Rajasri Bhattacharyya

Domain analysis of a groundnut calcium-dependent protein kinase: nuclear localization sequence in the junction domain is coupled with nonconsensus calcium binding domains.

The signature of calcium-dependent protein kinases (CDPKs) is a C-terminal calmodulin-like domain (CaMLD) with four consensus calcium-binding sites. A junction domain (JD) joins the kinase with CaMLD and interacts with them through its autoinhibitory and CaMLD binding subdomains, respectively. We noted several CDPKs additionally have a bipartite nuclear localization signal (NLS) sequence as a subdomain in their JD, and this feature is obligatorily coupled with the absence of consensus calcium-binding sites in their respective CaMLDs. These predicted features are substantiated by undertaking investigations on a CDPK (gi:67479988) isolated from cultured groundnut (Arachis hypogea) cells. This kinase can bind 3.1 mol of Ca2+ under saturating conditions with a considerably high Kd of 392 μm as compared with its canonical counterparts. CD spectroscopic analysis, however, indicates the intramolecular structural changes accompanied with calcium binding to be similar to canonical CDPKs. Attesting to the presence of NLS in the JD, the endogenous kinase is localized in the nucleus of osmotically stressed Arachis cells, and in vitro binding assays indicate the NLS in the JD to interact with nuclear transport factors of the importin family. Homology modeling also indicates the feasibility of interaction of importins with the NLS present in the JD of such CDPKs in their activated form. The possible significance of obligatory coupling between the presence of NLS in the junction domain and atypical calcium binding properties of these CDPKs is discussed in the light of the known mechanisms of activation of these kinases.

In vitro multiplication of Coleus forskohlii Briq. : An approach towards shortening the protocol

SummaryA protocol has been developed that leads to the development of complete plantlets of Coleus forskohlii within 35–40 d by culturing stem tip explants in MS medium containing 0.57 μM indole-3-acetic acid and 0.46 μM kinetin through direct multiplication at the rate of 12.5 shoots per explant. About 100% shoots rooted and micropropagated plants were successfully established in soil after hardening with a high survival rate. The significance of the present micropropagation protocol of C. forskohlii is the formulation of growth regulators which effected very fast multiplication of the plant (time reduced to one-third of the hitherto known methods).

Interaction geometry involving planar groups in protein–protein interfaces

The geometry of interactions of planar residues is nonrandom in protein tertiary structures and gives rise to conventional, as well as nonconventional (XH···π, XH···O, where X = C, N, or O) hydrogen bonds. Whether a similar geometry is maintained when the interaction is across the protein–protein interface is addressed here. The relative geometries of interactions involving planar residues, and the percentage of contacts giving rise to different types of hydrogen bonds are quite similar in protein structures and the biological interfaces formed by protein chains in homodimers and protein–protein heterocomplexes—thus pointing to the similarity of chemical interactions that occurs during protein folding and binding. However, the percentage is considerably smaller in the nonspecific and nonphysiological interfaces that are formed in crystal lattices of monomeric proteins. The CH···O interaction linking the aromatic and the peptide groups is quite common in protein structures as well as the three types of interfaces. However, as the interfaces formed by crystal contacts are depleted in aromatic residues, the weaker hydrogen bond interactions would contribute less toward their stability. Proteins 2007;

DEVELOPMENT OF A POTENT IN VITRO SOURCE OF PHYLLANTHUS AMARUS ROOTS WITH PRONOUNCED ACTIVITY AGAINST SURFACE ANTIGEN OF THE HEPATITIS B VIRUS

