and 8,328 duplicates, respectively. Mendelian-inheritance errors were identified by PedManager software, and concordance was determined for the duplicate samples. Concordance checking identifies only human errors, whereas Mendelian-inheritance-error checking is capable of detection of additional errors, such as mutations and null alleles. Neither strategy is able to detect all errors. Inheritance checking of the commercial marker data identified that the results contained 0.13% human errors and 0.12% other errors (0.25% total error), whereas concordance checking found 0.16% human errors. Similarly, Mendelian-inheritance-error checking of the custom-set data identified 1.37% errors, compared with 2.38% human errors identified by concordance checking. A greater variety of error types were detected by Mendelian-inheritance-error checking than by duplication of samples or by independent reanalysis of gels. These data suggest that Mendelian-inheritance-error checking is a worthwhile strategy for both types of genotyping data, whereas fine-mapping studies benefit more from concordance checking than do studies using commercial marker data. Maximization of error identification increases the likelihood of linkage when complex diseases are analyzed.     

An Optimal Free Energy Dissipation Strategy of the MinCDE Oscillator in Regulating Symmetric Bacterial Cell Division

Sustained molecular oscillations are ubiquitous in biology. The obtained oscillatory patterns provide vital functions as timekeepers, pacemakers and spacemarkers. Models based on control theory have been introduced to explain how specific oscillatory behaviors stem from protein interaction feedbacks, whereas the energy dissipation through the oscillating processes and its role in the regulatory function remain unexplored. Here we developed a general framework to assess an oscillator’s regulation performance at different dissipation levels. Using the Escherichia coli MinCDE oscillator as a model system, we showed that a sufficient amount of energy dissipation is needed to switch on the oscillation, which is tightly coupled to the system’s regulatory performance. Once the dissipation level is beyond this threshold, unlike stationary regulators’ monotonic performance-to-cost relation, excess dissipation at certain steps in the oscillating process damages the oscillator’s regulatory performance. We further discovered that the chemical free energy from ATP hydrolysis has to be strategically assigned to the MinE-aided MinD release and the MinD immobilization steps for optimal performance, and a higher energy budget improves the robustness of the oscillator. These results unfold a novel mode by which living systems trade energy for regulatory function.     

Conformational search of antisense nucleotides

A preliminary MMFF implementation of selenium atom parameters necessary to model the nucleoside 1 is reported. X-ray structures of two compounds 1 and 2 have been used as references. Ab initio methods have been adopted for checking torsional energy profile and charge distribution. Monte Carlo calculations and energy minimization in solvation complete the conformational search.     

Pragmatic ab initio prediction of enthalpies of formation for large molecules: accuracy of MP2 geometries and frequencies using CCSD(T) correlation energies

We have addressed the accuracy of calculating the enthalpy of formation of an arbitrary single reference molecule using practical ab initio methodologies. It is known that MP2 geometries with a triple zeta basis set are almost as reliable as CCSD(T) geometries. It is also known that CCSD(T) correlation energies, with basis extrapolation, feature chemical accuracy for single-reference molecules. We investigate what accuracy one might expect in enthalpies of formation from a MP2 geometry, MP2 harmonic vibrational frequencies, a CCSD(T) correlation energy using triple zeta basis sets. It is far from obvious, a priori, as to which error source contributes most significantly. We observe that the accuracy in calculating enthalpies of formation of single-reference molecules with this protocol is 4 kcal mol^-1; our error analysis shows this comes almost exclusively from the correlation energy basis extrapolation, rather than errors intrinsic to MP2.     

Quantifying robustness and dissipation cost of yeast cell cycle network: the funneled energy landscape perspectives

We study the origin of robustness of yeast cell cycle cellular network through uncovering its underlying energy landscape. This is realized from the information of the steady-state probabilities by solving a discrete set of kinetic master equations for the network. We discovered that the potential landscape of yeast cell cycle network is funneled toward the global minimum, G1 state. The ratio of the energy gap between G1 and average versus roughness of the landscape termed as robustness ratio (RR) becomes a quantitative measure of the robustness and stability for the network. The funneled landscape is quite robust against random perturbations from the inherent wiring or connections of the network. There exists a global phase transition between the more sensitive response or less self-degradation phase leading to underlying funneled global landscape with large RR, and insensitive response or more self-degradation phase leading to shallower underlying landscape of the network with small RR. Furthermore, we show that the more robust landscape also leads to less dissipation cost of the network. Least dissipation and robust landscape might be a realization of Darwinian principle of natural selection at cellular network level. It may provide an optimal criterion for network wiring connections and design.     

