The competition between protein folding and aggregation: off-lattice minimalist model studies

Protein aggregation has been associated with a number of human diseases, and is a serious problem in the manufacture of recombinant proteins. Of particular interest to the biotechnology industry is deleterious aggregation that occurs during the refolding of proteins from inclusion bodies. As a complement to experimental efforts, computer simulations of multi-chain systems have emerged as a powerful tool to investigate the competition between folding and aggregation. Here we report results from Langevin dynamics simulations of minimalist model proteins. Order parameters are developed to follow both folding and aggregation. By mapping natural units to real units, the simulations are shown to be carried out under experimentally relevant conditions. Data pertaining to the contacts formed during the association process show that multiple mechanisms for aggregation exist, but certain pathways are statistically preferred. Kinetic data show that there are multiple time scales for aggregation, although most association events take place at times much shorter than those required for folding. Last, we discuss results presented here as a basis for future work aimed at rational design of mutations to reduce aggregation propensity, as well as for development of small-molecular weight refolding enhancers.     

Bioluminescence Spectra of Native and Mutant Firefly Luciferases as a Function of pH

Bioluminescence spectra of the wild-type recombinant Luciola mingrelica firefly luciferase and its mutant form with the His433Tyr point mutation were obtained within the pH 5.6-10.2 interval. The spectra are shown to be a superposition of the spectra of the three forms of the electronically excited reaction product oxyluciferin: ketone (lambdamax = 618 nm), enol (lambdamax = 587 nm), and enolate-ion (lambdamax = 556 nm). The shift in lambdamax by 40 nm to the red region in the mutant luciferase bioluminescence at the pH optimum of enzyme activity (pH 7.8) is explained by the change in the relative content of different oxyluciferin forms due to the shift in the ketone <--> enol <--> enolate equilibria. A computer model of the luciferase-oxyluciferin-AMP complex was constructed and the structure of amino acid residues participating in the equilibrium is proposed. Computer models of the protein region near the His433 residue for the wild type and mutant luciferases are also proposed. Comparison of the models shows that the His433Tyr mutation increases flexibility of the polypeptide loop that binds the N and C domains of luciferase. As a result, the flexibility of the C domain amino acid residues in the emitter microenvironment increases, and this increase may be the reason for the observed differences in the bioluminescence spectra of the native and mutant luciferases.     

超声彩色血流成像的计算机快速仿真方法

研究超声彩色血流成像的快速仿真方法,克服原先仿真方法非常耗时的缺点。方法超声彩色血流成像计算机仿真中,血流信号是对成像区间内所有点散射体的回波信号累加而得到的。通过引入新的等效散射体模型,可以大大降低散射体的密度,从而减少计算回波信号所需时间。在计算机上用Matlab编程来进行仿真实验,对以往仿真方法和基于等效散射体模型方法的性能进行比较。结果实验表明:基于等效散射体模型的仿真,在保证相同流速精度的前提下,仿真速度比传统方法提高了10倍以上。结论基于等效散射体模型的仿真方法能极大地提高超声彩色血流成像的仿真速度,可以为超声彩色血流成像的方法研究提供便利。     

Two distinct heme distal site states define Cerebratulus lacteus mini-hemoglobin oxygen affinity

The nerve tissue hemoglobin of Cerebratulus lacteus (CerHb) is the smallest naturally occurring known hemoglobin. Stabilization of the diatomic bound species (e.g., O(2)) is achieved through a network of hydrogen bonds based on three key residues TyrB10, GlnE7, and ThrE11. The first two residues are typically associated in hemoglobins with enhanced O(2) affinity, related to hydrogen bond stabilization of the heme-bound O(2) resulting in a decrease of the ligand dissociation rates. In contrast to the above observations, the affinity of CerHb for O(2) is only moderate, and the rate of O(2) dissociation is unexpectedly high. To gain insight on the diverse molecular mechanisms controlling ligand affinities, we have analyzed w.t. CerHb and its ThrE11-->Val mutant by means of joint molecular dynamics and quantum mechanics simulation techniques, complementing recent site-directed mutagenesis experiments. Our results suggest that the observed O(2) dissociation rates can only be explained through a dynamic equilibrium between high and low affinity states of the w.t. CerHb heme distal site.     

