Quantitative parameters for amino acid-base interaction: implications for prediction of protein-DNA binding sites.

Inspection of the amino acid-base interactions in protein-DNA complexes is essential to the understanding of specific recognition of DNA target sites by regulatory proteins. The accumulation of information on protein-DNA co-crystals challenges the derivation of quantitative parameters for amino acid-base interaction based on these data. Here we use the coordinates of 53 solved protein-DNA complexes to extract all non-homologous pairs of amino acid-base that are in close contact, including hydrogen bonds and hydrophobic interactions. By comparing the frequency distribution of the different pairs to a theoretical distribution and calculating the log odds, a quantitative measure that expresses the likelihood of interaction for each pair of amino acid-base could be extracted. A score that reflects the compatibility between a protein and its DNA target can be calculated by summing up the individual measures of the pairs of amino acid-base involved in the complex, assuming additivity in their contributions to binding. This score enables ranking of different DNA binding sites given a protein binding site and vice versa and can be used in molecular design protocols. We demonstrate its validity by comparing the predictions using this score with experimental binding results of sequence variants of zif268 zinc fingers and their DNA binding sites.     

Text mining improves prediction of protein functional sites

We present an approach that integrates protein structure analysis and text mining for protein functional site prediction, called LEAP-FS (Literature Enhanced Automated Prediction of Functional Sites). The structure analysis was carried out using Dynamics Perturbation Analysis (DPA), which predicts functional sites at control points where interactions greatly perturb protein vibrations. The text mining extracts mentions of residues in the literature, and predicts that residues mentioned are functionally important. We assessed the significance of each of these methods by analyzing their performance in finding known functional sites (specifically, small-molecule binding sites and catalytic sites) in about 100,000 publicly available protein structures. The DPA predictions recapitulated many of the functional site annotations and preferentially recovered binding sites annotated as biologically relevant vs. those annotated as potentially spurious. The text-based predictions were also substantially supported by the functional site annotations: compared to other residues, residues mentioned in text were roughly six times more likely to be found in a functional site. The overlap of predictions with annotations improved when the text-based and structure-based methods agreed. Our analysis also yielded new high-quality predictions of many functional site residues that were not catalogued in the curated data sources we inspected. We conclude that both DPA and text mining independently provide valuable high-throughput protein functional site predictions, and that integrating the two methods using LEAP-FS further improves the quality of these predictions.     

Selective prediction of interaction sites in protein structures with THEMATICS

Background  

Methods are now available for the prediction of interaction sites in protein 3D structures. While many of these methods report high success rates for site prediction, often these predictions are not very selective and have low precision. Precision in site prediction is addressed using Theoretical Microscopic Titration Curves (THEMATICS), a simple computational method for the identification of active sites in enzymes. Recall and precision are measured and compared with other methods for the prediction of catalytic sites.     

Fast dynamics perturbation analysis for prediction of protein functional sites

Background  

We present a fast version of the dynamics perturbation analysis (DPA) algorithm to predict functional sites in protein structures. The original DPA algorithm finds regions in proteins where interactions cause a large change in the protein conformational distribution, as measured using the relative entropy D _x . Such regions are associated with functional sites.     

Exploiting structural and topological information to improve prediction of RNA-protein binding sites

Background  

RNA-protein interactions are important for a wide range of biological processes. Current computational methods to predict interacting residues in RNA-protein interfaces predominately rely on sequence data. It is, however, known that interface residue propensity is closely correlated with structural properties. In this paper we systematically study information obtained from sequences and structures and compare their contributions in this prediction problem. Particularly, different geometrical and network topological properties of protein structures are evaluated to improve interface residue prediction accuracy.     

The use of functional domains to improve transmembrane protein topology prediction

Transmembrane proteins affect vital cellular functions and pathogenesis, and are a focus of drug design. It is difficult to obtain diffraction quality crystals to study transmembrane protein structure. Computational tools for transmembrane protein topology prediction fill in the gap between the abundance of transmembrane proteins and the scarcity of known membrane protein structures. Their prediction accuracy is still inadequate: TMHMM, the current state-of-the-art method, has less than 52% accuracy in topology prediction on one set of transmembrane proteins of known topology. Based on the observation that there are functional domains that occur preferentially internal or external to the membrane, we have extended the model of TMHMM to incorporate functional domains, using a probabilistic approach originally developed for computational gene finding. Our extension is better than TMHMM in predicting the topology of transmembrane proteins. As prediction of functional domain improves, our system's prediction accuracy will likely improve as well.     

