猪-C反应蛋白的分离纯化及其性质的研究

以猪血清为材料,通过磷酸乙醇胺—琼脂糖亲和层析,Sepharose 4B柱层析和Sephacryl—S300凝胶过滤,获得了猪C—反应蛋白的结晶。猪C—反应蛋白可与肺炎球菌壁C多糖发生特异的沉淀反应,这种结合是依赖钙离子的。EDTA和一些磷脂代谢产物如磷酸胆碱,磷酸乙醇胺等,能抑制猪C—反应蛋白与C多糖的结合。在SDS—聚丙烯酰胺凝胶电泳及梯度聚丙烯酰胺凝胶电泳中,猪C—反应蛋白表现出与人C—反应蛋白相同的行为,亚基是一条分子量为23.5kD的肽链,全分子的表观分子量为150kD。猪C—反应蛋白与兔抗人C—反应的蛋白的抗血清能发生免疫交叉反应。     

Model-free Analysis of Protein Dynamics: Assessment of Accuracy and Model Selection Protocols Based on Molecular Dynamics Simulation

The popular model-free approach to analyze NMR relaxation measurements has been examined using artificial amide (15)N relaxation data sets generated from a 10 nanosecond molecular dynamics trajectory of a dihydrofolate reductase ternary complex in explicit water. With access to a detailed picture of the underlying internal motions, the efficacy of model-free analysis and impact of model selection protocols on the interpretation of NMR data can be studied. In the limit of uncorrelated global tumbling and internal motions, fitting the relaxation data to the model-free models can recover a significant amount of quantitative information on the internal dynamics. Despite a slight overestimation, the generalized order parameter is quite accurately determined. However, the model-free analysis appears to be insensitive to the presence of nanosecond time scale motions with relatively small magnitude. For such cases, the effective correlation time can be significantly underestimated. As a result, proteins appear to be more rigid than they really are. The model selection protocols have a major impact on the information one can reliably obtain. The commonly employed protocol based on step-up hypothesis testing has severe drawbacks of oversimplification and underfitting. The consequences are that the order parameter is more severely overestimated and the correlation time more severely underestimated. Instead, model selection based on Bayesian Information Criteria (BIC), recently introduced to the model-free analysis by d'Auvergne and Gooley (2003), provides a better balance between bias and variance. More appropriate models can be selected, leading to improved estimate of both the order parameter and correlation time. In addition, the computational cost is significantly reduced and subjective parameters such as the significance level are unnecessary.     

Obesity Associated Molecular Forms of C-Reactive Protein in Human

Objective

To describe novel C-reactive protein (CRP) molecular forms (mf) in human plasma.

Design and Methods

Five novel CRP-mfs, disctinct from the previously described native (nCRP) and modified (mCRP) C-reactive proteins, were separated from human plasma by polyacrylamide gel electrophoresis and immunodetected by western blot in subjects with or without increased BMI, cardiovascular disease (CVD), and diabetes (n = 1800).

Results

Three of the five CRP-mfs were present in all samples. One, CRPmf-4, was present in a subgroup of subjects and its presence was associated with elevated body mass index (BMI). CRP-mf-5 was present in about 2% of the subjects and was not associated with any other parameters. The presence or distribution of the 5 CRP-mfs were not Ca²⁺-dependent. Crossed immuno-localization experiments indicated that none of the CRP-mfs were complexed with any of the lipoprotein classes or with signature proteins of the complement-factor. Moreover, the distribution of CRP-mfs were not significantly correlated with plasma CRP levels. CRP-mf-4 was significantly associated with increased BMI, but not with other parameters of the metabolic syndrome (HDL-C and triglyceride levels, and diabetes).

Conclusions

We have identified five new CRP-mfs out of which CRP-mf-4 was significantly associated with obesity. We have shown that oligomerization of CRP was not calcium dependent. We hypothesize that adipose tissue produces a factor which influences the formation of CRP mf-4. CRP-mfs might be used as an obesity-associated inflammatory marker.     

