Assessment of a rigorous transitive profile based search method to detect remotely similar proteins

Profile-based sequence search procedures are commonly employed to detect remote relationships between proteins. We provide an assessment of a Cascade PSI-BLAST protocol that rigorously employs intermediate sequences in detecting remote relationships between proteins. In this approach we detect using PSI-BLAST, which involves multiple rounds of iteration, an initial set of homologues for a protein in a 'first generation' search by querying a database. We propagate a 'second generation' search in the database, involving multiple runs of PSI-BLAST using each of the homologues identified in the previous generation as queries to recognize homologues not detected earlier. This non-directed search process can be viewed as an iteration of iterations that is continued to detect further homologues until no new hits are detectable. We present an assessment of the coverage of this 'cascaded' intermediate sequence search on diverse folds and find that searches for up to three generations detect most known homologues of a query. Our assessments show that this approach appears to perform better than the traditional use of PSI-BLAST by detecting 15% more relationships within a family and 35% more relationships within a superfamily. We show that such searches can be performed on generalized sequence databases and non-trivial relationships between proteins can be detected effectively. Such a propagation of searches maximizes the chances of detecting distant homologies by effectively scanning protein "fold space".     

Light-dependent and tissue-specific expression of the H-protein of the glycine decarboxylase complex.

Glycine decarboxylase is a mitochondrial enzyme complex, which is the site of photorespiratory CO2 and NH3 release. Although the proteins that constitute the complex are located within the mitochondria, because of their intimate association with photosynthesis their expression is controlled by light. Comparisons of the kinetics of mRNA accumulation between the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and the H-protein of glycine decarboxylase during the greening of etiolated Arabidopsis thaliana suggest that their expression is controlled in parallel. A genomic clone for the H-protein (gdcH) was isolated from Arabidopsis and sequenced. The upstream region from -856 to +62 was fused to the beta-glucuronidase (GUS) reporter gene, and this construct was transformed into tobacco. This 5' upstream regulatory region appears to control GUS expression in a manner very similar to that of the endogenous H-protein gene. Constructs with deletions in the 5' upstream region were transformed into tobacco. These deletions revealed that light-dependent and tissue-specific expression was largely controlled by a 259-bp region between -376 and -117 bp. This region contains several putative GT boxes with the GGTTAA consensus core sequence. Once these strong light-dependent elements were removed, a second level of control was revealed. In constructs in which the gdcH 5' regulatory region was shortened to -117 bp or less, there was more GUS activity in the roots than in the leaves, and in dark-grown plants than in light-grown plants. This suggests that more proximal control elements may be responsible for the constitutive low levels of gene expression noted in all nonphotosynthetic tissues.     

Over-expression of OSRIP18 increases drought and salt tolerance in transgenic rice plants

Both drought and high salinity stresses are major abiotic factors that limit the yield of agricultural crops. Transgenic techniques have been regarded as effective ways to improve crops in their tolerance to these abiotic stresses. Functional characterization of genes is the prerequisite to identify candidates for such improvement. Here, we have investigated the biological functions of an Oryza sativa Ribosome-inactivating protein gene 18 (OSRIP18) by ectopically expressing this gene under the control of CaMV 35S promoter in the rice genome. We have generated 11 independent transgenic rice plants and all of them showed significantly increased tolerance to drought and high salinity stresses. Global gene expression changes by Microarray analysis showed that more than 100 probe sets were detected with up-regulated expression abundance while signals from only three probe sets were down-regulated after over-expression of OSRIP18. Most of them were not regulated by drought or high salinity stresses. Our data suggested that the increased tolerance to these abiotic stresses in transgenic plants might be due to up-regulation of some stress-dependent/independent genes and OSRIP18 may be potentially useful in further improving plant tolerance to various abiotic stresses by over-expression.     

