Optimizing substitution matrices by separating score distributions

MOTIVATION: Homology search is one of the most fundamental tools in Bioinformatics. Typical alignment algorithms use substitution matrices and gap costs. Thus, the improvement of substitution matrices increases accuracy of homology searches. Generally, substitution matrices are derived from aligned sequences whose relationships are known, and gap costs are determined by trial and error. To discriminate relationships more clearly, we are encouraged to optimize the substitution matrices from statistical viewpoints using both positive and negative examples utilizing Bayesian decision theory. RESULTS: Using Cluster of Orthologous Group (COG) database, we optimized substitution matrices. The classification accuracy of the obtained matrix is better than that of conventional substitution matrices to COG database. It also achieves good performance in classifying with other databases.     

A periplasmic fluorescent reporter protein and its application in high-throughput membrane protein topology analysis

We have developed a periplasmic fluorescent reporter protein suitable for high-throughput membrane protein topology analysis in Escherichia coli. The reporter protein consists of a single chain (scFv) antibody fragment that binds to a fluorescent hapten conjugate with high affinity. Fusion of the scFv to membrane protein sites that are normally exposed in the periplasmic space tethers the scFv onto the inner membrane. Following permealization of the outer membrane to allow diffusion of the fluorescent hapten into the periplasm, binding to the anchored scFv renders the cells fluorescent. We show that cell fluorescence is an accurate and sensitive reporter of the location of residues within periplasmic loops. For topological analysis, a set of nested deletions in the membrane protein gene is employed to construct two libraries of gene fusions, one to the scFvand one to the cytoplasmic reporter green fluorescent protein (GFP). Fluorescent clones are isolated by flow cytometry and the sequence of the fusion junctions is determined to identify amino acid residues within periplasmic and cytoplasmic loops, respectively. We applied this methodology to the topology analysis of E. coli TatC protein for which previous studies had led to conflicting results. The ease of screening libraries of fusions by flow cytometry enabled the rapid identification of almost 90 highly fluorescent scFv and GFP fusions, which, in turn, allowed the fine mapping of TatC membrane topology.     

Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes

We describe and validate a new membrane protein topology prediction method, TMHMM, based on a hidden Markov model. We present a detailed analysis of TMHMM's performance, and show that it correctly predicts 97-98 % of the transmembrane helices. Additionally, TMHMM can discriminate between soluble and membrane proteins with both specificity and sensitivity better than 99 %, although the accuracy drops when signal peptides are present. This high degree of accuracy allowed us to predict reliably integral membrane proteins in a large collection of genomes. Based on these predictions, we estimate that 20-30 % of all genes in most genomes encode membrane proteins, which is in agreement with previous estimates. We further discovered that proteins with N(in)-C(in) topologies are strongly preferred in all examined organisms, except Caenorhabditis elegans, where the large number of 7TM receptors increases the counts for N(out)-C(in) topologies. We discuss the possible relevance of this finding for our understanding of membrane protein assembly mechanisms. A TMHMM prediction service is available at http://www.cbs.dtu.dk/services/TMHMM/.     

Determination of membrane protein topology by red-edge excitation shift analysis: application to the membrane-bound colicin E1 channel peptide

A new approach for the determination of the bilayer location of Trp residues in proteins has been applied to the study of the membrane topology of the channel-forming bacteriocin, colicin E1. This method, red-edge excitation shift (REES) analysis, was initially applied to the study of 12 single Trp-containing channel peptides of colicin E1 in the soluble state in aqueous medium. Notably, REES was observed for most of the channel peptides in aqueous solution upon low pH activation. The extent of REES was subsequently characterized using a model membrane system composed of the tripeptide, Lys-Trp-Lys, bound to dimyristoyl-sn-glycerol-3-phosphatidylserine liposomes. Subsequently, data accrued from the model peptide-lipid system was used to interpret information obtained on the channel peptides when bound to dioleoyl-sn-glycerol-3-phosphatidylcholine/dioleoyl-sn-glycerol-3-phosphatidylglycerol membrane vesicles. The single Trp mutant peptides were divided into three categories based on the change in the REES values observed for the Trp residues when the peptides were bound to liposomes as compared to the REES values measured for the soluble peptides. F-404 W, F-413 W, F-443 W, F-484 W, and W-495 peptides exhibited small and/or insignificant REES changes (Delta REES) whereas W-424, F-431 W, and Y-507 W channel peptides possessed modest REES changes (3 nm< or = Delta REES< or = 7 nm). In contrast, wild-type, Y-367 W, W-460, Y-478 W, and I-499 W channel peptides showed large Delta REES values upon membrane binding (7 nm< Delta REES< or =12 nm). The REES data for the membrane-bound structure of the colicin E1 channel peptide proved consistent with previous data for the topology of the closed channel state, which lends further credence to the currently proposed channel model. In conclusion, the REES method provides another source of topological data for assignment of the bilayer location for Trp residues within membrane-associated proteins; however, it also requires careful interpretation of spectral data in combination with structural information on the proteins being investigated.     

