Biomedical subjects

Phang C Tai

Publications and source records attributed to Phang C Tai.

13 recordsLinked to original sources

PubMed2006

Molecular basis for differential nucleotide binding of the nucleotide-binding domain of ABC-transporter CvaB.

The cytoplasmic membrane protein CvaB, involved in colicin V secretion in Escherichia coli, belongs to the ABC-transporter family in which ATP hydrolysis is typically the driving force for substrate transport. However, our previous studies indicated that the nucleotide-binding domain of CvaB could also bind and hydrolyze GTP and, indeed, highly preferred GTP over ATP at low temperatures. In this study, we have examined the molecular basis of this preference. Sequence alignment and homology modeling of the CvaB nucleotide-binding domain predicted that the aromatic stacking region of CvaB (Y501DSQ loop) had a role in the differential binding of nucleotides, and Ser503 and Gln504 provided potential hydrogen bonds to GTP but not to ATP. Site-directed mutagenesis of the Y501DSQ loop, mutations S503A, Q504L, and double mutation S503A/Q504L, was made to test the predicted hydrogen bonds with GTP. The double mutation S503A/Q504L increased the affinity for ATP by 6-fold, whereas the affinity for GTP was reduced slightly: the ATP/GTP-binding ratio increased about 10-fold. The temperature effect assays on nucleotide binding and hydrolysis further indicated that the double mutant protein had largely eliminated the difference for substrates ATP and GTP, and behaved more similarly to the NBD of typical ABC-transporter HlyB. Therefore, we conclude that Ser503 and Gln504 in aromatic stacking region of CvaB block the ATP binding and are important for the GTP-binding preference.

ATP-Binding Cassette Transporters↗

PubMed2006

Design, synthesis, and evaluation of efflux substrate-metal chelator conjugates as potential antimicrobial agents.

Maintaining a proper balance of metal concentrations is critical to the survival of bacteria. We have designed and synthesized a series of conjugates of metal chelators and efflux transporter substrates aimed at disrupting bacterial metal homeostasis to achieve bacterial killing. Biological studies showed that two of the compounds had very significant antimicrobial effect with an MIC value of 7.8 microg/mL against Gram-positive Bacillus subtilis.

Anti-Bacterial Agents↗

PubMed2006

Expression, purification, and characterization of Pseudomonas aeruginosa SecA.

A secA gene from Pseudomonas aeruginosa PAO1 was amplified and expressed in Escherichia coli BL21.19 (secA13) under conditions where E. coli SecA was depleted. The binding of P. aeruginosa SecA (PaSecA) to the SP-Sepharose column was facilitated by ammonium sulfate fractionation but was not necessary for E. coli SecA (EcSecA) as the later bound more efficiently. PaSecA and EcSecA were purified by the single chromatographic step to greater than 98% purity and had a recovery of more than 20 and 40%, respectively, from the soluble fraction. This simple step purification obtained a higher homogeneity than previously reported. Cross-reactivity by immunoblotting showed that the purified PaSecA contained little EcSecA if any. The purified PaSecA is a dimer in solution, as judged by size exclusion chromatography, and is slightly larger than its counterpart EcSecA with an estimated molecular weight of 240 kDa. Further studies by the sedimentation velocity method indicate that PaSecA tends to remain as a monomer in solution. The purified PaSecA possessed ATPase activity; the intrinsic and liposome-stimulated ATPase specific activities of PaSecA were approximately 50% of EcSecA.

Adenosine Triphosphatases↗

PubMed2006

Rule generation for protein secondary structure prediction with support vector machines and decision tree.

Support vector machines (SVMs) have shown strong generalization ability in a number of application areas, including protein structure prediction. However, the poor comprehensibility hinders the success of the SVM for protein structure prediction. The explanation of how a decision made is important for accepting the machine learning technology, especially for applications such as bioinformatics. The reasonable interpretation is not only useful to guide the "wet experiments," but also the extracted rules are helpful to integrate computational intelligence with symbolic AI systems for advanced deduction. On the other hand, a decision tree has good comprehensibility. In this paper, a novel approach to rule generation for protein secondary structure prediction by integrating merits of both the SVM and decision tree is presented. This approach combines the SVM with decision tree into a new algorithm called SVM_ DT, which proceeds in three steps. This algorithm first trains an SVM. Then, a new training set is generated through careful selection from the output of the SVM. Finally, the obtained training set is used to train a decision tree learning system and to extract the corresponding rule sets. The results of the experiments of protein secondary structure prediction on RS126 data set show that the comprehensibility of SVM_DT is much better than that of the SVM. Moreover, the generalization ability of SVM_DT is better than that of C4.5 decision trees and is similar to that of the SVM. Hence, SVM_DT can be used not only for prediction, but also for guiding biological experiments.