Isoleucyl-tRNA synthetase from Escherichia coli MRE 600: discrimination between isoleucine and valine with modulated accuracy

Discrimination between isoleucine and valine is achieved with different accuracies by isoleucyl-tRNA synthetase from E. coli MRE 600. The recognition process consists of two initial discrimination steps and a pretransfer and a posttransfer proofreading event. The overall discrimination factors D were determined from kcat and Km values observed in aminoacylation of tRNA(Ile)-C-C-A with isoleucine and valine. From aminoacylation of the modified tRNA species tRNA(Ile)-C-C-A(3'NH2) initial discrimination factors I1 and pretransfer proofreading factors II1 were calculated. Factors I1 were computed from ATP consumption and D1, the overall discrimination in aminoacylation of the modified tRNA; factors II1 were calculated as quotient of AMP formation rates. Initial discrimination factors I2 and posttransfer proofreading factors II2 were determined from AMP formation rates observed in aminoacylation of tRNA(Ile)-C-C-A. The observed overall discrimination varies up to a factor of about four according to conditions. Under standard assay conditions 72,000, under optimal conditions 144,000 correct aminoacyl-tRNAs are produced per one error while 1.1 or 1.7 ATPs are consumed. A comparison with isoleucyl-tRNA synthetase from yeast shows that both enzymes act principally with the same recognition mechanism, but the enzyme from E. coli MRE 600 exhibits higher specificity and lower energy dissipation and does not show such high variation of accuracy as observed with the enzyme from yeast.     

On the relevance of structure preservation to simulations of muscle actuated movements

In this work, we implement a typical nonlinear Hill-type muscle model in a structure-preserving simulation framework and investigate the differences to standard simulations of muscle-actuated movements with MATLAB/Simulink. The latter is a common tool to solve dynamical problems, in particular, in biomechanic investigations. Despite the simplicity of the examples used for comparison, it becomes obvious that the MATLAB/Simulink integrators artificially loose or gain energy and angular momentum during dynamic simulations. The relative energy error of the MATLAB/Simulink integrators related to a very low actual muscle work can naturally reach large values, even higher than 100%. But also during periods with large muscle work, the relative energy error reaches up to 2%. Even in simulations with very small time steps, energy and angular momentum errors are still present using MATLAB/Simulink and can (at least partially) be responsible for phase errors in long-term simulations. This typical behaviour of commercial integrators is known to increase for more complex models or for computations with larger time steps, whose use is crucial for efficiency, especially in the context of optimal control simulations. In contrast to that, time-stepping schemes being derived from a discrete variational principle yield discrete analogues of the Euler-Lagrange equations and Noethers theorem. This ensures that the structure of the system is preserved, i.e. the simulation results are symplectic and momentum consistent and exhibit a good energy behaviour (no drift).     

The illusion of distribution-free small-sample classification in genomics

Classification has emerged as a major area of investigation in bioinformatics owing to the desire to discriminate phenotypes, in particular, disease conditions, using high-throughput genomic data. While many classification rules have been posed, there is a paucity of error estimation rules and an even greater paucity of theory concerning error estimation accuracy. This is problematic because the worth of a classifier depends mainly on its error rate. It is common place in bio-informatics papers to have a classification rule applied to a small labeled data set and the error of the resulting classifier be estimated on the same data set, most often via cross-validation, without any assumptions being made on the underlying feature-label distribution. Concomitant with a lack of distributional assumptions is the absence of any statement regarding the accuracy of the error estimate. Without such a measure of accuracy, the most common one being the root-mean-square (RMS), the error estimate is essentially meaningless and the worth of the entire paper is questionable. The concomitance of an absence of distributional assumptions and of a measure of error estimation accuracy is assured in small-sample settings because even when distribution-free bounds exist (and that is rare), the sample sizes required under the bounds are so large as to make them useless for small samples. Thus, distributional bounds are necessary and the distributional assumptions need to be stated. Owing to the epistemological dependence of classifiers on the accuracy of their estimated errors, scientifically meaningful distribution-free classification in high-throughput, small-sample biology is an illusion.     

