Circular dichroism and structure- function relationships in cloacin DF13- immunity protein complex

Comparison of the circular dichroism (CD), of cloacin-immunity protein complex with that of cloacin and of a mutant cloacin lacking the ability to bind immunity protein, shows that the binding of immunity protein imposes a definite structure on the cloacin molecule. It is discussed that this structure probably is a prerequisite for an effective killing activity of the bacteriocin. The cloacin molecule itself probably has two domains, as was found by limited proteolysis. Comparison of the structure of two of the proteolytic fragments with that of the intact molecule by means of circular dichroism also suggests that cloacin is made up of a part without much periodic structure and of a part with more helicity. The former part being rather sensitive to proteolysis, the latter being comparatively insensitive.     

A unification of models for meta-analysis of diagnostic accuracy studies

Studies of diagnostic accuracy require more sophisticated methods for their meta-analysis than studies of therapeutic interventions. A number of different, and apparently divergent, methods for meta-analysis of diagnostic studies have been proposed, including two alternative approaches that are statistically rigorous and allow for between-study variability: the hierarchical summary receiver operating characteristic (ROC) model (Rutter and Gatsonis, 2001) and bivariate random-effects meta-analysis (van Houwelingen and others, 1993), (van Houwelingen and others, 2002), (Reitsma and others, 2005). We show that these two models are very closely related, and define the circumstances in which they are identical. We discuss the different forms of summary model output suggested by the two approaches, including summary ROC curves, summary points, confidence regions, and prediction regions.     

A quantitative method for measuring protein phosphorylation

We have developed a novel method for quantitating protein phosphorylation by a variety of protein kinases. It can be used with purified kinases and their substrates in vitro or in combination with cell extracts. The method is based on the knowledge that protein kinase C (PKC) adds three phosphates to each molecule of its preferred substrate, myelin basic protein (MBP). A time course is performed in which a kinase is allowed to phosphorylate its preferred substrate or the protein under investigation in the presence of [gamma-32P]ATP. At the same time PKC is allowed to fully phosphorylate MBP. After resolving the products by SDS-PAGE, electrophoretic transfer, and determining the degree of incorporation of 32P by phosphorImager analysis, the data are converted to moles phosphate/mole protein by normalization with phosphorylated MBP. The method is both sensitive and relatively rapid and all the steps are commonly available in the biochemistry laboratory. We have used this method to confirm and extend information on the relationship of MEK1 and MAPK/Erk2 in rat lung fibroblasts exposed to V(2)O(5). A 4-h exposure to V(2)O(5) results in partial phosphorylation of MAPK/Erk2 such that 25% of the potential phosphorylation sites are occupied. We also demonstrate that despite multiple potential phosphorylation sites, recombinant human AP endonuclease is weakly phosphorylated in vitro (4% at best) by PKC, cGMP-dependent protein kinase, casein kinase II, and casein kinase I and not at all phosphorylated by MAPK. Furthermore we are unable to demonstrate phosphorylation in cell extracts from HeLa cells, mouse fibroblasts after oxidative damage with H(2)O(2) or alkylation damage with methylmethane sulfonate, or rat lung fibroblasts after oxidative damage with V(2)O(5).     

A general method for hyperquenching protein crystals

During flash cooling of protein crystals in liquid cryogens, cooling rates are determined by sample size, choice of cooling liquid, and by the thickness of the cold gas layer that forms above the liquid. We describe an experimental protocol for ultra-rapid cooling of protein crystals. This protocol requires no complex apparatus, and yields ice-ring-free diffraction without the use of penetrating cryoprotectants.     

A structure-based method for protein sequence alignment

MOTIVATION: With the continuing rapid growth of protein sequence data, protein sequence comparison methods have become the most widely used tools of bioinformatics. Among these methods are those that use position-specific scoring matrices (PSSMs) to describe protein families. PSSMs can capture information about conserved patterns within families, which can be used to increase the sensitivity of searches for related sequences. Certain types of structural information, however, are not generally captured by PSSM search methods. Here we introduce a program, Structure-based ALignment TOol (SALTO), that aligns protein query sequences to PSSMs using rules for placing and scoring gaps that are consistent with the conserved regions of domain alignments from NCBI's Conserved Domain Database. RESULTS: In most cases, the alignment scores obtained using the local alignment version follow an extreme value distribution. SALTO's performance in finding related sequences and producing accurate alignments is similar to or better than that of IMPALA; one advantage of SALTO is that it imposes an explicit gapping model on each protein family. AVAILABILITY: A stand-alone version of the program that can generate global or local alignments is available by ftp distribution (ftp://ftp.ncbi.nih.gov/pub/SALTO/), and has been incorporated to Cn3D structure/alignment viewer. CONTACT: bryant@ncbi.nlm.nih.gov.     