Algorithms↗

PubMed2006

Nucleotide-dependent dimerization of the C-terminal domain of the ABC transporter CvaB in colicin V secretion.

The cytoplasmic membrane proteins CvaB and CvaA and the outer membrane protein TolC constitute the bacteriocin colicin V secretion system in Escherichia coli. CvaB functions as an ATP-binding cassette transporter, and its C-terminal domain (CTD) contains typical motifs for the nucleotide-binding and Walker A and B sites and the ABC signature motif. To study the role of the CvaB CTD in the secretion of colicin V, a truncated construct of this domain was made and overexpressed. Different forms of the CvaB CTD were found during purification and identified as monomer, dimer, and oligomer forms by gel filtration and protein cross-linking. Nucleotide binding was shown to be critical for CvaB CTD dimerization. Oligomers could be converted to dimers by nucleotide triphosphate-Mg, and nucleotide release from dimers resulted in transient formation of monomers, followed by oligomerization and aggregation. Site-directed mutagenesis showed that the ABC signature motif was involved in the nucleotide-dependent dimerization. The spatial proximity of the Walker A site and the signature motif was shown by disulfide cross-linking a mixture of the A530C and L630C mutant proteins, while the A530C or L630C mutant protein did not dimerize on its own. Taken together, these results indicate that the CvaB CTD formed a nucleotide-dependent head-to-tail dimer.

ATP-Binding Cassette Transporters↗

PubMed2006

Packaging of chemicals in the defensive secretory glands of the sea hare Aplysia californica.

Sea hares protect themselves from predatory attacks with several modes of chemical defenses. One of these is inking, which is an active release of a protective fluid upon predatory attack. In many sea hares including Aplysia californica and A. dactylomela, this fluid is a mixture of two secretions from two separate glands, usually co-released: ink, a purple fluid from the ink gland; and opaline, a white viscous secretion from the opaline gland. These two secretions are mixed in the mantle cavity and directed toward the attacking predator. Some of the chemicals in these secretions and their mechanism of action have been identified. In our study, we used western blots, immunocytochemistry, amino acid analysis, and bioassays to examine the distribution of these components: (1) an L-amino acid oxidase called escapin for A. californica and dactylomelin-P for A. dactylomela, which has antimicrobial activity but we believe its main function is in defending sea hares against predators that evoke its release; and (2) escapin's major amino acid substrates--L-lysine and L-arginine. Escapin is exclusively produced in the ink gland and is not present in any other tissues or secretions. Furthermore, escapin is only sequestered in the amber vesicles of the ink glandand not in the red-purple vesicles, which contain algal-derived chromophores that give ink its distinctive purple color. The concentration of escapin and dactylomelin-P in ink, both in the gland and after its release, is as high as 2 mg ml(-1), or 30 micromol ml(-1), which is well above its antimicrobial threshold. Lysine and arginine (and other amino acids) are packaged into vesicles in the ink and opaline glands, but arginine is present in ink and opaline at <1 mmol l(-1) and lysine is present in ink at <1 mmol l(-1) but in opaline at 65 mmol l(-1). Our previous results showed that both lysine and arginine mediate escapin's bacteriostatic effects, but only lysine mediates its bactericidal effects. Given that escapin's antimicrobial effects require concentrations of lysine and/or arginine >1 mmol l(-1), our data lead us to conclude that lysine in opaline is the primary natural substrate for escapin in ink. Furthermore, packaging of the enzyme escapin and its substrate lysine into two separate glands and their co-release and mixing at the time of predatory attack allows for the generation of bioactive defensive compounds from innocuous precursors at the precise time they are needed. Whether lysine and/or arginine are substrates for escapin's antipredatory functions remains to be determined.

Amino Acid Oxidoreductases↗

PubMed2005

Expression and purification of Pseudomonas aeruginosa SecA N-terminal domain: stimulation of ATPase activity of the SecAL43P mutant protein.

Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium which secretes a wide range of hydrolytic enzymes, toxins, and virulence factors into the extracellular medium. Although P. aeruginosa possesses numerous specific systems for the export of proteins across its double-membrane envelopes, the Sec system is still the major and essential mechanism. However, very little is known about its molecular basis. We constructed, cloned, and expressed the N-terminal 236 amino acids of PaSecA domain (PaSecAN236), and SecAL43P mutants of P. aeruginosa in Escherichia coli BL21.19 (secA(ts)). Here, we describe the purification of PaSecAN236 by using osmotic shock as the first step to efficiently release targeted protein from cells, followed by cation-exchange and size exclusion columns to obtain homogeneous PaSecAN236. The purified PaSecA N-terminal domain was functional in stimulating the ATPase activity of mutant SecAL43P protein of P. aeruginosa.