Computational method for prediction of protein functional sites using specificity determinants

The current available data on protein sequences largely exceeds the experimental capabilities to annotate their function. So annotation in silico, i.e. using computational methods becomes increasingly important. This annotation is inevitably a prediction, but it can be an important starting point for further experimental studies. Here we present a method for prediction of protein functional sites, SDPsite, based on the identification of protein specificity determinants. Taking as an input a protein sequence alignment and a phylogenetic tree, the algorithm predicts conserved positions and specificity determinants, maps them onto the protein's 3D structure, and searches for clusters of the predicted positions. Comparison of the obtained predictions with experimental data and data on performance of several other methods for prediction of functional sites reveals that SDPsite agrees well with the experiment and outperforms most of the previously available methods. SDPsite is publicly available under http://bioinf.fbb.msu.ru/SDPsite.     

Ultrasound-based subject-specific parameters improve fascicle behaviour estimation in Hill-type muscle model

The estimation of muscle fascicle behaviour is decisive in a Hill-type model as they are related to muscle force by the force–length–velocity relationship and the tendon force–strain relationship. This study was aimed at investigating the influence of subject-specific tendon force–strain relationship and initial fascicle geometry (IFG) on the estimation of muscle forces and fascicle behaviour during isometric contractions. Ultrasonography was used to estimate the in vivo muscle fascicle behaviour and compare the muscle fascicle length and pennation angle estimated from the Hill-type model. The calibration–prediction process of the electromyography-driven model was performed using generic or subject-specific tendon definition with or without IFG as constraint. The combination of subject-specific tendon definition and IFG led to muscle fascicle behaviour closer to ultrasound data and significant lower forces of the ankle dorsiflexor and plantarflexor muscles compared to the other conditions. Thus, subject-specific ultrasound measurements improve the accuracy of Hill-type models on muscle fascicle behaviour.     

Equilibrium studies of the cyclic AMP receptor protein-DNA interaction

The binding of the Escherichia coli cyclic AMP receptor protein (CAP) to restriction fragments containing the lac promoter-operator region has been investigated as a function of cAMP concentration, using a sensitive gel electrophoresis assay. Under standard conditions (13 mM ionic strength), the equilibrium constant for CAP binding to its primary site on a 203 base-pair lac promoter fragment is 6.3 X 10(8) M-1 at 0.2 microM-cAMP, and increases to 8.4 X 10(10) M-1 at 5.0 microM-cAMP. The latter is about 10(5) times larger than the equilibrium constant for binding to an isolated, non-specific site. The L8 mutation, which renders the lac promoter unresponsive to CAP in vivo, lowers this binding affinity by five- to tenfold. Analysis of the cAMP dependency of binding over the concentration range of 0.2 microM to 10 microM reveals that uptake of a single equivalent of cAMP is required for site-specific binding. Similarly, the transfer of CAP from a non-specific DNA site to a specific site requires the net uptake of a single molecule of cAMP. In contrast, co-operative non-specific binding to DNA was found to be independent of cAMP concentration with an equilibrium binding constant of 6 X 10(6) M-1. We conclude that the cAMP affinity of the two CAP subunits in the specific promoter complex is not equal, and that the complex structure therefore deviates significantly from twofold symmetry. A model for the regulation of the lac promoter by the intracellular cAMP concentration is proposed on the basis of the equilibrium binding results.     

Detection of protein-DNA interaction and regulation using gold nanoparticles

The interaction between protein and DNA is usually regulated by a third species, an effector, which can be either a protein or a small molecule. Convenient methods capable of detecting protein-DNA interaction and its regulation are highly desirable research tools. In the current study, we developed a method to directly “visualize” the interaction between a protein-DNA pair and its effector through the coupling with gold nanoparticles (AuNPs). As a proof-of-concept experiment, we constructed a model system based on the interaction between the lac repressor (protein) and operator (DNA) and its interplay with the lac operon inducer isopropyl β-d-1-thiogalactopyranoside (IPTG, which inhibits the interaction between the lac repressor and operator). We coated AuNPs with the lac operator sequences and mixed them with the lac repressor. Because the lac repressor homotetramer contains two DNA binding modules, it bridged the particles and caused them to aggregate. We demonstrated that the assembly of DNA-modified AuNPs correlated with the presence of the corresponding protein and effector in a concentration-dependent manner. This AuNP-based platform has the potential to be generalized in the creation of reporter and detection systems for other interacting protein-DNA pairs and their effectors.     

