生物大分子的自由能计算方法

在本文中,我们总结了计算生物物理中广泛使用的经典的自由能计算方法,主要可以归纳为三大类:第一类是基于过渡态理论,利用路径系综得到自由能的方法;第二类方法采用一些经验势来补偿自由能表面达到均匀采样的目标;最后一种是将整个转换径分段计算,然后拼接完成得到完整自由能的方法.通过详细介绍自由能计算的重要性和实施细节,希望能够让...     

Hysteresis and Statistical Errors in Free Energy Perturbation L to D Amino Acid Conversion

Abstract

A theory based on a Langevin equation along the reaction coordinate is developed to explain and calculate systematic and statistical errors in free energy perturbation simulations. The errors are calculated exactly when both the perturbation potential and the mean potential from the surrounding degrees of freedom are harmonic in the reaction coordinate. The effect of the mean potential is small as long as the force constant is small compared to the force constant of the perturbation potential. This indicates that the results obtained with zero mean force may still be valid as long as the second derivate of the mean potential is small compared to that of the perturbation potential. The theory is applied to conversion between L and D amino acids by changing the position of the minimum of the harmonic improper dihedral potential between ±35.264 degrees. For phenylalanine bound in the active site of a protein (thermolysin) we find from 20 psec. simulations statistical errors and hysteresis that both are about 2.5 kJ/mol in agreement with what is obtained from the theoretical predictions. The statistical errors are proportional to the square root of the coupling to the heat bath and inversely proportional to the square root of integration time while the (positive) hysteresis due to that the reaction coordinate lags behind is linear in the same quantities. This shows that the systematic errors will dominate in short simulations while the statistical ones will dominate for long simulations. The treatment is based on that the systematic influence of the surroundings can be represented by a mean force upon the reaction coordinate. If the relaxation processes of the environment are slow this may not be true. Then additional errors have to be considered.     

不同生长期白羊草中游离氨基酸的含量

本文对不同生长期白羊草（Ｂｏｔｈｒｉｏｃｈｌｏａｉｓｃｈａｅｍｕｍ）中氨基酸成分进行了分析,共检出白羊草中氨基酸１６种,以８月份生长的白羊草氨基酸的含量最高,为５．８１６ｍｇ／ｇ。     

代谢工程改造微生物高产氨基酸的策略

氨基酸作为一类营养物质在维持机体正常的生理生化反应方面具有重要的功能,常用作食品、药品和化妆品等的添加剂。氨基酸的生产主要依靠微生物发酵,产氨基酸菌的选育却是制约大规模工业生产氨基酸的重要因素。随着微生物分子育种技术的发展和运用,利用代谢工程改造细胞本身固有的代谢网络,指导氨基酸高产菌的选育已成为当前研究的热点。以谷氨酸棒杆菌(Corynebacterium glutamicum)为例,就该菌株代谢网络的特征以及高产氨基酸的代谢工程策略和应用进行综述。     

Subcellular Distribution of Free Amino Acids in Relation to Protein Synthesis in Cells of Chara corallina

The influence of protein-synthesis inhibitors on the subcellular distribution of free amino acids was studied in internodal cells of Chara corallina. Use of an intracellular perfusion technique allowed separate measurements of amino acids in the vacuole, in the flowing sol endoplasm and in the gel layer. The sol endoplasm predominantly represents the cytosol, while the gel layer is occupied, for the most part, by chloroplasts. When cells were treated with 0.5 mM chloramphenicol (CRP) in the dark, both the total concentration of amino acids and the subcellular distribution were almost the same as in cells without treatment. In the light, however, the subcellular distribution changed dramatically, although the total concentration of amino acids was unchanged. The vacuolar concentration of amino acids was 3 times greater in CRP-treated cells than in the control. The concentrations of amino acids in the sol endoplasm and in the gel layer were only half of those in the control. Amino acid permeability of the chloroplast envelope, measured using the perfused internodal cells, slightly increased after the CRP treatment in the light. Time-dependent changes in concentrations of amino acids in the CRP-treated cells were also measured in the light. The total concentration of amino acids in the cytoplasm gradually decreased, while that in the vacuole increased commensurately. The concentration and/or subcellular distribution of alanine, glutamine, glutamate and glycine changed dramatically. The concentration of alanine increased considerably both in the vacuole and in the cytoplasm. The cytoplasmic concentration of glutamine increased transiently within 1 ?2 h after treatment with CRP. The cytoplasmic concentrations of glutamate and glycine decreased. Although the concentrations of some amino acids changed so markedly both in the vacuole and cytoplasm, only small differences in the activities of glutamic-pyruvic transaminase, glutamic-oxaloacetic transaminase and glutamine synthetase were detected between the control and the CRP-treated cells.     