A new method for protein domain recognition

A fuzzy cluster method is presented to recognize protein domains. This algorithm can identify domains globally. A protein structure set was used to test the algorithm. Among 219 proteins, 66.7% yielded results that agreed with the reference definitions, 30.6% showed minor differences, and only 2.7% (six proteins) showed major differences with the reference. The new method is more than 20 times fast than previous algorithms. Received: 9 November 1998 / Revised version: 20 December 1999 / Accepted: 20 December 1999     

OptGraft: A computational procedure for transferring a binding site onto an existing protein scaffold

One of the many challenging tasks of protein design is the introduction of a completely new function into an existing protein scaffold. In this study, we introduce a new computational procedure OptGraft for placing a novel binding pocket onto a protein structure so as its geometry is minimally perturbed. This is accomplished by introducing a two‐level procedure where we first identify where are the most appropriate locations to graft the new binding pocket into the protein fold by minimizing the departure from a set of geometric restraints using mixed‐integer linear optimization. On identifying the suitable locations that can accommodate the new binding pocket, CHARMM energy calculations are employed to identify what mutations in the neighboring residues, if any, are needed to ensure that the minimum energy conformation of the binding pocket conserves the desired geometry. This computational framework is benchmarked against the results available in the literature for engineering a copper binding site into thioredoxin protein. Subsequently, OptGraft is used to guide the transfer of a calcium‐binding pocket from thermitase protein (PDB: 1thm) into the first domain of CD2 protein (PDB:1hng). Experimental characterization of three de novo redesigned proteins with grafted calcium‐binding centers demonstrated that they all exhibit high affinities for terbium (K_d ～ 22, 38, and 55 μM) and can selectively bind calcium over magnesium.     

A unification of multivariate methods for meta-analysis of genetic association studies

Methods for multivariate meta-analysis of genetic association studies are reviewed, summarized and presented in a unified framework. Modifications of standard models are described in detail in order to be applied in genetic association studies. The model based on summary data is uniformly defined for both discrete and continuous outcomes and analytical expressions for the covariance of the two jointly modeled outcomes are derived for both cases. The models based on the binary nature of the data are fitted using both prospective and retrospective likelihood. Furthermore, formal tests for assessing the genetic model of inheritance are developed based on standard normal theory. The general model is compared to the recently proposed genetic model-free bivariate approach (either using summary or binary data), and it is clearly shown that the estimates provided by this approach are nearly identical to the estimates derived by the general bivariate model using the aforementioned tests for the genetic model. The methods developed here as well as the tests, are easily implemented in all major statistical packages, escaping the need of self written software. The methods are applied in several already published meta-analyses of genetic association studies (with both discrete and continuous outcomes) and the results are compared against the widely used univariate approach as well as against the genetic model free approaches. Illustrative examples of code in Stata are given in the appendix. It is anticipated that the methods developed in this work will be widely applied in the meta-analysis of genetic association studies.     

A generic protein purification method for protein complex characterization and proteome exploration.

We have developed a generic procedure to purify proteins expressed at their natural level under native conditions using a novel tandem affinity purification (TAP) tag. The TAP tag allows the rapid purification of complexes from a relatively small number of cells without prior knowledge of the complex composition, activity, or function. Combined with mass spectrometry, the TAP strategy allows for the identification of proteins interacting with a given target protein. The TAP method has been tested in yeast but should be applicable to other cells or organisms.     

一种新的蛋白质结构类预测方法

基于氨基酸的16种分类模型,给出蛋白质序列的派生序列,进而结合加权拟熵和LZ复杂度构造出34维特征向量来表示蛋白质序列。借助于贝叶斯分类器对同源性不超过25%的640数据集进行蛋白质结构类预测,准确度达到71.28%。     

A complexity-based method for predicting protein subcellular location

A complexity-based approach is proposed to predict subcellular location of proteins. Instead of extracting features from protein sequences as done previously, our approach is based on a complexity decomposition of symbol sequences. In the first step, distance between each pair of protein sequences is evaluated by the conditional complexity of one sequence given the other. Subcellular location of a protein is then determined using the k-nearest neighbor algorithm. Using three widely used data sets created by Reinhardt and Hubbard, Park and Kanehisa, and Gardy et al., our approach shows an improvement in prediction accuracy over those based on the amino acid composition and Markov model of protein sequences.     