Predicting the three-dimensional structure of human P-glycoprotein in absence of ATP by computational techniques embodying crosslinking data: insight into the mechanism of ligand migration and binding sites

P-glycoprotein is a membrane protein involved in the phenomenon of multidrug resistance. Its activity and transport function have been largely characterized by various biochemical studies and a low-resolution image has been obtained by electron microscopy. Obtaining a high-resolution structure is, however, still remote due to the inherent difficulties in the experimental determination of membrane protein structures. We present here a three-dimensional (3D) atomic model of P-glycoprotein in absence of ATP. This model was obtained using a combination of computational techniques including comparative modeling and rigid body dynamics simulations that embody all available cysteine disulfide crosslinking data characterizing the whole protein in absence of ATP. The model features rather well most of the experimental interresidue distances derived both in the transmembrane domains and in the nucleotide binding domains. The model is also in good agreement with electron microscopy data, particularly in terms of size and topology. It features a large cavity detected in the protein core into which seven ligands were successfully docked. Their predicted affinity correlates well with experimental values. Locations of docked ligands compare favorably with those suggested by cysteine-scanning data. The finding of different positions both for a single ligand and for different ligands corroborates the experimental evidence indicating the existence of multiple drug binding sites. The interactions identified between P-glycoprotein and the docked ligands reveal that different types of interactions such as H-bonds, pi-pi and cation-pi interactions occur in agreement with a recently proposed pharmacophore model of P-glycoprotein ligands. Furthermore, the model also displays a lateral opening located in the transmembrane domains connecting the lipid bilayer to the central cavity. This feature supports rather well the commonly admitted mechanism of substrate uptake from the lipid bilayer. We propose that this 3D model may be an important tool to understand the structure-function relationship of P-glycoprotein.     

Protein under pressure: molecular dynamics simulation of the arc repressor

Experimental nuclear magnetic resonance results for the Arc Repressor have shown that this dimeric protein dissociates into a molten globule at high pressure. This structural change is accompanied by a modification of the hydrogen-bonding pattern of the intermolecular beta-sheet: it changes its character from intermolecular to intramolecular with respect to the two monomers. Molecular dynamics simulations of the Arc Repressor, as a monomer and a dimer, at elevated pressure have been performed with the aim to study this hypothesis and to identify the major structural and dynamical changes of the protein under such conditions. The monomer appears less stable than the dimer. However, the complete dissociation has not been seen because of the long timescale needed to observe this phenomenon. In fact, the protein structure altered very little when increasing the pressure. It became slightly compressed and the dynamics of the side-chains and the unfolding process slowed down. Increasing both, temperature and pressure, a tendency of conversion of intermolecular into intramolecular hydrogen bonds in the beta-sheet region has been detected, supporting the mentioned hypothesis. Also, the onset of denaturation of the separated chains was observed.     

Life Cycle Assessment of a Personal Computer and its Effective Recycling Rate (7 pp)

Background, Aims and Scope Telecommunication and information technology, dramatically emerged during the last decade, has generated environmental problems by accelerating mass production, mass consumption, and mass disposal of personal computers (PCs) in Korea. In addition, it has led the Korean new economy. The Korean government has encouraged researchers and industry to study the environmental impact, adequate disposal treatment, and the reasonable recycling rate of an end-of-life personal computer. The main purpose of this research is to investigate the life cycle environmental impact of PCs and to determine the desirable or feasible recycle rate of an end-of-life PC. An LCA on a PC was performed based on different recycling scenario. Target audiences are new product developers, designers, product recovery managers and environmental policy makers who are interested in the environmental impact of PCs and recycling of end-of-life products. Methods A target product is a Pentium IV personal computer made in Korea in 2001, excluding the monitor and peripheral equipment. The procedure of the LCA followed the ISO14040 series. System boundary includes the entire life cycle of the product, including pre-manufacturing (the electrical parts and components manufacturing), manufacturing, transportation, use, and disposal. The LCI and impact assessment database for a PC was constructed using SIMAPRO version 4.0 software and LCI information was compiled by site-specific data and the Korean national database. The LCA was performed on different recycling scenarios: one being that of the current recycling rate of 46%, and the other being the ideal condition of a 100% recycling rate. Results and Discussion Abiotic depletion, global warming, ecotoxicity, human toxicity, acidification, ozone layer depletion, photo-oxidant formation, and eutrophication are adopted as the impact categories. The pre-manufacturing stage was a significant stage for all of the environmental parameters, besides human toxicity potential. PC manufacturing consists of rather simple processes such as assembly and packaging. For improving the environmental performance of PCs, environmental management approaches of design for the environment and green procurement are recommended. The use stage had a significant potential due to the electricity consumption produced by burning fossil fuel. The disposal stage's contribution to environmental impact was largest in human toxicity, and second largest in ozone layer depletion potential. The PC recycling was shown to inhibit all environmental impacts with the exception of the ozone depletion and ecotoxicity potential. The increase of light oil, nitric acid, sulfuric acid, and deoxidating agent consumption during the recycling process contributes to the environmental impact of ozone and ecotoxicity parameters. Current recovery and recycling technologies should be taken into account for enhancing the benefits of recycling. Anyway, the effectiveness of recycling was highlighted by this study. PC recycling reduces the total environmental impact of the product. The PC recycling is recommended to be raised up to at least 63% in order to reduce the environmental burdens of a PC in other life cycle stages. Conclusion and Recommendation This study implies that design for the environment (DfE) in the product design stage and green procurement are recommended for improving the entire environmental performance of electronic equipment such as PCs. The recycling of waste PCs clearly reduces the environmental burden. There are, however, trade-offs among environmental parameters according to the PC recycling rate. Current recycling methods are not effective in reducing ozone depletion and ecotoxicity environmental impact. The product recovery is another key for efficient recycling. Efficient reverse logistics to collect and transport end-of-life PCs should be taken into account to enhance recycling effects. There were several electrical parts not included in this assessment, due to the unavailability of adequate data. Further studies with more detail and reliable inventories for electrical parts and sub-components are recommended. Furthermore, costs of recycling should also be treated in further research.     