LRP-1 Mediated Endocytosis of EFE Across the Blood–Brain Barrier; Protein–Protein Interaction and Molecular Dynamics Analysis

International Journal of Peptide Research and Therapeutics - Additional biological protection; blood brain barrier (BBB) to neuronal tissue is essential against invading infections and unwanted...     

REACH Coarse-Grained Normal Mode Analysis of Protein Dimer Interaction Dynamics

The REACH (realistic extension algorithm via covariance Hessian) coarse-grained biomolecular simulation method is a self-consistent multiscale approach directly mapping atomistic molecular dynamics simulation results onto a residue-scale model. Here, REACH is applied to calculate the dynamics of protein-protein interactions. The intra- and intermolecular fluctuations and the intermolecular vibrational densities of states derived from atomistic molecular dynamics are well reproduced by the REACH normal modes. The phonon dispersion relations derived from the REACH lattice dynamics model of crystalline ribonuclease A are also in satisfactory agreement with the corresponding all-atom results. The REACH model demonstrates that increasing dimer interaction strength decreases the translational and rotational intermolecular vibrational amplitudes, while their vibrational frequencies are relatively unaffected. A comparative study of functionally interacting biological dimers with crystal dimers, which are formed artificially via crystallization, reveals a relation between their static structures and the interprotein dynamics: i.e., the consequence of the extensive interfaces of biological dimers is reduction of the intermonomer translational and rotational amplitudes, but not the frequencies.     

Mechanism of MicroRNA-Target Interaction: Molecular Dynamics Simulations and Thermodynamics Analysis

MicroRNAs (miRNAs) are endogenously produced ∼21-nt riboregulators that associate with Argonaute (Ago) proteins to direct mRNA cleavage or repress the translation of complementary RNAs. Capturing the molecular mechanisms of miRNA interacting with its target will not only reinforce the understanding of underlying RNA interference but also fuel the design of more effective small-interfering RNA strands. To address this, in the present work the RNA-bound (Ago-miRNA, Ago-miRNA-target) and RNA-free Ago forms were analyzed by performing both molecular dynamics simulations and thermodynamic analysis. Based on the principal component analysis results of the simulation trajectories as well as the correlation analysis in fluctuations of residues, we discover that: 1) three important (PAZ, Mid and PIWI) domains exist in Argonaute which define the global dynamics of the protein; 2) the interdomain correlated movements are so crucial for the interaction of Ago-RNAs that they not only facilitate the relaxation of the interactions between residues surrounding the RNA binding channel but also induce certain conformational changes; and 3) it is just these conformational changes that expand the cavity of the active site and open putative pathways for both the substrate uptake and product release. In addition, by thermodynamic analysis we also discover that for both the guide RNA 5′-end recognition and the facilitated site-specific cleavage of the target, the presence of two metal ions (of Mg²⁺) plays a predominant role, and this conclusion is consistent with the observed enzyme catalytic cleavage activity in the ternary complex (Ago-miRNA-mRNA). Our results find that it is the set of arginine amino acids concentrated in the nucleotide-binding channel in Ago, instead of the conventionally-deemed seed base-paring, that makes greater contributions in stabilizing the binding of the nucleic acids to Ago.     