Determination of membrane protein topology by red-edge excitation shift analysis: application to the membrane-bound colicin E1 channel peptide

A new approach for the determination of the bilayer location of Trp residues in proteins has been applied to the study of the membrane topology of the channel-forming bacteriocin, colicin E1. This method, red-edge excitation shift (REES) analysis, was initially applied to the study of 12 single Trp-containing channel peptides of colicin E1 in the soluble state in aqueous medium. Notably, REES was observed for most of the channel peptides in aqueous solution upon low pH activation. The extent of REES was subsequently characterized using a model membrane system composed of the tripeptide, Lys-Trp-Lys, bound to dimyristoyl-sn-glycerol-3-phosphatidylserine liposomes. Subsequently, data accrued from the model peptide-lipid system was used to interpret information obtained on the channel peptides when bound to dioleoyl-sn-glycerol-3-phosphatidylcholine/dioleoyl-sn-glycerol-3-phosphatidylglycerol membrane vesicles. The single Trp mutant peptides were divided into three categories based on the change in the REES values observed for the Trp residues when the peptides were bound to liposomes as compared to the REES values measured for the soluble peptides. F-404W, F-413W, F-443W, F-484W, and W-495 peptides exhibited small and/or insignificant REES changes (ΔREES) whereas W-424, F-431W, and Y-507W channel peptides possessed modest REES changes (3 nm≤ΔREES≤7 nm). In contrast, wild-type, Y-367W, W-460, Y-478W, and I-499W channel peptides showed large ΔREES values upon membrane binding (7 nm<ΔREES≤12 nm). The REES data for the membrane-bound structure of the colicin E1 channel peptide proved consistent with previous data for the topology of the closed channel state, which lends further credence to the currently proposed channel model. In conclusion, the REES method provides another source of topological data for assignment of the bilayer location for Trp residues within membrane-associated proteins; however, it also requires careful interpretation of spectral data in combination with structural information on the proteins being investigated.     

Decoy models for protein structure comparison score normalisation

A method is described to construct sets of decoy models that can be used to generate a background score distribution for protein structure comparison. The models are derived directly from the two proteins being compared and retain all the essential properties of the structures, including length, density, shape and secondary structure composition but have different folds. As each comparison involves a pair of proteins of the same length, no explicit normalisation is required to adjust for the length of the proteins being compared. This allows substructure (or domain) matches to score almost equally to the comparison of isolated domains. A normalised probability measure was derived that allows joint family/family comparison. The method was applied to some of the CASP6 models for targets with new folds.     

Graph models of oncogenesis with an application to melanoma

We describe several analytical techniques for use in developing genetic models of oncogenesis including: methods for the selection of important genetic events, construction of graph models (including distance-based trees, branching trees, contingency trees and directed acyclic graph models) from these events and methods for interpretation of the resulting models. The models can be used to make predictions about: which genetic events tend to occur early, which events tend to occur together and the likely order of events. Unlike simple path models of oncogenesis, our models allow dependencies to exist between specific genetic changes and allow for multiple, divergent paths in tumor progression. A variety of genetic events can be used with the graph models including chromosome breaks, losses or gains of large DNA regions, small mutations and changes in methylation. As an application of the techniques, we use a recently published cytogenetic analysis of 206 melanoma cases [Nelson et al. (2000), Cancer Genet. Cytogenet.122, 101-109] to derive graph models for chromosome breaks in melanoma. Among our predictions are: (1) breaks in 6q1 and 1q1 are early events, with 6q1 preferentially occurring first and increasing the probability of a break in 1q1 and (2) breaks in the two sets [1p1, 1p2, 9q1] and [1q1, 7p2, 9p2] tend to occur together. This study illustrates that the application of graph models to genetic data from tumor sets provide new information on the interrelationships among genetic changes during tumor progression.     