Modelling the role of intracolonial genetic diversity on regulation of brood temperature in honey bee (Apis mellifera L.) colonies

In polyandrous social insects such as honey bees, a worker’s affinity for a particular task may be genetically infl uenced and so some patrilines may have lower stimulus thresholds for commencing a task than others. We used simulation models to investigate the effects of intracolonial diversity in the task thresholds that stimulate workers to engage in heating and cooling during nest thermoregulation. First, we simulated colonies comprised of one or 15 patrilines that were engaged in heating the brood nest, and observed that single patriline colonies maintained, on average, less stable brood nest temperatures than multiple patriline colonies. Second we simulated colonies with five patrilines that were engaged in cooling their nest, recording the proportions of bees of different patrilines that engaged in nest cooling in response to changing temperatures. Both of our simulations show remarkably similar qualitative patterns to those that we have previously observed empirically. This provides further support for the hypothesis that geneticallybased variability in task thresholds among patrilines within honey bee colonies is an important contributor to the ability of colonies to precisely thermoregulate their nests, and we suggest that diversity is important for optimal expression of a range of other colony-level phenotypes. Received 17 June 2005; revised 27 October 2005; accepted 23 December 2005.     

Stochastic computer model of the cell microtubule dynamics

To study the effect of various factors on the microtubule system, one of the main cytoskeletal elements in the cell, which organizes the intracellular transport of different organelles and is necessary for mitosis and meiosis, a computer model of this system is created. Using a stochastic approach, the model describes the microtubule assembly/disassembly as a set of chemical reactions with certain rate constants. Microtubules are visualized in the computer program field, which makes the model vivid. The program imitates the dynamics and structure of the microtubule system with high reliability. The parameters calculated by the model correlate with the corresponding parameters of microtubules in living cells. This approach to modeling microtubules and similar systems continues to be developed so that the models would better describe living systems and the effect of a still broader range of factors could be studied.     

Modeling H3 histone N-terminal tail and linker DNA interactions

Molecular dynamics computer simulations were performed for the 25-residue N-terminal tail of the H3 histone protein in the proximity of a DNA segment of 10 base pairs (bp), representing a model for the linker DNA in chromatin. Several least biased configurations were used as initial configurations. The secondary structure content of the protein was increased by the presence of DNA close to it, but the locations of the secondary motifs were different for different initial orientations of the DNA grooves with respect to the protein. As a common feature to all simulations, the electrostatic attraction between negatively charged DNA and positively charged protein was screened by the water solvent and counterbalanced by the intrinsic compaction of the protein due to hydrophobic effects. The protein secondary structure limited the covering of DNA by the protein to 4-5 bp. The degree of compaction and charge density of the bound protein suggests a possible role of H3 tail in a nonspecific bending and plasticity of the linker DNA when the protein is located in the crowded dense chromatin.