SummaryIn vitro culture of hairy roots of Phyllanthus amarus induced by Agrobacterium rhizogenes was established. Their growth and ability for in vitro inactivation of hepatitis B virus surface antigen was studied and compared with adventitious roots grown in vitro. The selected hairy root clone HR-1 was capable of growing at a very fast rate, and an approximately 900-fold increase in weight of root biomass was achieved after 4 wk of culture in hormone-free quarter-strength liquid Murashige and Skoog medium with continuous agitation. Non-transformed roots cultured in the presence of 1.0 mg l−1 (5.71 μM) indole-3-acetic acid increased by 330-fold. The immuno-inactive property of roots was maximal in the crude extract. The hairy roots were shown to possess 85% inhibition (in contrast to 15% in the control) in binding of hepatitis B surface antigen (HBsAg) to its antibody (anti-HBs) after 24 h of incubation with HbsAg-positive sera in vitro at 37°C. Out of three fractions selected on the basis of molecular weight components of the extract, the Fraction III containing comparatively lower molecular weight substances (≤3500) yielded the highest activity. The extract from non-transformed roots was found to possess similar efficiency (87% inhibition). The levels of activity in both types of in vitro-raised roots were higher than those of naturally occurring roots and leafy shoots. The ability of P. amarus hairy root cultures to yield high biomass with the anti-viral property at high levels may provide an alternative source of raw material for more detailed study in the field of pharmaceutical research.

Conservation and documentation of the medicinal plant resources of India

In India, activities in the field of medicinal plants, including conservation of germplasm, have been enhanced significantly during the past couple of decades and a huge volume of data is being generated out of these works. For maintaining the records in a consolidated form, documentation is required to store and manage all information on the related studies. In accordance with the implementation of various plans and programmes, some pioneer organisations started developing databases on medicinal plants. Based on the knowledge on contemporary works, as collected from published literature and websites, this article presents information on current activities in India in two important aspects of the field, namely, (1) conservation of medicinal plants; and (2) management of data generated from such studies. Another important aspect of the article is the announcement of a plant conservation related software, ‘PlantCon’. This digitised database contains data of 40 selected nationally prioritised medicinal plants (list enclosed). The notable difference of ‘PlantCon’ from other databases lies in its conservation-related information which is up-to-date and covers a wide area of Indian geographical sites. The database provides information in a user-friendly manner.

Secondary structures at polypeptide-chain termini and their features

An analysis of secondary structures (alpha-helices and beta-strands) in the two terminal regions of polypeptide chains reveals features different from those observed over the whole protein structure. Compared with the overall distribution, the helices in the N-terminal region tend to be smaller and have higher propensities to contain Gln and Leu, while the C-terminal helices are longer and have a greater proportion of Lys and Glu. As a strand, the C-terminal region is never found in the interior of parallel beta-sheets and has a higher propensity to be at the edge of antiparallel beta-sheets. In contrast, compared with the whole structure the N-terminal region has a higher propensity to be in the interior of parallel beta-sheets. Compared with the overall distributions, terminal helices and strands show distinct periodicities in length. The Schellman motif, which is a prevalent C-capping motif in helices, is not common in C-terminal helices. There are other observations that can be used in the design of helical peptides: more residues beyond the C-terminus of helices are used for capping interactions than residues before the N-terminus. Consideration of the distribution of terminal strands in the interior and at the edge of beta-sheets suggests a sequential folding mechanism beginning at the N-terminus of the polypeptide chain.

Environment and susceptibility of disulfide bonds towards radiation damage

Radiation induced damage to protein crystals during data collection is a major impediment for obtaining accurate structural information on macromolecules. Some of the specific impairments inflicted upon highly brilliant X-ray irradiation are disulfide bond cleavage and the loss of the integrity of carboxyl groups of acidic residues. There are results to indicate that not all disulfide bridges are equally susceptible to damage. Consideration of the disulfide bonds in the structures of elastase, lysozyme and acetylcholinesterase indicate that the S-S bonds which have a close contact with a carbonyl oxygen atom along the extension of the bond are more susceptible to breakage. Such an arrangement predisposes electron transfer to occur from the oxygen atom to the disulfide bond leading to its reduction. The energetics and charge transfer in S···O interactions were quantified based on ab initio calculations, using 6-31G(d,p)++ basis set on models generated mimicking protein structures. The charge transfer not only depends on distance but also on geometric orientation of carbonyl O atom with respect to the S atom involved in disulphide bond. Interaction between a nucleophile and electrophile, akin to hydrogen bonding, stabilizes protein structures; but this also provides a pathway of electron transfer to the S-S bond leading to its reduction during exposure of the protein crystal to intense X-ray beam.