The ups and downs of protein topology; rapid comparison of protein structure

Protein topology can be described at different levels. At the most fundamental level, it is a sequence of secondary structure elements (a "primary topology string"). Searching predicted primary topology strings against a library of strings from known protein structures is the basis of some protein fold recognition methods. Here a method known as TOPSCAN is presented for rapid comparison of protein structures. Rather than a simple two-letter alphabet (encoding strand and helix), more complex alphabets are used encoding direction, proximity, accessibility and length of secondary elements and loops in addition to secondary structure. Comparisons are made between the structural information content of primary topology strings and encodings which contain additional information ("secondary topology strings"). The algorithm is extremely fast, with a scan of a large domain against a library of more than 2000 secondary structure strings completing in approximately 30 s. Analysis of protein fold similarity using TOPSCAN at primary and secondary topology levels is presented.     

Graph analysis of functional brain network topology using minimum spanning tree in driver drowsiness

A large number of traffic accidents due to driver drowsiness have been under more attention of many countries. The organization of the functional brain network is associated with drowsiness, but little is known about the brain network topology that is modulated by drowsiness. To clarify this problem, in this study, we introduce a novel approach to detect driver drowsiness. Electroencephalogram (EEG) signals have been measured during a simulated driving task, in which participants are recruited to undergo both alert and drowsy states. The filtered EEG signals are then decomposed into multiple frequency bands by wavelet packet transform. Functional connectivity between all pairs of channels for multiple frequency bands is assessed using the phase lag index (PLI). Based on this, PLI-weighted networks are subsequently calculated, from which minimum spanning trees are constructed—a graph method that corrects for comparison bias. Statistical analyses are performed on graph-derived metrics as well as on the PLI connectivity values. The major finding is that significant differences in the delta frequency band for three graph metrics and in the theta frequency band for five graph metrics suggesting network integration and communication between network nodes are increased from alertness to drowsiness. Together, our findings also suggest a more line-like configuration in alert states and a more star-like topology in drowsy states. Collectively, our findings point to a more proficient configuration in drowsy state for lower frequency bands. Graph metrics relate to the intrinsic organization of functional brain networks, and these graph metrics may provide additional insights on driver drowsiness detection for reducing and preventing traffic accidents and further understanding the neural mechanisms of driver drowsiness.     

IMPALA: matching a protein sequence against a collection of PSI-BLAST-constructed position-specific score matrices

MOTIVATION: Many studies have shown that database searches using position-specific score matrices (PSSMs) or profiles as queries are more effective at identifying distant protein relationships than are searches that use simple sequences as queries. One popular program for constructing a PSSM and comparing it with a database of sequences is Position-Specific Iterated BLAST (PSI-BLAST). RESULTS: This paper describes a new software package, IMPALA, designed for the complementary procedure of comparing a single query sequence with a database of PSI-BLAST-generated PSSMs. We illustrate the use of IMPALA to search a database of PSSMs for protein folds, and one for protein domains involved in signal transduction. IMPALA's sensitivity to distant biological relationships is very similar to that of PSI-BLAST. However, IMPALA employs a more refined analysis of statistical significance and, unlike PSI-BLAST, guarantees the output of the optimal local alignment by using the rigorous Smith-Waterman algorithm. Also, it is considerably faster when run with a large database of PSSMs than is BLAST or PSI-BLAST when run against the complete non-redundant protein database.     

Transmembrane protein topology mapping by the substituted cysteine accessibility method (SCAM(TM)): application to lipid-specific membrane protein topogenesis

We provide an overview of lipid-dependent polytopic membrane protein topogenesis, with particular emphasis on Escherichia coli strains genetically altered in their lipid composition and strategies for experimentally determining the transmembrane organization of proteins. A variety of reagents and experimental strategies are described including the use of lipid mutants and thiol-specific chemical reagents to study lipid-dependent and host-specific membrane protein topogenesis by substituted cysteine site-directed chemical labeling. Employing strains in which lipid composition can be controlled temporally during membrane protein synthesis and assembly provides a means to observe dynamic changes in protein topology as a function of membrane lipid composition.     

