Molecular dynamics model structures for the molten globule state of {alpha}-lactalbumin: aromatic residue clusters I and II

To model the molten globule structure of ALT="{alpha}" BORDER="0">-lactalbumin, moleculardynamics (MD) simulations were carried out for the protein inexplicit water at high temperature. In these simulations, long-rangeCoulomb interactions were evaluated explicitly with an originalmethod (particle–particle and particle–cell: PPPC)to avoid artifacts caused by the cut-off. The MD simulationswere started from two initial conditions to verify that similarresults would be obtained. From the last 150 ps trajectoriesof the two MD simulations, two partially unfolded average structureswere obtained. These structures had the following common structuralfeatures which are characteristic of the molten globule state.The radii of gyration for these conformations were 7.4 and 9.6%larger than that of the native state. These values were almostthe same as the experimental value (9.6%) observed recentlyby small-angle X-ray scattering (Kataoka,M., Kuwajima,K., Tokunaga,F.and Goto,Y., 1997, Protein Sci., 6, 422–430). Furthermore,aromatic residues of clusters I and II in these structures werefar apart from each other except for Try103–Trp104. Thisresult is in good agreement with NMR experimental results forthe acid-denatured molten globule state (Alexandrescu et al.,1992, 1993); that is, NOE signals between the aromatic residueswere not observed, except for that of Try103–Trp104 inthe molten globule state. Other structural features of thesemodels for the molten globule state are discussed with referenceto native state structures.     

A holistic approach to protein structure alignment

A method of protein structure comparison developed previouslyis extended to incorporate other aspects of protein structurein addition to the inter-atomic vectors on which it was originallybased. Each additional aspect, which included hydrogen bonding,solvent exposure, torsional angles and sequence, was introducedseparately and evaluated for its ability to improve alignmentquality. The components were then combined, suitably weighted,to produce a more holistic comparison method. The method wastested on a group of remotely related ß/ALT="{alpha}" BORDER="0"> type proteinsthat share a common feature in their overall chain fold. Theresults indicated that while the original inter-atomic vectorcomponent was sufficient to give the correct alignment of mostpairs of topologically equivalent proteins, the inclusion ofhydrogen bonds, torsion angles and a measure of solvent exposureled to improvements in the more difficult comparisons. Considerationof amino acid properties, including hydrophobicity, had no beneficialeffect. The failure of the latter component was not unexpectedconsidering the almost total lack of sequence similarity amongthe proteins considered.     

Calcium binding mode of {gamma}-carboxyglutamic acids in conantokins

Conantokin-T (con-T) and conantokin-G (con-G) are two highlyhomologous peptide toxins found in Conus venom. The former isa 21-residue peptide with four ALT="{gamma}" BORDER="0">-carboxyglutamic acid (Gla) residues(at positions 3, 4, 10 and 14), while the latter is a 17-residuepeptide with five ALT="{gamma}" BORDER="0">-carboxyglutamic acid residues (at positions3, 4, 7, 10 and 14). Despite the apparent similarity in numberand relative positions of the ALT="{gamma}" BORDER="0">-carboxyglutamic acid residues,¹¹³Cd-NMR studies indicated a distinct metal binding behaviorfor con-G and con-T. There appears to be four binding sitesin con-G in contrast to one metal binding site in con-T. Toelucidate the mode of calcium binding by the ALT="{gamma}" BORDER="0">-carboxyglutamicacid residues in these conantokins, we designed various analogouspeptides with their ALT="{gamma}" BORDER="0">-carboxyglutamic acid replaced by otheramino acid residues. ¹¹³Cd-NMR experiments on conantokin analoguesreveal that the major difference in the number of metal bindingsites between con-G and con-T is due to the residue at position7. We also performed molecular simulations to calculate therelative binding free energies of several potential bindingsites. Based on our theoretical and experimental results, wepropose a `four-site' binding model for conantokin-G and a `single-site'binding model for conantokin-T.     

Molecular modelling of Staphylococcal {delta}-toxin ion channels by restrained molecular dynamics

ALT="{delta}" BORDER="0">-Toxin is a 26-residue channel-forming peptide from Staphylococcusaureus which forms an amphipathic ALT="{alpha}" BORDER="0">-helix in a membrane environment.Channel formation in planar bilayers suggests that an averageof six ALT="{delta}" BORDER="0">-toxin helices self-assemble to form transbilayer pores.Molecular models for channels formed by ALT="{delta}" BORDER="0">-toxin and by a syntheticanalogue have been generated using a simulated annealing protocolapplied via restrained molecular dynamics. These models areanalysed in terms of the predicted geometric and energetic propertiesof the transbilayer pores. Pore radius calculations of the modelsdemonstrate that rings of channel-lining residues contributea series of constrictions along the pore. Electrostatic propertiesof the pores are determined both by pore-lining charged sidechains and by the aligned helix dipoles of the parallel helixbundle. Molecular dynamics simulations (100 ps) of ALT="{delta}" BORDER="0">-toxin modelscontaining intra-pore water were performed. Analysis of theresultant dynamics trajectories further supports the proposalthat alternative conformations of pore-constricting side chainsmay be responsible for the observed conductance heterogeneityof ALT="{delta}" BORDER="0">-toxin ion channels.     