Biophysical characterization of anticoagulant hemextin AB complex from the venom of snake Hemachatus haemachatus

Hemextin AB complex from the venom of Hemachatus haemachatus is the first known natural anticoagulant that specifically inhibits the enzymatic activity of blood coagulation factor VIIa in the absence of factor Xa. It is also the only known heterotetrameric complex of two three-finger toxins. Individually only hemextin A has mild anticoagulant activity, whereas hemextin B is inactive. However, hemextin B synergistically enhances the anticoagulant activity of hemextin A and their complex exhibits potent anticoagulant activity. In this study we characterized the nature of molecular interactions leading to the complex formation. Circular dichroism studies indicate the stabilization of β-sheet in the complex. Hemextin AB complex has an increased apparent molecular diameter in both gas and liquid phase techniques. The complex formation is enthalpically favorable and entropically unfavorable with a negative change in the heat capacity. Thus, the anticoagulant complex shows less structural flexibility than individual subunits. Both electrostatic and hydrophobic interactions are important for the complexation; the former driving the process and the latter helping in the stabilization of the tetramer. The tetramer dissociates into dimers and monomers with the increase in the ionic strength of the solution and also with increase in the glycerol concentration in the buffer. The two dimers formed under each of these conditions display distinct differences in their apparent molecular diameters and anticoagulant properties. Based on these results, we have proposed a model for this unique anticoagulant complex.     

Analysis on sliding helices and strands in protein structural comparisons: a case study with protein kinases

Protein structural alignments are generally considered as 'golden standard' for the alignment at the level of amino acid residues. In this study we have compared the quality of pairwise and multiple structural alignments of about 5900 homologous proteins from 718 families of known 3-D structures. We observe shifts in the alignment of regular secondary structural elements (helices and strands) between pairwise and multiple structural alignments. The differences between pairwise and multiple structural alignments within helical and beta-strand regions often correspond to 4 and 2 residue positions respectively. Such shifts correspond approximately to "one turn" of these regular secondary structures. We have performed manual analysis explicitly on the family of protein kinases. We note shifts of one or two turns in helix-helix alignments obtained using pairwise and multiple structural alignments. Investigations on the quality of the equivalent helix-helix, strand-strand pairs in terms of their residue side-chain accessibilities have been made. Our results indicate that the quality of the pairwise alignments is comparable to that of the multiple structural alignments and, in fact, is often better. We propose that pairwise alignment of protein structures should also be used in formulation of methods for structure prediction and evolutionary analysis.     

Cardiac mitochondrial preconditioning by Big Ca2+-sensitive K+ channel opening requires superoxide radical generation

ATP-sensitive K+ channel opening in inner mitochondrial membranes protects hearts from ischemia-reperfusion (I/R) injury. Opening of the Big conductance Ca2+-sensitive K+ channel (BK(Ca)) is now also known to elicit cardiac preconditioning. We investigated the role of the pharmacological opening of the BK(Ca) channel on inducing mitochondrial preconditioning during I/R and the role of O2-derived free radicals in modulating protection by putative mitochondrial (m)BK(Ca) channel opening. Left ventricular (LV) pressure (LVP) was measured with a balloon and transducer in guinea pig hearts isolated and perfused at constant pressure. NADH, reactive oxygen species (ROS), principally superoxide (O2(-*)), and m[Ca2+] were measured spectrophotofluorometrically at the LV free wall using autofluorescence and fluorescent dyes dihydroethidium and indo 1, respectively. BK(Ca) channel opener 1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl-2(3H)benzimid-axolone (NS; NS-1619) was given for 15 min, ending 25 min before 30 min of global I/R. Either Mn(III)tetrakis(4-benzoic acid)porphyrin (TB; MnTBAP), a synthetic dismutator of O2(-*), or an antagonist of the BK(Ca) channel paxilline (PX) was given alone or for 5 min before, during, and 5 min after NS. NS pretreatment resulted in a 2.5-fold increase in developed LVP and a 2.5-fold decrease in infarct size. This was accompanied by less O2(-*) generation, decreased m[Ca2+], and more normalized NADH during early ischemia and throughout reperfusion. Both TB and PX antagonized each preconditioning effect. This indicates that 1) NS induces a mitochondrial-preconditioned state, evident during early ischemia, presumably on mBK(Ca) channels; 2) NS effects are blocked by BK(Ca) antagonist PX; and 3) NS-induced preconditioning is dependent on the production of ROS. Thus NS may induce mitochondrial ROS release to initiate preconditioning.