Biophysical Characterization and Folding Studies of Plant Protease,Wrightin: Identification of Folding Intermediate Under Different Conditions

Wrightin, a serine protease from Wrightia tinctoria, has been used as model system to examine structure-function and stability. Our studies show high stability of the enzyme with major elements of secondary structure being β-sheets. Under neutral conditions the enzyme is stable in 8 M urea and high temperature. GuHCl induced unfolding of wrightin at lower pH cannot be satisfactorily fit to a two state model for unfolding. Multiple intermediates were identified during unfolding of wrightin. Further, two intermediates, early and late are identified in the urea induced unfolding pathway at pH 3.0. Spectroscopic properties of intermediate states are analyzed and interpreted.     

Interaction between AR signalling and CRKL bypasses casodex inhibition in prostate cancer

The underlying mechanism of failed androgen ablation therapy is unknown. It is recognised that under therapeutic conditions the androgen receptor (AR) remains functionally active independent of hormone stimulation and may function through an alternative pathway. We report a novel cooperative interaction between CRKL (an intracellular signalling adaptor protein) and the AR. We demonstrate by biochemical and genetic approaches that CRKL is associated with the AR complex and is localised in the nucleus of prostate cancer cells and patient tissue biopsies. The interaction between CRKL and the AR is functionally relevant as demonstrated by its presence on the enhancer region of an androgen regulated gene (human Kallikrein-2), its upregulation of PSA, and reduction in AR transactivation following its disruption by siRNA knockdown. In the presence of the AR inhibitor casodex, the expression of CRKL co-stimulated by growth factors is able to rescue AR activity independent of hormone. Our data provides insight on how a non-nuclear factor such as CRKL may interact with the AR complex to bypass hormone dependency by using an alternative growth factor signalling pathway in advanced prostate cancer.     

Analysis of the key active subsites of glycoside hydrolase 13 family members

α-Amylase, pullulanase, neopullulanase, cyclomaltodextrinase (CDase), cyclomaltodextin glucanotransferase (CGTase), etc. are some of the amylolytic enzymes that act on polysaccharides. These enzymes differ from each other with respect to substrate and linkage specificities. These enzymes have been grouped into the GH13 (GH, Glycoside Hydrolase) family in the CAZy database on the basis of similarity in amino acid sequence. Members of this family share three domains viz., A, B, and C, which have several binding subsites to accommodate monomeric units of the polysaccharide substrate. Among these subsites, −2, −1, +1, and +2 subsites are the most critical subsites for catalytic activity. In the present study, the substrate analog-, inhibitor-, or product-bound 3-D structures of 24 members of GH13 family have been analyzed to identify the features of the −2, −1, +1, and +2 subsites shared by all the members for recognition of the common substrate. It is found that neither the number nor the nature of the potential hydrogen bond-forming residues is conserved with the exception of the presence of tyrosine as a stacking residue in the −1 subsite. The relative spatial disposition of the conserved subsite residues are conserved as judged by distance matrices. The backbone of the −2, −1, +1, and +2 subsites does not undergo conformational change for the recognition of the substrate. This analysis suggests that these enzymes recognize their substrate on the basis of shape of the substrate rather than on the basis of specific interactions within the binding site.     

Procerain,a stable cysteine protease from the latex of Calotropis procera

A protease was purified to homogeneity from the latex of medicinal plant Calotropis procera (Family-Asclepiadaceae). The molecular mass and isoelectric point of the enzyme are 28.8 kDa and 9.32, respectively. Hydrolysis of azoalbumin by the enzyme was optimal in the range of pH 7.0-9.0 and temperature 55-60 degree C. The enzyme hydrolyses denatured natural substrates like casein, azoalbumin, and azocasein with high specific activity. Proteolytic and amidolytic activities of the enzyme were activated by thiol protease activators and inhibited by thiol protease inhibitors, indicating the enzyme to be a cysteine protease. The enzyme named as procerain, cleaves N-succinyl-Ala-Ala-Ala-p-nitroanilide but not -Ala-Ala-p-nitroanilide, -Ala p-nitroanilide and N-d-Benzoyl--Arg-p-nitroanilide and appears to be peptide length dependent. The extinction coefficient (epsilon 1% 280 nm) of the enzyme was 24.9 and it had no detectable carbohydrate moiety. Procerain contains eight tryptophan, 20 tyrosine and seven cysteine residues forming three disulfide bridges, and the remaining one being free. Procerain retains full activity over a broad range of pH 3.0-12.0 and temperatures up to 70 degree C, besides being stable at very high concentrations of chemical denaturants and organic solvents. Polyclonal antibodies against procerain do not cross-react with other related proteases. Procerain unlike most of the plant cysteine proteases has blocked N-terminal residue.     

Polyamine metabolism in various tissues during pathogenesis of chloroquine-susceptible and resistant malaria

The pathophysiological impact of infections with chloroquine-susceptible (CQS) and chloroquine-resistant (CQR) strains of Plasmodium berghei in Mastomys natalensis was studied with respect to changes in polyamine profiles in various tissues. Both CQS and CQR infections produced similar changes in polyamine profiles of various tissues. Maximum increase was recorded in spleen followed by liver and lungs. Renal, cardiac and cerebral tissues did not register significant changes. An increase in spermidine level was more prominent as compared to putrescine and spermine, leading to an overall increase in spermidine/spermine ratio. This ratio is an important index of cellular proliferation. Liver did not show considerable change in the activities of ornithine decarboxylase and S-adenosyl methionine decarboxylase, the regulatory enzymes of the polyamine biosynthetic pathway. Spleen however, registered marked induction of both the enzymes which was more prominent in the CQS infection than CQR. Normal erythrocytes contained traces of polyamine while the erythrocytes loaded with P. berghei parasites exhibited appreciably higher polyamine levels. Spermidine was detected in about five-fold higher concentrations than putrescine and spermine which were detected in equimolar levels. Again, CQS as well as CQR P. berghei, exhibited qualitatively and quantitatively similar polyamine profiles thus ruling out a role of polyamines in CQ-resistance in malaria. © 1997 John Wiley & Sons, Ltd.     