Structure, Dynamics, and RNA Interaction Analysis of the Human SBDS Protein

Shwachman-Bodian-Diamond syndrome is an autosomal recessive genetic syndrome with pleiotropic phenotypes, including pancreatic deficiencies, bone marrow dysfunctions with increased risk of myelodysplasia or leukemia, and skeletal abnormalities. This syndrome has been associated with mutations in the SBDS gene, which encodes a conserved protein showing orthologs in Archaea and eukaryotes. The Shwachman-Bodian-Diamond syndrome pleiotropic phenotypes may be an indication of different cell type requirements for a fully functional SBDS protein. RNA-binding activity has been predicted for archaeal and yeast SBDS orthologs, with the latter also being implicated in ribosome biogenesis. However, full-length SBDS orthologs function in a species-specific manner, indicating that the knowledge obtained from model systems may be of limited use in understanding major unresolved issues regarding SBDS function, namely, the effect of mutations in human SBDS on its biochemical function and the specificity of RNA interaction. We determined the solution structure and backbone dynamics of the human SBDS protein and describe its RNA binding site using NMR spectroscopy. Similarly to the crystal structures of Archaea, the overall structure of human SBDS comprises three well-folded domains. However, significant conformational exchange was observed in NMR dynamics experiments for the flexible linker between the N-terminal domain and the central domain, and these experiments also reflect the relative motions of the domains. RNA titrations monitored by heteronuclear correlation experiments and chemical shift mapping analysis identified a classic RNA binding site at the N-terminal FYSH (fungal, Yhr087wp, Shwachman) domain that concentrates most of the mutations described for the human SBDS.     

Conformational Dynamics of Protein Side Chains and Enzyme-Substrate Interaction

Abstract

The electric interaction of charged and polar protein side chains with dipole moments of the bonds of substrate molecules bound in enzyme active sites is considered. The conformational motion of the side chains leads to the fact that the electric interaction, besides a constant (electrostatic) part, contains a fluctuating one, which is a random force (noise) exerted on the substrate molecule. On the time scale of enzyme turnovers this noise can be considered as the white one with good approximation. The noise is external, and the explicit expression for its intensity has been obtained. The possible functional role of the noise as an activating factor in enzyme catalysis is discussed.     

Multiplexed-Replica Exchange Molecular Dynamics Method for Protein Folding Simulation

Simulating protein folding thermodynamics starting purely from a protein sequence is a grand challenge of computational biology. Here, we present an algorithm to calculate a canonical distribution from molecular dynamics simulation of protein folding. This algorithm is based on the replica exchange method where the kinetic trapping problem is overcome by exchanging noninteracting replicas simulated at different temperatures. Our algorithm uses multiplexed-replicas with a number of independent molecular dynamics runs at each temperature. Exchanges of configurations between these multiplexed-replicas are also tried, rendering the algorithm applicable to large-scale distributed computing (i.e., highly heterogeneous parallel computers with processors having different computational power). We demonstrate the enhanced sampling of this algorithm by simulating the folding thermodynamics of a 23 amino acid miniprotein. We show that better convergence is achieved compared to constant temperature molecular dynamics simulation, with an efficient scaling to large number of computer processors. Indeed, this enhanced sampling results in (to our knowledge) the first example of a replica exchange algorithm that samples a folded structure starting from a completely unfolded state.     

Communication of γ Phage Lysin plyG Enzymes Binding toward SrtA for Inhibition of Bacillus Anthracis: Protein–Protein Interaction and Molecular Dynamics Study

Abstract

Bacillus anthracis is a pathogenic, Gram-positive bacterium which chiefly affects the livestock of animals and humans through acute disease anthrax. All around the globe this bio-threat organism damages millions of lives in every year and also most of the drugs were not responding properly in inhibition against this diseased pathogen. In recent development, phage therapy is considered as alternative solution to treat this serious infectious disease. In this study, we elucidated the binding of γ phage lysin plyG enzymes toward the SrtA along with its activator peptide LPXTG. Through protein–protein docking and molecular dynamics simulation studies, we showed the distinguished structure complementarity of SrtA and plyG complex. Especially, MD simulation relates strong and stable interaction occurs between the protein complex structures. These results suggest that additional experimental studies on our approach will lead to availability of better inhibitor against the SrtA.     

Agar-Gel Precipitin-Inhibition Technique for C-Reactive Protein Determinations: III. Quantitation of C-Reactive Protein in Serum Specimens

A quantitative C-reactive protein serological procedure has been developed. By use of this method, which is performed in agar-gel plates, from 2 to 654 μg of C-reactive protein per ml of titrated human serum can be detected. The method is based on the inhibition of a specific C-reactive protein antigen-antibody precipitate formed in agar-gel by the minimal reactive dilutions of each reagent in 48 hr. It is simple, sensitive, and readily reproducible.     

