Stepwise assembling of polypeptide chain energy distributions

The principles and application of conformational analysis software that makes use of a new algorithm are described. It is known that the existence of a local energy minimum in the energy landscape is in general related to the clustering of polypeptide chain conformations near that energy value or, in other words, to a high density of states. A criterion based on this principle is part of an algorithm employed to select subsets of polypeptide chain conformations in broad energy ranges. Chain fragments belonging to these subsets are then combined to build larger polypeptide chains and the corresponding energy distributions. The functionality of the various operations employed in the process is described and the FORTRAN 77 source code that defines the algorithm is listed. The methodology is illustrated with a calculation involving three chain fragments belonging to the cellular prion protein (PrP(C)).     

Non-equilibrium proteins.

There exist no methodical studies concerning non-equilibrium systems in cellular biology. This paper is an attempt to partially fill this shortcoming. We have undertaken an extensive data-mining operation in the existing scientific literature to find scattered information about non-equilibrium subcellular systems, in particular concerning fast proteins, i.e. those with short turnover half-time. We have advanced the hypothesis that functionality in fast proteins emerges as a consequence of their intrinsic physical instability that arises due to conformational strains resulting from co-translational folding (the interdependence between chain elongation and chain folding during biosynthesis on ribosomes). Such intrinsic physical instability, a kind of conformon (Klonowski-Klonowska conformon, according to Ji, (Molecular Theories of Cell Life and Death, Rutgers University Press, New Brunswick, 1991)) is probably the most important feature determining functionality and timing in these proteins. If our hypothesis is true, the turnover half-time of fast proteins should be positively correlated with their molecular weight, and some experimental results (Ames et al., J. Neurochem. 35 (1980) 131) indeed demonstrated such a correlation. Once the native structure (and function) of a fast protein macromolecule is lost, it may not be recovered--denaturation of such proteins will always be irreversible; therefore, we searched for information on irreversible denaturation. Only simulation and modeling of protein co-translational folding may answer the questions concerning fast proteins (Ruggiero and Sacile, Med. Biol. Eng. Comp. 37 (Suppl. 1) (1999) 363). Non-equilibrium structures may also be built up of protein subunits, even if each one taken by itself is in thermodynamic equilibrium (oligomeric proteins; sub-cellular sol-gel dissipative network structures).     

Incorporation and use of modified nucleotides in aqueous DNA computing

The concept of aqueous computing involves the use of large numbers of initially identical molecules to serve as memory registers in a fluid environment. Here, we test a new approach to aqueous computing where modified nucleotides are used to “write” on double-stranded DNA molecules to establish the logical values of true or false for a set of clauses. We introduce an implementation scenario where binding proteins specific to each modification can be used to selectively isolate DNA fragments with these modified nucleotides. In addition, we present initial results showing successful incorporation and detection of modifications. We have successfully labeled DNA fragments with four modifications, specifically Alexa Fluor-488, BODIPY-FL, biotin, and digoxigenin using polymerase chain reaction. The first two produce fluorescent molecules that can be distinguished by their color. We have confirmed that binding proteins or antibodies to these four modifications are specific and do not detect the other modifications. We have also successfully separated the DNAs labeled with Alexa Fluor and biotin using binding proteins. We present attempts at rebinding these modified molecules to a second binding protein; the equivalent of applying more than one clause to a set of values. We have found some challenges with this approach that likely can be resolved with further work. As there are millions of molecules with corresponding binding proteins, this approach has the potential to yield unlimited computing power as compared with other aqueous computing methods.     

A new algorithm for analysis of the homology in protein primary structure

A new algorithm for analysis of the homology and genetic semihomology in protein sequence is described. It assumes the close relation between the compared amino acids and their codons in related proteins. The algorithm is based on the network of the genetic relationship between amino acids and, thus differs from the commonly used statistical matrices. The results obtained by using this method are more comprehensive than used at present, and reflect the actual mechanism of protein differentiation and evolution. They concern: (1) location of homologous and semihomologous sites in compared proteins; (2) precise estimation of insertion/deletion gaps in non-homologous fragments; (3) analysis of internal homology and semihomology; (4) precise location of domains in multidomain proteins; (5) estimation of genetic code of non-homologous fragments; (6) construction of genetic probes; (7) studies on differentiation processes among related proteins; (8) estimation of the degree of relationship among related proteins; (9) studies on the evolution mechanism within homologous protein families and (10) confirmation of actual relationship of sequences showing low degree of homology.     