Adenosine Triphosphatases↗

PubMed2005

Improved K-means clustering algorithm for exploring local protein sequence motifs representing common structural property.

Information about local protein sequence motifs is very important to the analysis of biologically significant conserved regions of protein sequences. These conserved regions can potentially determine the diverse conformation and activities of proteins. In this work, recurring sequence motifs of proteins are explored with an improved K-means clustering algorithm on a new dataset. The structural similarity of these recurring sequence clusters to produce sequence motifs is studied in order to evaluate the relationship between sequence motifs and their structures. To the best of our knowledge, the dataset used by our research is the most updated dataset among similar studies for sequence motifs. A new greedy initialization method for the K-means algorithm is proposed to improve traditional K-means clustering techniques. The new initialization method tries to choose suitable initial points, which are well separated and have the potential to form high-quality clusters. Our experiments indicate that the improved K-means algorithm satisfactorily increases the percentage of sequence segments belonging to clusters with high structural similarity. Careful comparison of sequence motifs obtained by the improved and traditional algorithms also suggests that the improved K-means clustering algorithm may discover some relatively weak and subtle sequence motifs, which are undetectable by the traditional K-means algorithms. Many biochemical tests reported in the literature show that these sequence motifs are biologically meaningful. Experimental results also indicate that the improved K-means algorithm generates more detailed sequence motifs representing common structures than previous research. Furthermore, these motifs are universally conserved sequence patterns across protein families, overcoming some weak points of other popular sequence motifs. The satisfactory result of the experiment suggests that this new K-means algorithm may be applied to other areas of bioinformatics research in order to explore the underlying relationships between data samples more effectively.

Algorithms↗

PubMed2005

Cloning, characterization and expression of escapin, a broadly antimicrobial FAD-containing L-amino acid oxidase from ink of the sea hare Aplysia californica.

A 60 kDa monomeric protein isolated from the defensive purple ink secretion of the sea hare Aplysia californica was cloned and sequenced, and is the first sea hare antimicrobial protein to be functionally expressed in E. coli. Sequence analysis suggested that this protein is a flavin-containing l-amino acid oxidase (LAAO), with one predicted potential glycosylation site, although the glycosylation could not be experimentally confirmed. This protein, which we call ;escapin', has high sequence similarity to several other gastropod proteins. Escapin was verified by NMR, mass spectroscopy and HPLC to have FAD as its flavin cofactor. Escapin's antimicrobial effects, bacteriostasis and bactericidal, were determined using a combination of two assays: (1) incubation of bacteria on solid media followed by assessment of inhibition by direct observation of zones of inhibition or by turbidity measurements; and (2) incubation of bacteria in liquid media followed by counting viable colonies after growing on agar plates. Native escapin inhibited the growth of Gram-positive and Gram-negative bacteria, including marine bacteria (Vibrio harveyii and Staphylococcus aureus) and pathogenic bacteria (Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa). Escapin also inhibited the growth of yeast and fungi, with different efficacies. Escapin's antimicrobial activity was concentration dependent and did not decrease when stored for more than 5 months at room temperature. Escapin was bacteriostatic and not bactericidal in minimal media (e.g. salt media) with glucose, yeast extract, and a mixture of 20 amino acids each at 50 micromol l(-1), but was bactericidal in media enriched with Tryptone Peptone. Escapin was also strongly bactericidal in media with l-lysine at concentrations as low as 3 mmol l(-1) and slightly bactericidal in 50 mmol l(-1) l-arginine, but not in most other amino acids even at 50 mmol l(-1). Escapin had high oxidase activity (producing hydrogen peroxide) with either l-arginine or l-lysine as a substrate and little to no oxidase activity with other l-amino acids. Hydrogen peroxide alone (without escapin or amino acids) was strongly bacteriostatic but poorly bactericidal, similar in this respect to l-arginine but different from l-lysine in the presence of escapin. Together these results suggest that there are multiple mechanisms to escapin's antimicrobial effects, with bacteriostasis resulting largely or entirely from the effects of hydrogen peroxide produced by escapin's LAAO activity, but bactericidal effects resulting from lysine-dependent mechanisms not directly involving hydrogen peroxide. Recombinant escapin expressed in bacteria was also active against Gram-positive and Gram-negative bacteria, suggesting that glycosylation is not essential for antimicrobial activity.

Amino Acid Sequence↗

PubMed2004

Serine proteases in the spiny lobster olfactory organ: their functional expression along a developmental axis, and the contribution of a CUB-serine protease.