Changes in the Subcellular Distribution of Free Amino Acids in Relation to Light Conditions in Cells of Chara corallina

Subcellular concentrations of free amino acids in internodal cells of a Characeae, Chara corallina, were measured in the dark and in the light. Using an intracellular perfusion technique, we measured concentrations of amino acids in the vacuole, in the flowing sol endoplasm and in the cortical gel layer. The sol endoplasm was predominantly the cytosol. On the basis of microscopic observations, the gel layer appeared to be occupied predominantly by a layer of chloroplasts, while the sol endoplasm was free from chloroplasts. Both in the light and in darkness, the major amino acids in the internodal cells were isoasparagine, glutamic acid, aspartic acid, serine, glycine and alanine, as reported by Sakano and Tazawa (1984). The same major amino acids are found in each of the three compartments. The pattern of distribution of amino acids in the vacuole was similar to that in the sol endoplasm, but quite different from that in the gel layer. The total level of amino acids in the light was lower than that in darkness. The amino acid composition did not change very much, but the subcellular distribution of amino acids differed significantly between cells subjected to illumination and those kept in the dark. Concentrations of amino acids in both the vacuole and the gel layer decreased, whereas those in the sol endoplasm were almost constant.     

Concentration of Free Amino Acids in Primary Cultures of Neurones and Astrocytes

The cellular distribution of free amino acids was estimated in primary cultures (14 days in vitro) composed principally of cerebellar interneurones or cerebellar and forebrain astrocytes. In cultured neural cells, the overall concentration of amino acids resembled that found in brain at the corresponding age in vivo. In the two neural cell types, there were marked differences in the distribution of amino acids, in particular, those associated with the metabolic compartmentation of glutamate. In neuronal cell cultures, the concentrations of glutamate, aspartate, and gamma-aminobutyric acid were, respectively, about three, four, and seven times greater than in astrocytes. By contrast, the amount of glutamine was approximately 65% greater in astroglial cell cultures than in interneurone cultures. An unexpected finding was a very high concentration of glycine in astrocytes derived from 8-day-old cerebellum, but the concentrations of both serine and glycine were greater in nerve cell cultures than in forebrain astrocytes. The essential amino acids threonine, valine, isoleucine, leucine, tyrosine, phenylalanine, histidine, lysine, and arginine were all present in the growth medium, and small cellular changes in the contents of some of these amino acids may relate to differences in their influx and efflux during culturing and washing procedures. The present results, together with our previous findings, provide further support for the model assigning the "small" compartment of glutamate to glial cells and the "large" compartment to neurones, and also underline the metabolic interaction between these two cell types in the brain.     

Comparison of Protein Precipitants for the Determination of Free Amino Acids in Plasma

The present study was undertaken to select the most preferred deproteinizing agent for microbiological or chromatographic determination of free amino acids in plasma. The highest quantities of amino acids were obtained from samples treated with picric acid, ethanol or sulfosalicylic acid. It was also found from examining the recoveries of ¹⁴C-labeled amino acids added to plasma following treatment with picric acid, ethanol and sulfosalicylic acid that one kind of deproteinizing agent is not equally effective with all amino acids. Picric acid treatment gave the most reliable results for determination of amino acids except the basic amino acids, alanine and tryptophan. Sulfosalicylic acid was the most recommended deproteinizing agent for basic amino acids and ethanol was good for the assay of alanine and tryptophan. The response of d-amino acids to the deproteinizing agents tested was not always similar to that of their l-isomers.     

高效液相色谱法分析虾头中的氨基酸

采用OPA柱前衍生氨基酸,用高效液相色谱法,经多步洗脱,用荧光检测器检测,分析虾头中各类氨基酸。用时短,样品用量少,灵敏度高,适用于分析大量样品     

A Free Energy Based Computational Pathway from Chemical Templates to Lead Compounds: A Case Study of COX-2 Inhibitors

Abstract

Automation of lead compound design in silico given the structure of the protein target and a definition of its active site vies for the top of the wish list in any drug discovery programme. We present here an enumeration of steps starting from chemical templates and propose a solution at the state of the art, in the form of a system independent comprehensive computational pathway. This methodology is illustrated with cyclooxygenase-2 (COX-2) as a target. We built candidate molecules including a few Non Steroidal Anti-inflammatory Drugs (NSAIDs) from chemical templates, passed them through empirical filters to assess drug-like properties, optimized their geometries, derived partial atomic charges via quantum calculations, performed Monte Carlo docking, carried out molecular mechanics and developed free energy estimates with Molecular Mechanics Generalized Born Solvent Accessibility (MMGBSA) methodology for each of the candidate molecules. For the case of aspirin, we also conducted molecular dynamics on the enzyme, the drug and the complex with explicit solvent followed by binding free energy analysis. Collectively, the results obtained from the above studies viz. sorting of drugs from non-drugs, semi-quantitative estimates of binding free energies, amply demonstrate the viability of the strategy proposed for lead selection/design for biomolecular targets.     