A computational method for NMR-constrained protein threading.

Protein threading provides an effective method for fold recognition and backbone structure prediction. But its application is currently limited due to its level of prediction accuracy and scope of applicability. One way to significantly improve its usefulness is through the incorporation of underconstrained (or partial) NMR data. It is well known that the NMR method for protein structure determination applies only to small proteins and that its effectiveness decreases rapidly as the protein mass increases beyond about 30 kD. We present, in this paper, a computational framework for applying underconstrained NMR data (that alone are insufficient for structure determination) as constraints in protein threading and also in all-atom model construction. In this study, we consider both secondary structure assignments from chemical shifts and NOE distance restraints. Our results have shown that both secondary structure assignments and a small number of long-range NOEs can significantly improve the threading quality in both fold recognition and threading-alignment accuracy, and can possibly extend threading's scope of applicability from homologs to analogs. An accurate backbone structure generated by NMR-constrained threading can then provide a great amount of structural information, equivalent to that provided by many NMR data; and hence can help reduce the number of NMR data typically required for an accurate structure determination. This new technique can potentially accelerate current NMR structure determination processes and possibly expand NMR's capability to larger proteins.     

A novel method for sampling alpha-helical protein backbones

We present a novel technique of sampling the configurations of helical proteins. Assuming knowledge of native secondary structure, we employ assembly rules gathered from a database of existing structures to enumerate the geometrically possible three-dimensional arrangements of the constituent helices. We produce a library of possible folds for 25 helical protein cores. In each case, our method finds significant numbers of conformations close to the native structure. In addition, we assign coordinates to all atoms for four of the 25 proteins and show that this has a small effect on the number of near-native conformations. In the context of database driven exhaustive enumeration our method performs extremely well, yielding significant percentages of conformations (between 0.02% and 82%) within 6 A of the native structure. The method's speed and efficiency make it a valuable tool for predicting protein structure.     

A pentapeptide-based method for protein secondary structure prediction

We present a new method for protein secondary structure prediction, based on the recognition of well-defined pentapeptides, in a large databank. Using a databank of 635 protein chains, we obtained a success rate of 68.6%. We show that progress is achieved when the databank is enlarged, when the 20 amino acids are adequately grouped in 10 sets and when more pentapeptides are attributed one of the defined conformations, alpha-helices or beta-strands. The analysis of the model indicates that the essential variable is the number of pentapeptides of well-defined structure in the database. Our model is simple, does not rely on arbitrary parameters and allows the analysis in detail of the results of each chosen hypothesis.     

A method for predicting protein structure from sequence

BACKGROUND: The ability to predict the native conformation of a globular protein from its amino-acid sequence is an important unsolved problem of molecular biology. We have previously reported a method in which reduced representations of proteins are folded on a lattice by Monte Carlo simulation, using statistically-derived potentials. When applied to sequences designed to fold into four-helix bundles, this method generated predicted conformations closely resembling the real ones. RESULTS: We now report a hierarchical approach to protein-structure prediction, in which two cycles of the above-mentioned lattice method (the second on a finer lattice) are followed by a full-atom molecular dynamics simulation. The end product of the simulations is thus a full-atom representation of the predicted structure. The application of this procedure to the 60 residue, B domain of staphylococcal protein A predicts a three-helix bundle with a backbone root mean square (rms) deviation of 2.25-3 A from the experimentally determined structure. Further application to a designed, 120 residue monomeric protein, mROP, based on the dimeric ROP protein of Escherichia coli, predicts a left turning, four-helix bundle native state. Although the ultimate assessment of the quality of this prediction awaits the experimental determination of the mROP structure, a comparison of this structure with the set of equivalent residues in the ROP dime- crystal structure indicates that they have a rms deviation of approximately 3.6-4.2 A. CONCLUSION: Thus, for a set of helical proteins that have simple native topologies, the native folds of the proteins can be predicted with reasonable accuracy from their sequences alone. Our approach suggest a direction for future work addressing the protein-folding problem.     

A simple method for constructing a tagged protein

We have developed a simple method for preparing a tagged protein by PCR. With this method any protein sequence can be easily tagged. The techniques include three steps of DNA restriction, ligation and PCR. We could obtain a DNA construct containing SUMO-1 gene with His6 tag sequence with high efficiency by the next day.