Investigation of an albumin‐enriched fraction of human serum and its albuminome

The removal of albumin and other high abundance proteins is a routine first step in the analysis of serum and plasma proteomes. However, as albumin can bind proteins and peptides, there is a universal concern as to how the serum proteome is changed by the removal of albumin. To address this concern, the current study was designed to identify proteins and peptides removed from the serum during albumin depletion; to determine which of these are bound to albumin (rather than copurified) and whether the bound proteins are intact proteins or peptide fragments. Sequential, independent analyses including both anti‐albumin antibody (anti‐HSA) affinity chromatography and SEC were used to isolate albumin‐bound proteins. RP‐HPLC and 1‐D SDS‐PAGE were then used to further separate the proteins prior to identification by MS/MS. Finally, whole protein molecular weight (MW) MS measurements coupled with protein coverage obtained by MS were combined to assess whether the bound proteins were intact or peptide fragments. Combining the results from multiple approaches, 35 proteins, of which 24 are intact, were found to be associated with albumin, and they include both known high and low abundance proteins.     

纵向产品差异的闭环供应链定价策略研究

为了解决闭环供应链中的定价策略问题,根据单一制造商和单一零售商构成的二级闭环供应链系统,应用博弈理论研究新产品与再造品之间存在纵向产品差异时闭环供应链中制造商和零售商的定价策略。得出了一个合作博弈的均衡解（合作决策）和一个非合作博弈（均衡解斯坦克尔伯格）的均衡解（分散决策）,并对两种定价策略作了进一步的比较,得出了一些有价值的结论。     

Barnase thermal titration via molecular dynamics simulations: detection of early denaturation sites

The thermal stability of barnase has been studied using constant pressure and temperature (CPT) molecular dynamics at different temperatures. Barnase X-ray coordinates were obtained from the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Bank (PDB code:1rnb). Simulations were performed at 285, 295, 300, 335, 345, and 395 K in explicit water under periodic boundary conditions for 280 ps. For each simulation, conformations were saved every 0.2 ps. Root mean square deviation (RMSD) values were calculated relative to the starting structure at 300 K and at time t = 0. Root mean square fluctuation (RMSF) values were calculated relative to the average structure obtained from the 300K simulation. Both root mean square deviation and fluctuation analysis indicated the presence of discrete regions of hyper-sensitivity along the barnase polypeptide chain. These regions exhibited spikes in flexibility prior to any global structural changes. The specific changes in barnase backbone flexibility are accompanied by increased phi/psi angle fluctuations. These results suggest the presence of early denaturation sites or denaturation nuclei whose local structure is disrupted prior to global structure disruption. Identification of denaturation nuclei suggests that appropriate amino acid replacements at these sites may lead to the design and development of more stable barnase mutants. This strategy of identifying denaturation nuclei in protein structures may represent a first step in the design of more stable protein structures.     

Towards defining the whole salivary peptidome

We report the identification of 2294 peptides/proteins in whole saliva in the mass range between 700 and 16 000 Da by LC‐MS and MS/MS using a MALDI‐TOF/TOF mass spectrometer. Most of the identified peptides/proteins are originated from cellular debris or plasma components while only 26% (n = 607) correspond to salivary peptides/proteins species. In spite of the presence of the major salivary peptides in all samples from the six subjects analyzed, each individual presents a different pattern of fragments, many deriving from the same protein sequence. All our data, in particular the large number of fragments found, suggest high proteolytic activity insight the oral cavity. The analysis of samples by gelatin zymography showed that all saliva donors displayed multiple proteolytic bands, two identified as cathepsin D and G by MS. Analysis of the cleavage site distribution on the main peptide sequences based on contingency tables shows that the predominant cleavages occur between Gln‐Gly or Tyr‐Gly. These cleavages are largely associated with proline‐rich proteins peptides and with histatin 1 and P‐B peptide, respectively. However, depending on the peptide class, different cleavage hits were observed suggesting the presence of a set of proteases acting in different ways according to different peptide sequences. Comparing the number of cleavages involving all residues, it is possible to observe that 44% (±10%) of the observed cleavages in histatin, statherin and P‐B peptide in all individuals may be explained by cathepsin D, suggesting a major role for this enzyme in oral cavity proteolysis.     