A Detailed Study of the Amino Acids Produced from the Vacuum UV Irradiation of Interstellar Ice Analogs

We present a detailed analysis of the variety, quantity and distribution of the amino acids detected in organic residues after acid hydrolysis. Such organic residues are produced in the laboratory after the vacuum ultraviolet (VUV) irradiation of several astrophysically relevant ice mixtures containing H(2)O, CO, CO(2), CH(3)OH, CH(4) and NH(3) at low temperature (10-80 K), and subsequent warm-up to room temperature. We explore five experimental parameters: the irradiation time, the temperature, the ice mixture composition, the photon dose per molecule and the substrate for the ice deposition. The amino acids were detected and identified by ex-situ liquid chromatography analysis of the organic residues formed after warming the photolysed ices up to room temperature. This study shows that in all experiments amino acids are formed. Their total quantities and distribution depend slightly on the experimental parameters explored in the present work, the important requirement to form such molecules being that the starting ice mixtures must contain the four elements C, H, O and N. We also discuss the effects of the chemical treatment needed to detect and identify the amino acids in the organic residues. Finally, these results are compared with meteoritic amino acid data from the carbonaceous chondrite Murchison, and the formation processes of such compounds under astrophysical conditions are discussed.     

The Response of Amino Acid Frequencies to Directional Mutation Pressure in Mitochondrial Genome Sequences Is Related to the Physical Properties of the Amino Acids and to the Structure of the Genetic Code

The frequencies of A, C, G, and T in mitochondrial DNA vary among species due to unequal rates of mutation between the bases. The frequencies of bases at fourfold degenerate sites respond directly to mutation pressure. At first and second positions, selection reduces the degree of frequency variation. Using a simple evolutionary model, we show that first position sites are less constrained by selection than second position sites and, therefore, that the frequencies of bases at first position are more responsive to mutation pressure than those at second position. We define a measure of distance between amino acids that is dependent on eight measured physical properties and a similarity measure that is the inverse of this distance. Columns 1, 2, 3, and 4 of the genetic code correspond to codons with U, C, A, and G in their second position, respectively. The similarity of amino acids in the four columns decreases systematically from column 1 to column 2 to column 3 to column 4. We then show that the responsiveness of first position bases to mutation pressure is dependent on the second position base and follows the same decreasing trend through the four columns. Again, this shows the correlation between physical properties and responsiveness. We determine a proximity measure for each amino acid, which is the average similarity between an amino acid and all others that are accessible via single point mutations in the mitochondrial genetic code structure. We also define a responsiveness for each amino acid, which measures how rapidly an amino acid frequency changes as a result of mutation pressure acting on the base frequencies. We show that there is a strong correlation between responsiveness and proximity, and that both these quantities are also correlated with the mutability of amino acids estimated from the mtREV substitution rate matrix. We also consider the variation of base frequencies between strands and between genes on a strand. These trends are consistent with the patterns expected from analysis of the variation among genomes. [Reviewing Editor: Dr. David Pollock]     

Extracellular Neuroactive Amino Acids in the Rat Striatum During Ischaemia: Comparison Between Penumbral Conditions and Ischaemia with Sustained Anoxic Depolarisation

Abstract: Changes in the extracellular levels of excitatory and inhibitory amino acid transmitters were studied in the rat striatum during penumbral ischaemia using intracerebral microdialysis. Effects of penumbral forebrain ischaemia were compared with those of ischaemia with sustained anoxic depolarisation and K⁺ (100 m M ). Comparisons were also made between different groups of animals at 2 and 24 h after dialysis probe implantation. The K⁺ stimulus did not provoke any release of excitatory amino acids in the 24-h group, probably reflecting a decrease of functional synapses adjacent to the probe. During 30 min of penumbral ischaemia, excitatory amino acids did not reach critical concentrations in the extracellular fluid, and increases in levels of inhibitory/modulatory amino acids were similar. On the other hand, severe transient ischaemia resulted in massive synchronous release of many neuroactive excitatory and inhibitory compounds, in both the 2- and 24-h groups. These and other data suggest that changes during severe ischaemia may arise from both neurotransmitter and metabolic pools. It is concluded that is- chaemic damage in the penumbra may not be related to extracellular neuroactive amino acid changes generated within this region.     