A wide repertoire of miRNA binding sites: prediction and functional implications

MOTIVATION: Over the past decade, deciphering the roles of microRNAs (miRNAs) has relied heavily upon the identification of their targets. Most of the targets that were computationally and experimentally characterized were evolutionarily conserved 'seed' targets, containing a perfect 6-8 nt match between the miRNA 5(')-region and the messenger RNA (mRNA). Gradually, it has become evident that other types of miRNA binding can confer target regulation, but their characterization has been lagging behind. RESULTS: Here, we complement the putative evolutionarily-conserved seed-containing targets by a wide repertoire of putative targets exhibiting a variety of miRNA binding patterns, predicted by our algorithm RepTar. These include non-conserved sites, 'seed' binding sites with G:U-wobbles within the seed, '3(') compensatory' sites and 'centered' sites. Apart from the centered sites, we demonstrate the functionality of these sites and characterize the target profile of a miRNA by the types of binding sites predicted in its target 3(') UTRs. We find that different miRNAs have individual target profiles, with some more inclined to seed binding and others more inclined to binding through 3(') compensatory sites. This diversity in targeting patterns is also evident within several miRNA families (defined by common seed sequences), leading to divergence in the target sets of members of the same family. The prediction of non-conventional miRNA targets is also beneficial in the search for targets of the non-conserved viral miRNAs. Analyzing the cellular targets of viral miRNAs, we show that viral miRNAs use various binding patterns to exploit cellular miRNA binding sites and suggest roles for these targets in virus-host interactions.     

Topography of simian virus 40 A protein-DNA complexes: arrangement of pentanucleotide interaction sites at the origin of replication.

Investigation of the DNA binding properties of the simian virus 40 (SV40) A protein (large T antigen) and the hybrid adenovirus-SV40 D2 protein revealed that both viral proteins protect similar regions of SV40 DNA from digestion by DNase I or methylation by dimethyl sulfate. However, the interaction of D2 protein with DNA was more sensitive to increases of NaCl concentration than was the interaction of wild-type SV40 A protein. Dimethylsulfate footprinting identified 13 DNA pentanucleotide contact sites at the viral origin of replication. The sequences of these sites corresponded to the consensus family 5'-(G greater than T) (A greater than G)GGC-3'. The pentanucleotides were distributed in three regions of origin DNA. Region I contained three pentanucleotide contact sites arranged as direct repetitions encompassing a span of 23 base pairs. In region II, four pentanucleotides were oriented as inverted repetitions that also spanned a total of 23 base pairs. Region III had six recognition pentanucleotides arranged as direct repetitions in a space of 59 base pairs. These fundamental variations in DNA arrangement are likely to determine different patterns of protein binding in each region.     

Identification of two interaction sites in SecY that are important for the functional interaction with SecA

The motor protein SecA drives the translocation of (pre-)proteins across the SecYEG channel in the bacterial cytoplasmic membrane by nucleotide-dependent cycles of conformational changes often referred to as membrane insertion/de-insertion. Despite structural data on SecA and an archaeal homolog of SecYEG, the identity of the sites of interaction between SecA and SecYEG are unknown. Here, we show that SecA can be cross-linked to several residues in cytoplasmic loop 5 (C5) of SecY, and that SecA directly interacts with a part of transmembrane segment 4 (TMS4) of SecY that is buried in the membrane region of SecYEG. Mutagenesis of either the conserved Arg357 in C5 or Glu176 in TMS4 interferes with the catalytic activity of SecA but not with binding of SecA to SecYEG. Our data explain how conformational changes in SecA could be directly coupled to the previously proposed opening mechanism of the SecYEG channel.     

A method for detecting protein-DNA interactions at sites of chromatin replication

Two versions of an approach to identify DNA-protein interactions at sites of DNA replication in HeLa cell nuclei are described. In this procedure, newly replicated DNA chains are first labeled and photosensitized in vitro by the incorporation of [α-³²P]dCTP and bromodeoxyuridine triphosphate, respectively. Irradiation with ultraviolet light is then used to covalently crosslink the proteins that are adjacent to the photosensitized and isotopically labeled strands of newly replicated DNA. After the bulk of the DNA is digested with nucleases, the crosslinked proteins—marked by short covalently linked radioactive DNA tags—are fractionated by electrophoresis in sodium dodecyl sulfate-polyacrylamide gels and detected by autoradiography. With this technology, certain proteins have been shown to associate selectively with newly replicated DNA. The method appears adaptable for application to a variety of problems involving DNA-protein association.     

捕食性昆虫的功能反应方程及其参数的估算

昆虫的功能反应是指每头捕食者在一定时间内的捕食量对猎物密度变化的反应。该文介绍描述这种功能反应的圆盘方程 (Ⅱ型 )的由来及其参数的估测方法 ,简要讨论了影响昆虫功能反应测定的主要因素。