Phosphate binding protein as the biorecognition element in a biosensor for phosphate

This work explores the potential use of a member of the periplasmic family of binding proteins, the phosphate binding protein (PBP), as the biorecognition element in a sensing scheme for the detection of inorganic phosphate (Pi). The selectivity of this protein originates from its natural role which, in Escherichia coli, is to serve as the initial receptor for the highly specific translocation of Pi to the cytoplasm. The single polypeptide chain of PBP is folded into two similar domains connected by three short peptide linkages that serve as a hinge. The Pi binding site is located deep within the cleft between the two domains. In the presence of the ligand, the two globular domains engulf the former in a hinge-like manner. The resultant conformational change constitutes the basis of the sensor development. A mutant of PBP (MPBP), where an alanine was replaced by a cysteine residue, was prepared by site-directed mutagenesis using the polymerase chain reaction (PCR). The mutant was expressed, from plasmid pSD501, in the periplasmic space of E. coli and purified in a single chromatographic step on a perfusion anion-exchange column. Site-specific labeling was achieved by attaching the fluorophore, N-[2-(1-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC), to the protein through the sulfhydryl group of the cysteine moiety. Steady-state fluorescence studies of the MPBP-MDCC conjugate showed a change in the intensity of the signal upon addition of Pi. Calibration curves for Pi were constructed by relating the intensity of the fluorescence signal with the amount of analyte present in the sample. The sensing system was first developed and optimized on a spectrofluorometer using ml volumes of sample. It was then adapted to be used on a microtiter plate arrangement with microliter sample volumes. The system's versatility was finally proven by developing a fiber optic fluorescence-based sensor for monitoring Pi. In all three cases the detection limits for the analyte were in the sub-microMolar range. It was also demonstrated that the sensing system was selective for phosphate over other structurally-similar anions, paving the way for the design and development of a new family of biosensors utilizing the specific binding properties of periplasmic proteins.     

基于微分对策的供应链合作广告决策研究

针对供应链系统中制造商和零售商的合作广告计划问题,利用微分对策构建动态模型．分别研究制造商和零售商在合作和非合作条件下的广告策略．运用动态规划原理。分别得出静态反馈Nash均衡和反馈Stackelberg均衡,将两种均衡策略加以比较,结果显示合作广告计划是供应链系统中的一种协调和激励机制,可以提高两个渠道成员以及整个供应链系统的利润。     

Differences in the proteome profile in placenta from normal term and preeclamptic preterm pregnancies

The aim of this study was to use proteomic approaches to examine differences in protein expression in placentae from normal term and preterm preeclamptic pregnancies and to validate the data thus obtained by other independent methods. Using 2-DE we found that 80% of the proteins were present in both normal and preeclamptic placentae. However, 26 proteins in the normal term placentae were not matched in the preterm preeclamptic group. Six proteins showed increased intensity and one protein was down-regulated in preeclampsia. Four of the seven proteins that were altered in preeclampsia were further analyzed by Western blot and immunohistochemistry. Identification by MS techniques revealed these proteins to be involved in regulatory pathways activated by stress. This is significant because preeclampsia is a multisystem disorder in human pregnancies that results in considerable oxidative and nitrative stress. Three proteins identified by MS to be Hsp27, catalase, and glucose-regulated protein were confirmed by Western blot analysis to be significantly up-regulated in preeclampsia. Endothelial monocyte-activating polypeptide was shown to be down-regulated in preeclampsia by 2-DE and MS.     

Identification of differentially expressed proteins in papillary thyroid carcinomas with V600E mutation of BRAF

BRAF, a serine/threonine kinase of the RAF family, is a downstream transducer of the RAS-regulated MAPK pathway. V600E mutation of BRAF protein is the most common genetic alteration occurring in papillary thyroid carcinomas and is prognostic of poor clinicopathological outcomes. Protein expression in the subclass of PTC bearing the BRAF(V600E) mutation was investigated by using 2-DE and MS/MS techniques and compared to that of matched normal thyroid tissues from seven patients. 2-D gel image analysis revealed that the expression of eight polypeptide spots, corresponding to five proteins, were significantly underexpressed in PTC bearing BRAF(V600E) mutation whereas 25 polypeptides, representing 19 distinct proteins, were significantly upregulated in tumour tissue, as compared to normal thyroid. Among the differentially expressed polypeptides, mitochondrial proteins, ROS-scavenger enzymes, apoptosis-related proteins as well as proteins involved in tumour cell proliferation were identified. Although dissimilarities between the present results and those previously reported can be ascribed to the use of different 2-DE techniques, the possibility that BRAF(V600E) mutation is responsible for changes in protein expression distinct from those induced by other oncogenes cannot be ruled out.     

Coupled model- and solution-adaptivity in the finite-element method

Modeling of elastic thin-walled beams, plates and shells as ID- and 2D-boundary value problems is valid in undisturbed subdomains. Disturbances near supports and free edges, in the vicinity of concentrated loads and at thickness jumps cannot be described in a sufficient way by 1D- and 2D-BVPs. In these disturbed subdomains dimensional (d)-adaptivity and model (m)-adaptivity have to be performed coupled with h- and/or p-adaptivity using hierarchically expanded test spaces in order to guarantee reliable and efficient overall results. The expansion strategy is applied for enhancing the spatial dimension and the model which is more efficient and evident for engineers than the reduction method.