Agar-Gel Precipitin-Inhibition Technique for C-Reactive Protein Determinations

An application of the agargel precipitin-inhibition technique of Ray and Kadull detects the C-reactive protein present in the acute-phase human sera. The sensitivity of this procedure is compared with the capillary tube-precipitin method of Selman and Halpern.     

血清C-反应蛋白(CRP)测定报告

通过２８４９例患者血清Ｃ－反应蛋白（ＣＲＰ）检测证实,ＣＲＰ是细菌性感染的重要指标,败血症增高率达９１．６％,肺炎为４８％,其它细菌感染性疾病也均有不同程度的增高,而病毒性心肌炎则不增高。ＣＲＰ是诊断恶性肿瘤的重要指标之一,在恶网、肝癌、膀胱癌中均为１００％增高,而肺癌增高率为６６．６％。ＣＲＰ在风湿病活动期很敏感,故用于诊断活动性ＲＡ、ＳＬＥ极有意义,ＣＲＰ也可作为白血病继发感染的诊断及急性组织损伤的可靠证据     

A Systematic Framework for Molecular Dynamics Simulations of Protein Post-Translational Modifications

By directly affecting structure, dynamics and interaction networks of their targets, post-translational modifications (PTMs) of proteins play a key role in different cellular processes ranging from enzymatic activation to regulation of signal transduction to cell-cycle control. Despite the great importance of understanding how PTMs affect proteins at the atomistic level, a systematic framework for treating post-translationally modified amino acids by molecular dynamics (MD) simulations, a premier high-resolution computational biology tool, has never been developed. Here, we report and validate force field parameters (GROMOS 45a3 and 54a7) required to run and analyze MD simulations of more than 250 different types of enzymatic and non-enzymatic PTMs. The newly developed GROMOS 54a7 parameters in particular exhibit near chemical accuracy in matching experimentally measured hydration free energies (RMSE = 4.2 kJ/mol over the validation set). Using this tool, we quantitatively show that the majority of PTMs greatly alter the hydrophobicity and other physico-chemical properties of target amino acids, with the extent of change in many cases being comparable to the complete range spanned by native amino acids.     

Protein Surface Dynamics: Interaction with Water and Small Solutes

Previous time resolved measurements had indicated that protons could propagate on the surface of a protein, or a membrane, by a special mechanism that enhances the shuttle of the proton towards a specific site [1]. It was proposed that a proper location of residues on the surface contributes to the proton shuttling function. In the present study, this notion was further investigated using molecular dynamics, with only the mobile charge replaced by Na⁺ and Cl⁻ ions. A molecular dynamics simulation of a small globular protein (the S6 of the bacterial ribosome) was carried out in the presence of explicit water molecules and four pairs of Na⁺ and Cl⁻ ions. A 10 ns simulation indicated that the ions and the protein's surface were in equilibrium, with rapid passage of the ions between the protein's surface and the bulk. Yet it was noted that, close to some domains, the ions extended their duration near the surface, suggesting that the local electrostatic potential prevented them from diffusing to the bulk. During the time frame in which the ions were detained next to the surface, they could rapidly shuttle between various attractor sites located under the electrostatic umbrella. Statistical analysis of molecular dynamics and electrostatic potential/entropy consideration indicated that the detainment state is an energetic compromise between attractive forces and entropy of dilution. The similarity between the motion of free ions next to a protein and the proton transfer on the protein's surface are discussed.     

High-Sensitivity C-Reactive Protein and Risk of Sepsis

Background

Conventional C-reactive protein assays have been used to detect or guide the treatment of acute sepsis. The objective of this study was to determine the association between elevated baseline high-sensitivity C-reactive protein (hsCRP) and the risk of future sepsis events.