Hydromechanical stress in shake flasks: correlation for the maximum local energy dissipation rate

Shake flasks are widely used in biotechnological process research. Bioprocesses for which hydromechanical stress may become the rate controlling parameter include those where oils are applied as carbon sources, biotransformation of compounds with low solubility in the aqueous phase, or processes employing animal, plant, or filamentous microorganisms. In this study, the maximum local energy dissipation rate as the measure for hydromechanical stress is characterized in shake flasks by measuring the maximum stable drop size. The theoretical basis for the method is that the maximum stable drop diameter in a coalescence inhibited liquid/liquid dispersion is only a function of the maximum local energy dissipation rate and not of the dispersing apparatus. The maximum local energy dissipation rate is obtained by comparing the drop diameters in shake flasks to those in a stirred tank reactor. At the same volumetric power consumption, the maximum energy dissipation rate in shake flasks is about 10 times lower than in stirred tank reactors explaining the common observation of considerable differences in the morphology of hydromechanically sensitive cells between these two reactor types. At the same volumetric power consumption, the maximum local energy dissipation rate in baffled and in unbaffled shake flasks is very similar. A correlation is presented to quantify the maximum local energy dissipation rate in shake flasks as a function of the operating conditions. Non-negligible drop viscosity may be considered by known literature correlations. Further, from dispersion experiments a critical Reynolds number of about 60,000 is proposed for turbulent flow in unbaffled shake flasks.     

Characterization of the nuclear protein import mechanism using Ran mutants with altered nucleotide binding specificities.

The small nuclear GTP binding protein Ran is required for transport of nuclear proteins through the nuclear pore complex (NPC). Although it is known that GTP hydrolysis by Ran is essential for this reaction, it has been unclear whether additional energy-consuming steps are also required. To uncouple the energy requirements for Ran from other nucleoside triphosphatases, we constructed a mutant derivative of Ran that has an altered nucleotide specificity from GTP to xanthosine 5' triphosphate. Using this Ran mutant, we demonstrate that nucleotide hydrolysis by Ran is sufficient to promote efficient nuclear protein import in vitro. Under these conditions, protein import could no longer be inhibited with non-hydrolysable nucleotide analogues, indicating that no Ran-independent energy-requiring steps are essential for the protein translocation reaction through the NPC. We further provide evidence that nuclear protein import requires Ran in the GDP form in the cytoplasm. This suggests that a coordinated exchange reaction from Ran-GDP to Ran-GTP at the pore is necessary for translocation into the nucleus.     

Methods of automatic nucleotide-sequence analysis. Multicomponent spectrophotometric analysis of mixtures of nucleic acid components by a least-squares procedure

1. A theoretical analysis of the errors in multicomponent spectrophotometric analysis of nucleoside mixtures, by a least-squares procedure, has been made to obtain an expression for the error coefficient, relating the error in calculated concentration to the error in extinction measurements. 2. The error coefficients, which depend only on the `library' of spectra used to fit the experimental curves, have been computed for a number of `libraries' containing the following nucleosides found in s-RNA: adenosine, guanosine, cytidine, uridine, 5-ribosyluracil, 7-methylguanosine, 6-dimethylaminopurine riboside, 6-methylaminopurine riboside and thymine riboside. 3. The error coefficients have been used to determine the best conditions for maximum accuracy in the determination of the compositions of nucleoside mixtures. 4. Experimental determinations of the compositions of nucleoside mixtures have been made and the errors found to be consistent with those predicted by the theoretical analysis. 5. It has been demonstrated that, with certain precautions, the multicomponent spectrophotometric method described is suitable as a basis for automatic nucleotide-composition analysis of oligonucleotides containing nine nucleotides. Used in conjunction with continuous chromatography and flow chemical techniques, this method can be applied to the study of the sequence of s-RNA.     