Using local residual error estimators of the primal problem in the energy norm by solving Dirichlet-problems on element patches, an efficient integrated adaptive calculation of the discretization—and the dimensional error is possible and reasonable, demonstrated by examples.

We also present an error estimator of the dual problem, namely a posterior equilibrium method (PEM) for calculation of the interface tractions on local patches with Neumann boundary conditions, using orthogonality conditions. These tractions are equilibrated with respect to the global equilibrium condition of the stress resultants. An upper bound error estimator based on differences between the new tractions and the discontinuous tractions calculated from the stresses of the current finite element solution. The introduction of new element boundary tractions yields a method which can be regarded as a stepwise hybrid displacement method or as Trefftz method for local Neumann problems of element patches.

An important advantage of PEM is the coupled computation of local discretization, dimensional- and model errors by an additive split.     

Cys(x)His(y)-Zn2+ interactions: possibilities and limitations of a simple pairwise force field

In zinc proteins, the Zn2+ cation frequently binds with a tetrahedral coordination to cysteine and histidine side chains. We examine the possibilities and limitations of a classical, pairwise force field for molecular dynamics of such systems. Hartree Fock and density functional calculations are used to obtain geometries, charge distributions, and association energies of side chain analogues bound to Zn2+. Both ionized and neutral cysteines are considered. Two parameterizations are obtained, then tested and compared through molecular dynamics simulations of two small, homologous proteins in explicit solvent: Protein Kinase C and the Cysteine Rich Domain (CRD) of Raf, which have two Cys3His-Zn2+ groups each. The lack of explicit polarizability and charge transfer in the force field leads to poor accuracy for the association energies, and to parameters--including the zinc charge, that depend on the number of bound cysteines and their protonation state. Nevertheless, the structures sampled with the best parameterization are in good overall agreement with experiment, and have zinc coordination geometries compatible with related structures in the Cambridge Structural Database and the Protein Data Bank. Non-optimized parameters lead to poorer structures. This suggests that while a simple force field is not appropriate for processes involving exchange between water and amino acids in the zinc coordination sphere (e.g. protein unfolding), it can be useful for equilibrium simulations of stable Cys3His zinc fingers.     

A Hidden Markov Model applied to the protein 3D structure analysis

Understanding and predicting protein structures depend on the complexity and the accuracy of the models used to represent them. A Hidden Markov Model has been set up to optimally compress 3D conformation of proteins into a structural alphabet (SA), corresponding to a library of limited and representative SA-letters. Each SA-letter corresponds to a set of short local fragments of four C_α similar both in terms of geometry and in the way in which these fragments are concatenated in order to make a protein. The discretization of protein backbone local conformation as series of SA-letters results on a simplification of protein 3D coordinates into a unique 1D representation. Some evidence is presented that such approach can constitute a very relevant way to analyze protein architecture in particular for protein structure comparison or prediction.     

一种蛋白质点突变计算机预测的并行模型

认识和预测蛋白质天然构象的波动对蛋白质-蛋白质对接和设计等应用是非常重要的.但是许多骨架柔性的方法会导致骨架较大幅度的波动.Backrub模型能够对骨架进行微小的扰动,符合高分辨率晶体结构中观察到的构象的微妙变化.本文提出了一种基于Backrub的并行扰动骨架和侧链的模型,可以对天然构象的等价状态进行模拟.这种并行扰动方式更加接近于真实情况下蛋白质构象的运动方式,更好地模拟了实验数据.通过预测10个点突变实例,相比串行随机扰动模型产生的构象,并行模型不仅从时间上提高了产生构象的速度,更提高了侧链的预测精度.     

Predicting function from structure: examples of the serine protease inhibitor canonical loop conformation found in extracellular proteins.

The prediction of protein function from structure is becoming of growing importance in the age of structural genomics. We have focused on the problem of identifying sites of potential serine protease inhibitor interactions on the surface of proteins of known structure. Given that there is no sequence conservation within canonical loops from different inhibitor families we first compare representative loops to all fragments of equal length among proteins of known structure by calculating main-chain RMS deviation. Fragments with RMS deviation below a certain threshold (hits) are removed if residues have solvent accessibilities appreciably lower than those observed in the search structure. These remaining hits are further filtered to remove those occurring largely within secondary structure elements. Likely functional significance is restricted further by considering only extracellular protein domains. Also a test is performed to see if the loop can dock into the binding site of the serine protease trypsin without unacceptable steric clashes. By comparing different canonical loop structures to the protein structure database we show that the method was able to detect previously known inhibitors. In addition, we discuss potentially new canonical loop structures found in secreted hydrolases, toxins, viral proteins, cytokines and other proteins. We discuss the possible functional significance of several of the examples found.