Redesigning symmetry-related "mini-core" regions of FGF-1 to increase primary structure symmetry: thermodynamic and functional consequences of structural symmetry

Previous reports detailing mutational effects within the hydrophobic core of human acidic fibroblast growth factor (FGF-1) have shown that a symmetric primary structure constraint is compatible with a stably folded protein. In the present report, we investigate symmetrically related pairs of buried hydrophobic residues in FGF-1 (termed "mini-cores") that are not part of the central core. The effect upon the stability and function of FGF-1 mutations designed to increase primary structure symmetry within these "mini-core" regions was evaluated. At symmetry-related positions 22, 64, and 108, the wild-type protein contains either Tyr or Phe side chains. The results show that either residue can be readily accommodated at these positions. At symmetry-related positions 42, 83, and 130, the wild-type protein contains either Cys or Ile side chains. While positions 42 and 130 can readily accommodate either Cys or Ile side chains, position 83 is substantially destabilized by substitution by Ile. Tertiary structure asymmetry in the vicinity of position 83 appears responsible for the inability to accommodate an Ile side chain at this position, and is known to contribute to functional half-life. A mutant form of FGF-1 with enforced primary structure symmetry at positions 22, 64, and 108 (all Tyr) and 42, 83, and 130 (all Cys) is shown to be more stable than the reference FGF-1 protein. The results support the hypothesis that a symmetric primary structure within a symmetric protein superfold represents a solution to achieving a foldable, stable polypeptide, and highlight the role that function may play in the evolution of asymmetry within symmetric superfolds.     

Identification of the conserved spatial position of key active-site atoms in glycoside hydrolase 13 family members

A computational study on the glycoside hydrolase 13 (GH13) family of the CAZy database has been carried out at the atomic level in order to identify the conserved positions that may be responsible for recognition of the substrate. Analysis with substrate analog-, inhibitor-, or product-bound 3D structures was carried out to find the atomic spatial arrangement of the amino acids that make −2, −1, +1, and +2 subsites and water oxygen atoms around the ligand. The identified conserved positions of subsites were independent from the nature of the amino acid. The −1 and +1 subsites have more conserved positions than the −2 and +2 subsites. Some of the clusters of the −1 and +1 subsites have atoms of the same chemical nature. A spatially conserved position for water, which is stabilized by a hydrogen bond with the carboxyl group of a proton donor (Glu) and Asp of the catalytic triad, was found in the −1 subsite of 75% of the enzymes subjected to analysis. This position could be the region of hydrolytic water.     

Using Protein Dimers to Maximize the Protein Hybridization Efficiency with Multisite DNA Origami Scaffolds

DNA origami provides a versatile platform for conducting ‘architecture-function’ analysis to determine how the nanoscale organization of multiple copies of a protein component within a multi-protein machine affects its overall function. Such analysis requires that the copy number of protein molecules bound to the origami scaffold exactly matches the desired number, and that it is uniform over an entire scaffold population. This requirement is challenging to satisfy for origami scaffolds with many protein hybridization sites, because it requires the successful completion of multiple, independent hybridization reactions. Here, we show that a cleavable dimerization domain on the hybridizing protein can be used to multiplex hybridization reactions on an origami scaffold. This strategy yields nearly 100% hybridization efficiency on a 6-site scaffold even when using low protein concentration and short incubation time. It can also be developed further to enable reliable patterning of a large number of molecules on DNA origami for architecture-function analysis.     

Biochemical and thermodynamic characteristics of thermo-alkali-stable xylanase from a novel polyextremophilic Bacillus halodurans TSEV1

The purified extracellular xylanase of polyextremophilic Bacillus halodurans TSEV1 has been visualized as a single band on SDS-PAGE and eluted as single peak by gel filtration, with a molecular mass of 40 kDa. The peptide finger print and cloned xylanase gene sequence analyses indicate that this enzyme belongs to GH family 10. The active site carboxyl residues are mainly involved in catalysis, while tryptophan residues are involved in substrate binding. The enzyme is optimally active at 80 °C and pH 9.0, and stable in the pH range of 7.0–12.0 with T _1/2 of 35 min at 80 °C (pH 9.0). Activation energy for birch wood xylan hydrolysis is 30.51 kJ mol^?1. The K _m, V _max and k _cat (birchwood xylan) are 2.05 mg ml^?1, 333.33 μmol mg^?1 min^?1 and 3.33 × 10⁴ min^?1, respectively. The pKa₁ and pKa₂ of ionizable groups of the active site that influence V _max are 8.51 and 11.0. The analysis of thermodynamic parameters for xylan hydrolysis suggests this as a spontaneous process. The enzyme is resistant to chemical denaturants like urea and guanidinium-HCl. The site-directed mutagenesis of catalytic glutamic acid residues (E196 and E301) resulted in a complete loss of activity. The birch wood xylan hydrolyzate contained xylobiose and xylotriose as the main products without any trace of xylose, and the enzyme hydrolyzes xylotetraose and xylopentaose rapidly to xylobiose. Thermo-alkali-stability, resistance to various chemical denaturants and mode of action make it a useful biocatalyst for generating xylo-oligosaccharides from agro-residues and bleaching of pulp in paper industries.