Logical elements in living cells

Recognition processes with enhanced accuracy (as performed by structures like enzymes or ribosomes) are investigated using elementary ideas of statistical mechanics and related concepts of thermodynamics. The analysis starts from a formal definition of recognition and provides a correspondence with appropriate physical properties of the macromolecular logical elements. Transitions of the recognizing system between different modifications are a necessary feature of a more exacting recognition process. Rearrangement steps provide the process with higher accuracy by performing two physical operations: (1) rearranging the phase space of the system so that the "correct" states be better separated from the "wrong" states and the probability of occupation of the "correct" states be enhanced, (2) directing the process toward the more favourable modifications thus formed. Both operations are related to changes in the physical properties of the recognizing system. These changes can be expressed as differences of macromolecular Gibbs energy levels; if ligand binding or release participate in a step, directivity of the step depends also on the actual chemical potentials of the ligands in solution. The two operations just mentioned resemble two basic operations known to be necessary in electronic digital networks: directivity of control and signal standardization. An analysis of the entire reaction catalysed by a macromolecular logical element takes into account the requirements imposed by the logical functions as well as the need that the chemical potential of the product be not restricted to very low values. To satisfy these conditions, the reaction must be supported by a so-called non-specific reaction, usually implemented by the cleavage reaction of a nucleoside triphosphate.     

Computational active site analysis of molecular pathways to improve functional classification of enzymes

This study describes a method to computationally assess the function of homologous enzymes through small molecule binding interaction energy. Three experimentally determined X-ray structures and four enzyme models from ornithine cyclo-deaminase, alanine dehydrogenase, and mu-crystallin were used in combination with nine small molecules to derive a function score (FS) for each enzyme-model combination. While energy values varied for a single molecule-enzyme combination due to differences in the active sites, we observe that the binding energies for the entire pathway were proportional for each set of small molecules investigated. This proportionality of energies for a reaction pathway appears to be dependent on the amino acids in the active site and their direct interactions with the small molecules, which allows a function score (FS) to be calculated to assess the specificity of each enzyme. Potential of mean force (PMF) calculations were used to obtain the energies, and the resulting FS values demonstrate that a measurement of function may be obtained using differences between these PMF values. Additionally, limitations of this method are discussed based on: (a) larger substrates with significant conformational flexibility; (b) low homology enzymes; and (c) open active sites. This method should be useful in accurately predicting specificity for single enzymes that have multiple steps in their reactions and in high throughput computational methods to accurately annotate uncharacterized proteins based on active site interaction analysis.     

Non-additive interactions of nucleobases in model dinucleotide steps occurring in B-DNA crystals

Non-additivity of base-base interactions in all ten possible model dinucleotide steps were analyzed on MP2/aug-cc-pvDZ quantum chemistry level. Conformations of four nucleobases exactly matched to ones occurring in B-DNA crystals. In most of thw 162 analyzed tetramers both three- and four-body contributions are negligible except for d(GpG) steps. However, in these dinucleotides both contributions are always of opposite signs and in all cases the sum of all non-additive part of intermolecular interactions do not exceed 2.6 kcal mol^-1. This stands for less than 5% of the overall binding energy of dinucleotide steps. Also replacements of guanine with 8-oxoguanine in d(GpG) systems introduces non-additivity of the same magnitude as for canonical dinucleotides. It is observed linear relationships between values of total binding energy obtained in the tetramer basis set and estimated energy exclusively in dimers basis sets with assumption of pairwise additivities. For all analyzed dinucleotides steps there are also linear correlations between amount of non-additive contributions and magnitude of pairs interactions. Based on differences in electrostatic contribution to the total binding energy of four nucleobases and polarity of dinucleotide steps three distinct classes of dinucleotide steps were identified.     

Accurate gas phase acidities of carboxylic acids estimated by scaling the vibrational contribution of ab initio gibbs free energies

The gas phase Gibbs free energies deltaG(T) of dissociation reaction of 14 carboxylic acids were calculated on the SCF, as well as G3 and CBS-Q levels. Corresponding accuracies were critically compared with experimental data. Since all of the results suffer from systematic errors, the procedure of scaling of thermal contribution to Gibbs free energy was applied for minimizing differences between theoretical and experimental values of deltaG(T). Two parameters were adjusted, namely the scaling of thermal contribution to Gibbs free energy of neutral and anionic forms. The presented results suggest the great effectiveness of such a procedure since for all applied basis sets within the SCF framework the achieved accuracy was below the experimental error. Besides, the proposed low-cost approximation method leads to precision comparable to or even exceeding the quality offered by more sophisticated composite quantum chemistry methods. The extension of the set of training molecules up to 82 has an insignificant impact on the overall quality of deltaG(T) estimation, which suggests that a wisely chosen set of reference data may be used for the characteristics of the whole class of compounds. There is a straightforward way for the analysis of acidities/basicities of other classes of chemicals such as DNA bases, alcohols, phenols, amines, amino acids, etc.     