Analyses of Soluble Protein,Total and Free Amino Acids in Leaves of Different Ecotypes of Phragmites communis Growing in the Hexi Corridor

The difference of total and free amino acids and protein extracted from the leaves of four different reed ecotypes growing in Hexi corridor of Gansu Province were investigated. In all of the different reed ecotypes, the content of Asp, Glu, Gly, Leu and Ala in total amino acids were high, while the contents of Ala, Phe, Met and Thr, Pro in total amino acids varied among different reed ecotypes. Albeit Ala, Glu, Asp, Gly and Ser were the chief composition of free amino acids in leaves of all reed ecotypes, but temarkble difference was found in the content of each free amino acid from different reed ecotypes. The content of free Pro in leaves of salt meadow and salt meadow-sand dune transitional zone reed were 3.5 and 1.6 times respectively as much as in leaves of swamp reed. Swamp reed had 11 soluble proteins whereas other three reed ecotypes show that each has 13 soluble proteins. Three “salt adaptation proteins” (66 kD, 40.3 kD, 16.5 kD) were found in leaves of three reed ecotypes with varying degree of salt stress, however, the contents of 3 “salt adaptation protens” showed a negative correlation with the degree of salt stress. There was a large amount of “special protein” (11.7 kD) in leaves of sand dune reeds. These results suggest that the difference in cytogene expression takes a priority basis of adaptation of reed plants to different habitats, while a closer relationship of reeds tolerance to salt or drought stress with Pro accumulation in cells is seen than with the of accumulation stress adaptation protein.     

光质对水稻幼苗蛋白质、氨基酸含量的影响

蓝光对水稻幼苗可溶性蛋白积累的促进效应,在幼苗生长的初期比较明显;第５天,幼苗的可溶性蛋白质、蛋白氮、非蛋白氮以及Ａｓｐ、Ａｓｎ、Ｇｌｕ、Ｇｌｎ等游离氨基酸含量都远远高于白光或红光的处理。随着苗龄的增加,蓝光的促进作用减弱,到第１７天,各项指标都低于白光处理的幼苗。红光处理的幼苗可溶性蛋白始终低于白光或蓝光的处理,其Ａｓｎ、Ｇｌｎ两种酰胺含量在第１０天以后明显高于同期的白光或蓝光的处理。     

红菜薹6个品种氨基酸含量的分析比较

对红菜薹6个品种的氨基酸种类、含量进行分析测定,结果表明,红菜薹含有17种氨基酸,其中7种为人体必需氨基酸,各品种间各类氨基酸含量差异不显著;相对于茎干而言,红菜薹的薹叶具有更高的营养价值和药用价值;不同的采收温度对各类氨基酸含量的影响不同。     

Insights into the functional role of protonation states in the HIV-1 protease-BEA369 complex: molecular dynamics simulations and free energy calculations

The molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method combined with molecular dynamics (MD) simulations were used to investigate the functional role of protonation in human immunodeficiency virus type 1 (HIV-1) protease complexed with the inhibitor BEA369. Our results demonstrate that protonation of two aspartic acids (Asp25/Asp25′) has a strong influence on the dynamics behavior of the complex, the binding free energy of BEA369, and inhibitor–residue interactions. Relative binding free energies calculated using the MM-PBSA method show that protonation of Asp25 results in the strongest binding of BEA369 to HIV-1 protease. Inhibitor–residue interactions computed by the theory of free energy decomposition also indicate that protonation of Asp25 has the most favorable effect on binding of BEA369. In addition, hydrogen-bond analysis based on the trajectories of the MD simulations shows that protonation of Asp25 strongly influences the water-mediated link of a conserved water molecule, Wat301. We expect that the results of this study will contribute significantly to binding calculations for BEA369, and to the design of high affinity inhibitors.     

Continuous metadynamics in essential coordinates as a tool for free energy modelling of conformational changes

Modelling of conformational changes in biopolymers is one of the greatest challenges of molecular biophysics. Metadynamics is a recently introduced free energy modelling technique that enhances sampling of configurational (e.g. conformational) space within a molecular dynamics simulation. This enhancement is achieved by the addition of a history-dependent bias potential, which drives the system from previously visited regions. Discontinuous metadynamics in the space of essential dynamics eigenvectors (collective motions) has been proposed and tested in conformational change modelling. Here, we present an implementation of two continuous formulations of metadynamics in the essential subspace. The method was performed in a modified version of the molecular dynamics package GROMACS. These implementations were tested on conformational changes in cyclohexane, alanine dipeptide (terminally blocked alanine, Ace-Ala-Nme) and SH3 domain. The results illustrate that metadynamics in the space of essential coordinates can accurately model free energy surfaces associated with conformational changes. Figure The conformational free energy surface of cyclohexane in the space of the two most intensive collective motions.

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Infrared Methods for Monitoring the Protonation State of Carboxylic Amino Acids in the Photocycle of Bacteriorhodopsin

This review deals with the role of carboxylic amino acids in the proton-transport activity of bacteriorhodopsin. The main focus is on the infrared data, which allow direct monitoring of the protonation/deprotonation of specific residues during the proton movement in the course of the photocycle. Additional attention is paid to the potential use of carboxylic acids in proteins as internal sensors, based on the sensitivity of their IR frequencies to the immediate environment.