Several serine proteases and protease inhibitors have been identified in the crustacean olfactory organ, which is comprised of the lateral flagellum of the antennule and its aesthetascs sensilla that house olfactory receptor neurons and their supporting cells. The function of these proteases in the olfactory organ is unknown, but may include a role in perireception (e.g., odor activation or inactivation) or in the development or survival of olfactory receptor neurons. To examine directly the function of proteases in the olfactory organ of the Caribbean spiny lobster Panulirus argus, we used different tissue fractions from the lateral flagellum in an enzyme activity assay with a variety of protease substrates and inhibitors. Trypsin-like serine protease activity occurs throughout the lateral flagellum but is enriched in the cell membranes from aesthetascs. Cysteine- and metalloprotease activities also occur in olfactory tissue, but are more abundant in tissue fractions other than aesthetascs. To assess the contribution of one of the olfactory serine proteases--CUB-serine protease (Csp)--Csp was immunoprecipitated using an antibody; results with the remaining fraction suggest that Csp accounts for at least 40% of the total serine protease activity in the olfactory organ. The amount of total serine protease activity follows a developmental axis in the lateral flagellum. Total protease activity is lowest in the proximal zone, which lacks aesthetascs, and the proliferation zone, where olfactory receptor neurons and associated cells are born, and highest in aesthetascs of the distally-located senescence zone, which has the oldest olfactory tissue.

Animals↗

PubMed2004

Improved protein secondary structure prediction using support vector machine with a new encoding scheme and an advanced tertiary classifier.

Prediction of protein secondary structures is an important problem in bioinformatics and has many applications. The recent trend of secondary structure prediction studies is mostly based on the neural network or the support vector machine (SVM). The SVM method is a comparatively new learning system which has mostly been used in pattern recognition problems. In this study, SVM is used as a machine learning tool for the prediction of secondary structure and several encoding schemes, including orthogonal matrix, hydrophobicity matrix, BLOSUM62 substitution matrix, and combined matrix of these, are applied and optimized to improve the prediction accuracy. Also, the optimal window length for six SVM binary classifiers is established by testing different window sizes and our new encoding scheme is tested based on this optimal window size via sevenfold cross validation tests. The results show 2% increase in the accuracy of the binary classifiers when compared with the instances in which the classical orthogonal matrix is used. Finally, to combine the results of the six SVM binary classifiers, a new tertiary classifier which combines the results of one-versus-one binary classifiers is introduced and the performance is compared with those of existing tertiary classifiers. According to the results, the Q3 prediction accuracy of new tertiary classifier reaches 78.8% and this is better than the best result reported in the literature.

Algorithms↗

PubMed2003

Cys32 and His105 are the critical residues for the calcium-dependent cysteine proteolytic activity of CvaB, an ATP-binding cassette transporter.

CvaB, a member of the ATP-binding cassette transporter superfamily, is the central membrane transporter of the colicin V secretion system in Escherichia coli. Cys32 and His105 in the N-terminal domain of CvaB were identified as critical residues for both colicin V secretion and cysteine proteolytic activity. By inhibiting degradation with N-ethylmaleimide and a mixture of protease inhibitors, a stable wild-type N-terminal domain (which showed cysteine protease activity when activated) was purified. Such protease activity was Ca2+- and concentration-dependent and could be inhibited by antipain, N-ethylmaleimide, EDTA, and EGTA. At low concentrations, the Ca2+ analogs Tb3+ and La3+ (but not Fe3+) significantly enhanced proteolytic activity, suggesting that the size of the cations is important for activity. Together with comparisons of the sequences of members of the cysteine protease family, these results indicate that Cys32 and His105 are the critical residues in the CvaB N-terminal domain for the calcium-dependent cysteine protease activity and secretion of colicin V.

ATP-Binding Cassette Transporters↗

PubMed2003

Ring-like pore structures of SecA: implication for bacterial protein-conducting channels.

SecA, an essential component of the general protein secretion pathway of bacteria, is present in Escherichia coli as soluble and membrane-integral forms. Here we show by electron microscopy that SecA assumes two characteristic forms in the presence of phospholipid monolayers: dumbbell-shaped elongated structures and ring-like pore structures. The ring-like pore structures with diameters of 8 nm and holes of 2 nm are found only in the presence of anionic phospholipids. These ring-like pore structures with larger 3- to 6-nm holes (without staining) were also observed by atomic force microscopic examination. They do not form in solution or in the presence of uncharged phosphatidylcholine. These ring-like phospholipid-induced pore-structures may form the core of bacterial protein-conducting channels through bacterial membranes.

Adenosine Triphosphatases↗