Gynogenesis Assessment Using Microsatellite Genetic Markers in Turbot (<Emphasis Type="Italic">Scophthalmus maximus</Emphasis>)

Gynogenesis was assessed by different methods in 2 families of gynogenetic offspring in turbot (Scophthalmus maximus). Karyotype analysis in embryos and larvae demonstrated high accuracy in estimation of ploidy level, but performance was uneven given the low quality and number of plates obtained. The use of silver staining to estimate the number of nucleoli per nucleus resulted in a straightforward and easy method to evaluate the ploidy of the samples studied. However, the existence of a nucleolus organizer region polymorphism in turbot determined a small error in ploidy estimation, important when checking ploidy in specific individuals. The use of a set of 11 highly variable microsatellite loci proved to be a powerful method to confirm the exclusive maternal inheritance to gynogenetic offspring in turbot, with probabilities of detection of putative paternal genetic contribution above 99.99%.     

Realized Sampling Variances of Estimates of Genetic Parameters and the Difference between Genetic and Phenotypic Correlations

A data set of 1572 heritability estimates and 1015 pairs of genetic and phenotypic correlation estimates, constructed from a survey of published beef cattle genetic parameter estimates, provided a rare opportunity to study realized sampling variances of genetic parameter estimates. The distribution of both heritability estimates and genetic correlation estimates, when plotted against estimated accuracy, was consistent with random error variance being some three times the sampling variance predicted from standard formulae. This result was consistent with the observation that the variance of estimates of heritabilities and genetic correlations between populations were about four times the predicted sampling variance, suggesting few real differences in genetic parameters between populations. Except where there was a strong biological or statistical expectation of a difference, there was little evidence for differences between genetic and phenotypic correlations for most trait combinations or for differences in genetic correlations between populations. These results suggest that, even for controlled populations, estimating genetic parameters specific to a given population is less useful than commonly believed. A serendipitous discovery was that, in the standard formula for theoretical standard error of a genetic correlation estimate, the heritabilities refer to the estimated values and not, as seems generally assumed, the true population values.     

Step Detection and Activity Recognition Accuracy of Seven Physical Activity Monitors

The aim of this study was to compare the seven following commercially available activity monitors in terms of step count detection accuracy: Movemonitor (Mc Roberts), Up (Jawbone), One (Fitbit), ActivPAL (PAL Technologies Ltd.), Nike+ Fuelband (Nike Inc.), Tractivity (Kineteks Corp.) and Sensewear Armband Mini (Bodymedia). Sixteen healthy adults consented to take part in the study. The experimental protocol included walking along an indoor straight walkway, descending and ascending 24 steps, free outdoor walking and free indoor walking. These tasks were repeated at three self-selected walking speeds. Angular velocity signals collected at both shanks using two wireless inertial measurement units (OPAL, ADPM Inc) were used as a reference for the step count, computed using previously validated algorithms. Step detection accuracy was assessed using the mean absolute percentage error computed for each sensor. The Movemonitor and the ActivPAL were also tested within a nine-minute activity recognition protocol, during which the participants performed a set of complex tasks. Posture classifications were obtained from the two monitors and expressed as a percentage of the total task duration.The Movemonitor, One, ActivPAL, Nike+ Fuelband and Sensewear Armband Mini underestimated the number of steps in all the observed walking speeds, whereas the Tractivity significantly overestimated step count. The Movemonitor was the best performing sensor, with an error lower than 2% at all speeds and the smallest error obtained in the outdoor walking. The activity recognition protocol showed that the Movemonitor performed best in the walking recognition, but had difficulty in discriminating between standing and sitting. Results of this study can be used to inform choice of a monitor for specific applications.