Methods

We studied data from 30,239 community dwelling, black and white individuals, age ≥45 years old enrolled in the REasons for Geographic and Racial Differences in Stroke (REGARDS) cohort. Baseline hsCRP and participant characteristics were determined at the start of the study. We identified sepsis events through review of hospital records. Elevated hsCRP was defined as values >3.0 mg/L. Using Cox regression, we determined the association between elevated hsCRP and first sepsis event, adjusting for sociodemographic factors (age, sex, race, region, education, income), health behaviors (tobacco and alcohol use), chronic medical conditions (coronary artery disease, diabetes, dyslipidemia, hypertension, chronic kidney disease, chronic lung disease) and statin use.

Results

Over the mean observation time of 5.7 years (IQR 4.5–7.1), 974 individuals experienced a sepsis event, and 11,447 (37.9%) had elevated baseline hsCRP (>3.0 mg/L). Elevated baseline hsCRP was independently associated with subsequent sepsis (adjusted HR 1.56; 95% CI 1.36–1.79), adjusted for sociodemographics, health behaviors, chronic medical conditions and statin use.

Conclusion

Elevated baseline hsCRP was associated with increased risk of future sepsis events. hsCRP may help to identify individuals at increased risk for sepsis.     

Effect of Calcium Ions on Hydrodynamic Properties of Pentameric and Decameric C-Reactive Protein in Solution

The molecular mass and sedimentation coefficient of native C-reactive protein in solution were determined by analytical ultracentrifugation in the presence and absence of calcium ions. Pentameric C-reactive protein was shown to be the major macroscopic form of this protein in solution. The removal of calcium ions from solution caused decompaction of the protein accompanied by changes in its hydrodynamic parameters. The sedimentation coefficient s ⁰ _20,w of pentameric C-reactive protein in solution containing 2 mM Ca²⁺ (6.6S) exceeded that for C-reactive protein in solution containing 2 mM EDTA (6.4S). Analysis of average molecular masses M _w and M _z obtained from sedimentation data demonstrated that the solution of highly purified protein was not homogeneous. As shown by intermolecular crosslinking, the solution also contained the 241-kDa decamer of C-reactive protein (9.5S) as a separate macroscopic form, whose share hardly reached 10% in the presence of 2 mM Ca²⁺ and increased after removal of calcium ions. The decamers were shown to result from intermolecular association of the pentamers.     

壳聚糖与细菌细胞膜相互作用的分子动力学研究

目的 壳聚糖（chitosan,CS）是一种天然的广谱抗菌活性物质。现有研究表明，壳聚糖与细菌细胞膜的相互作用是其发挥抗菌功能的关键。受限于传统实验技术的表征能力，壳聚糖与细菌细胞膜相互作用的具体机制仍有待研究。本文旨在研究壳聚糖与细菌细胞膜相互作用的分子机制。方法 本研究利用全原子分子动力学模拟技术主要探究了完全脱乙酰化的不同聚合度壳聚糖（八聚糖、十二聚糖和十六聚糖）与革兰氏阴性菌外膜（outer membrane,OM）和革兰氏阳性菌质膜（cytoplasmic membrane,CM）相互作用的动态过程。结果 壳聚糖主要依靠其氨基、碳6位羟基和碳3位羟基与OM和CM的头部极性区发生快速结合。随后壳聚糖末端糖基单元倾向于插入OM内部，深度约1 nm，并与脂质分子脂肪酸链上的羰基形成稳定的氢键相互作用。与之相比，壳聚糖分子难以稳定地插入CM内部。壳聚糖结合对膜结构性质产生影响，主要表现在降低OM和CM的单分子脂质面积，显著减少OM和CM极性区的Ca2+和Na+数目，破坏阳离子介导的脂质间相互作用。结论 本研究证明，壳聚糖带正电的氨基基团是介导其与膜相互作用的关键，并破环脂质间的相互作...