Computer simulations of signal transduction mechanism in {alpha}1B-adrenergic and m3-muscarinic receptors

Molecular dynamices simulations of the hamster ALT="{alpha}" BORDER="0">_1Badrenergicand the rat m3-muscarinic seven-helix bundle receptor modelshave been carried out. The free, agonist-bound and antagonist-boundforms have been considered. Moreover, three mutant forms ofthe m3-muscarinic recep-tor (N507ALT="<-" BORDER="0">A, N507ALT="<-" BORDER="0">D and N507ALT="<-" BORDER="0">S) have alsobeen simulated; among these, the N507ALT="<-" BORDER="0">S mutant shows a constitutiveactivity. A comparative structural/dynamics analysis has beenperformed to elucidate (i) the perturbations induced by thefunctionally different ligands upon binding to their targetreceptor, (ii) the features of the three single-point mutantswith respect to the receptor wild type and (iii) the propertiesshared by the agonist-boundforms of the ALT="{alpha}" BORDER="0">_1B-adrenergic receptorand the m3-muscarinic receptor and by the constitutively activemutant N507ALT="<-" BORDER="0">S. The consistency obtained between the structuralrearrangement of the transmembrane seven-helix bundle modelsconsidered, and the experimental pharmacological efficaciesof the ligands and of the mutants, constitute an important validationof the 3-D models obtained and allow the inference of the mechanismof ligand- or mutation-induced receptor activation at the molecularlevel.     

Functionally essential, invariant glutamate near the C-terminus of strand {beta}5 in various ({alpha}/{beta})8-barrel enzymes as a possible indicator of their evolutionary relatedness

Twelve different (ALT="{alpha}" BORDER="0">/ß)₈-barrel enzymes belonging tothree structurally distinct families were found to contain,near the C-terminus of their strand ß5, a conservedinvariant glutamic acid residue that plays an important functionalrole in each of these enzymes. The search was based on the ideathat a conserved sequence region of an (ALT="{alpha}" BORDER="0">/ß)₈-barrelenzyme should be more or less conserved also in the equivalentpart of the structure of the other enzymes with this foldingmotif owing to their mutual evolutionary relatedness. For thispurpose, the sequence region around the well conserved fifthß-strand of a-amylase containing catalytic glutamate(Glu230, Aspergillus oryzae ALT="{alpha}" BORDER="0">-amylase numbering), was used asthe sequence-structural template. The isolated sequence stretchesof the 12 (ALT="{alpha}" BORDER="0">/ß)₈-barrels are discussed from both thesequence-structural and the evolutionary point of view, theinvariant glutamate residue being proposed to be a joining featureof the studied group of enzymes remaining from their ancestral(ALT="{alpha}" BORDER="0">/ß)₈-barrel     

基于抗体选择策略的克隆挖掘算法

针对如何有效地利用大量的原始数据分析现状来预测未来的问题,基于抗体选择策略提出一种克隆选择挖掘算法。通过评估抗体的支持度、可信度和亲和度,求得有效的关联规则。实验结果表明,该算法能较快地获得可理解的规则,并且具有较高的准确率。     

食品级纳米二氧化硅对小鼠器官功能影响的研究

目的:通过测定小鼠连续性口服食品级纳米二氧化硅（Nano silicon dioxide,Nano-SiO₂）12周后血清及肝、肾、睾丸组织中硅元素的含量,并对小鼠肝肾等器官进行观察,初步评价纳米二氧化硅对机体的安全性。方法:将雄性昆明小鼠随机分为对照组（0.05%BSA）、纳米SiO₂低剂量组（100 mg/kg/day）、中剂量组（500 mg/kg/day）、高剂量组（1000 mg/kg/day）和微米SiO₂组（1000 mg/kg/day）,各组按上述剂量分别连续灌胃二氧化硅84 d后全部处死,利用HE染色观察各组动物的肝脏、肾脏、睾丸等组织病理学变化;ICP-OES检测血清及肝、肾、睾丸组织中硅元素的含量;检测各组动物血清中的谷丙转氨酶（ALT）、谷草转氨酶（AST）和尿素氮（BUN）的含量。结果:病理学观察在纳米低、中、高剂量组均发现肝脏、肾脏和睾丸组织有形态上的异常改变,以高剂量组更严重;纳米组血清中ALT、AST和BUN的含量均显著增高（p<0.01或p<0.05）;同时在血清及肝、肾、睾丸组织中纳米组的硅离子含量增高并呈现剂量依赖性;上述指标中同等剂量纳米组和微米组相比,纳米组含量高且病理变化更明显。结论:较长时间口服摄入食品级纳米二氧化硅可能在机体的肝脏、肾脏、睾丸等脏器内蓄积,造成一定程度的肝脏、肾脏等的病理学损害并影响其功能。长期多量食入纳米二氧化硅可能会对机体造成不良影响。