Gene fusion analysis of membrane protein topology: a direct comparison of alkaline phosphatase and beta-lactamase fusions.

To compare two approaches to analyzing membrane protein topology, a number of alkaline phosphatase fusions to membrane proteins were converted to beta-lactamase fusions. While some alkaline phosphatase fusions near the N terminus of cytoplasmic loops of membrane proteins have anomalously high levels of activity, the equivalent beta-lactamase fusions do not. This disparity may reflect differences in the folding of beta-lactamase and alkaline phosphatase in the cytoplasm.     

The distribution of lipid attached spin probes in bilayers: application to membrane protein topology

The distribution of the lipid-attached doxyl electron paramagnetic resonance (EPR) spin label in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes has been studied by (1)H and (13)C magic angle spinning nuclear magnetic resonance relaxation measurements. The doxyl spin label was covalently attached to the 5th, 10th, and 16th carbons of the sn-2 stearic acid chain of a 1-palmitoyl-2-stearoyl-(5/10/16-doxyl)-sn-glycero-3-phosphocholine analog. Due to the unpaired electron of the spin label, (1)H and (13)C lipid relaxation rates are enhanced by paramagnetic relaxation. For all lipid segments the influence of paramagnetic relaxation is observed even at low probe concentrations. Paramagnetic relaxation rates provide a measure for the interaction strength between lipid segments and the doxyl group. Plotted along the membrane director a transverse distribution profile of the EPR probe is obtained. The chain-attached spin labels are broadly distributed in the membrane with a maximum at the approximate chain position of the probe. Both (1)H and (13)C relaxation measurements show these broad distributions of the doxyl group in the membrane indicating that (1)H spin diffusion does not influence the relaxation measurements. The broad distributions of the EPR label result from the high degree of mobility and structural heterogeneity in liquid-crystalline membranes. Knowing the distribution profiles of the EPR probes, their influence on relaxation behavior of membrane inserted peptide and protein segments can be studied by (13)C magic angle spinning nuclear magnetic resonance. As an example, the location of Ala residues positioned at three sites of the transmembrane WALP-16 peptide was investigated. All three doxyl-labeled phospholipid analogs induce paramagnetic relaxation of the respective Ala site. However, for well ordered secondary structures the strongest relaxation enhancement is observed for that doxyl group in the closest proximity to the respective Ala. Thus, this approach allows study of membrane insertion of protein segments with respect to the high molecular mobility in liquid-crystalline membranes.     

Transmembrane Nox4 topology revealed by topological determination by Ubiquitin Fusion Assay,a novel method to uncover membrane protein topology

The NADPH oxidase Nox4 is a multi-pass membrane protein responsible for the generation of reactive oxygen species that are implicated in cellular signaling but may also cause pathological situations when dysregulated. Although topological organization of integral membrane protein dictates its function, only limited experimental data describing Nox4's topology are available.To provide deeper insight on Nox4 structural organization, we developed a novel method to determinate membrane protein topology in their cellular environment, named Topological Determination by Ubiquitin Fusion Assay (ToDUFA). It is based on the proteolytic capacity of the deubiquitinase enzymes to process ubiquitin fusion proteins. This straightforward method, validated on two well-known protein's topologies (IL1RI and Nox2), allowed us to discriminate rapidly the topological orientation of protein's domains facing either the nucleocytosolic or the exterior/luminal compartments. Using this method, we were able for the first time to determine experimentally the topology of Nox4 which consists of 6 transmembrane domains with its N- and C-terminus moieties facing the cytosol. While the first, third and fifth loops of Nox4 protein are extracellular; the second and fourth loops are located in the cytosolic side. This approach can be easily extended to characterize the topology of all others members of the NADPH oxidase family or any multi-pass membrane proteins.Considering the importance of protein topology knowledge in cell biology research and pharmacological development, we believe that this novel method will represent a widely useful technique to easily uncover complex membrane protein's topology.     

Identification of transmembrane protein functions by binary topology patterns

We propose a novel method for identifying and classifying the functions of transmembrane (TM) proteins based on their TM topology [the number of TM segments (tms), the loop length and the N-terminus location]. In this method, the TM topology is expressed as a string of '0' and '1', and this is designated the binary topology pattern (BTP). We focused on TM proteins with up to 12 tms, with the exception of 1 and 9 tms, and classified them into 37 functional groups by the number of tms and the functional annotation. These grouped TM protein sequences were used to determine BTPs which are specific to the individual functional groups. Since the evaluated accuracies (sensitivity, specificity and self-consistency) of these patterns in functional identification were quite high overall, i.e. 0.940, 0.934 and 0.935, respectively, as averaged over the 37 functional groups, we confirmed that TM protein function can be identified by the number of tms and the characteristics of loop lengths, i.e. BTPs.     

Bayesian phylogenetic analysis reveals two-domain topology of S-adenosylhomocysteine hydrolase protein sequences

S-Adenosylhomocysteine hydrolase (SahH) is involved in the degradation of the compound which inhibits methylation reactions. Using a Bayesian approach and other methods, we reconstructed a phylogenetic tree of amino acid sequences of this protein originating from all three major domains of living organisms. The SahH sequences formed two major branches: one composed mainly of Archaea and the other of eukaryotes and majority of bacteria, clearly contradicting the three-domain topology shown by small subunit rRNA gene. This topology suggests the occurrence of lateral transfer of this gene between the domains. Poor resolution of eukaryotes and bacteria excluded an ultimate conclusion in which out of the two domains this gene appeared first, however, the congruence of the secondary branches with SS rRNA and/or concatenated ribosomal protein datasets phylogenies suggested an "early" acquisition by some bacterial and eukaryotic phyla. Similarly, the branching pattern of Archaea reflected the phylogenies shown by SS rRNA and ribosomal proteins. SahH is widespread in Eucarya, albeit, due to reductive evolution, it is missing in the intracellular parasite Encephalitozoon cuniculi. On the other hand, the lack of affinity to the sequences from the alpha-Proteobacteria and cyanobacteria excludes a possibility of its acquisition in the course of mitochondrial or chloroplast endosymbioses. Unlike Archaea, most bacteria carry MTA/SAH nucleosidase, an enzyme involved also in metabolism of methylthioadenosine. However, the double function of MTA/SAH nucleosidase may be a barrier to ensure the efficient degradation of S-adenosylhomocysteine, specially when the intensity of methylation processes is high. This would explain the presence of S-adenosylhomocysteine hydrolase in the bacteria that have more complex metabolism. On the other hand, majority of obligate pathogenic bacteria due to simpler metabolism rely entirely on MTA/SAH nucleosidase. This could explain the observed phenetic pattern in which bacteria with larger (>6 Mb-million base pairs) genomes carry SAH hydrolase, whereas bacteria that have undergone reductive evolution usually carry MTA/SAH nucleosidase. This suggests that the presence or acquisition of S-adenosylhomocysteine hydrolase in bacteria may predispose towards higher metabolic, and in consequence, higher genomic complexity. The good examples are the phototrophic bacteria all of which carry this gene, however, the SahH phylogeny shows lack of congruence with SSU rRNA and photosyntethic genes, implying that the acquisition was independent and presumably preceded the acquisition of photosyntethic genes. The majority of cyanobacteria acquired this gene from Archaea, however, in some species the sahH gene was replaced by a copy from the beta- or gamma-Proteobacteria.     

Graph theoretical analysis of the phytosociological structure of plant communities: An application to mixed forest

Summary A sampling method, compatible with the theory elaborated in the previous paper, was used to investigate the mixed-forest community of a woodlot in Southern Ontario. The results provide an illustration of the graph theoretical methods developed for the elucidation of a community's phytosociological structure. Certain conjectures about the community are tested and it is found that Goodall's hypothesis concerning the nature of a plant community is supported. The tests also show that the vegetation of the study area forms a single natural grouping despite the disparity of the position ofAcer saccharum and the polarity evident among the other tree species.Nomenclature follows Gleason (1952).From a thesis submitted to the University of Toronto in partial fulfilment of the requirements for the degree of Master of Science.I wish to thank Dr. G. A. Yarranton for his ideas and helpful supervision, and my father for his advice and encouragement. Thanks ag also due to Miss J. E. Ellard and Mr. S. Roy who helped prepare the figures. This research has been supported by a National Research Council of Canada and by NRCC grant A-2910.