The research and development efforts in our laboratory are directed towards understanding protein structure, protein–protein and protein–ligand interactions in the solution state through the use of high-resolution solution NMR spectroscopic methods. The structural and functional aspects of several enzymes and peptide toxins of animal origin are under investigation. A fusion protein system, based on the protein cytochrome b5 has been developed specifically for the production of isotopically enriched proteins for these NMR based investigations. In addition to NMR spectroscopy, the laboratory also uses Mass Spectrometry for characterization of natural peptide toxins.
A novel fusion protein system based on the highly soluble heme-binding domain of cytochrome b5 has been designed. The ability of cytochrome b5 to increase the levels of expression and solubility of target proteins has been tested by expressing several proteins and peptides. The fusion protein system has been designed to incorporate protease cleavage sites for commonly used proteases, viz., Enterokinase, Factor Xa, Thrombin and the Tobacco etch virus protease. Accumulation of expressed protein as a function of time may be visually ascertained from the bright red color of the cells during the course of induction. The proteins of interest may be cleaved from the parent protein by either chemical or enzymatic means. Several proteins and peptides have since been produced using this system. 1
The structural determinants that govern the catalytic activity and the mechanism of regulation of acetohydroxyacid synthase I (AHAS I),
an enzyme that catalyzes the first committed step in the biosynthesis of the branched-chain amino acids (isoleucine, leucine and valine), are under
investigation. A catalytically competent enzyme could be assembled from the individual domains of the catalytic subunit
(IlvB) and the activity significantly increased upon addition of the regulatory subunit (IlvN). Using solution NMR methods it was observed that
binding of IlvN to the FAD binding domain (~21 kDa) of IlvB results in an activation of the enzyme.
The tertiary structure of dimeric IlvN (~22 kDa) has been determined. IlvN was found to exist as a mixture of conformational states that are in equilibrium and that conformational exchange occurs at a rate that is intermediate on the NMR time scale. Binding of valine by IlvN, a key interaction that is responsible for the negative feedback regulation, caused a coil-to-helix conformational transition in the protein. The above studies have significantly improved our understanding of the structural and dynamic properties of the AHAS I isoenzyme. 4, 5
The enzyme pantothenate synthetase (panC) catalyzes the final step of the biosynthesis of vitamin b5 (pantothenic acid), the precursor for which is α-ketovalerate (produced in the branched-chain amino acid biosynthetic pathway). In a study of E.coli pantothenate synthetase, the solution structure of the dimeric N-terminal domain (NpanC, ~42 kDa) of the enzyme was determined. NMR studies showed that the NpanC is sufficient for substrate binding. Furthermore, at high concentrations, the substrate pantoate binds to its canonical binding site as well as to the ATP binding site. ATP, in stoichiometric excess could displace this bound pantoate. The X-ray crystal structure of the pantoate bound form of NpanC, provided unequivocal evidence for the homotropic inhibition of this enzyme by pantoate and this provides the rationale for the design of antimicrobial or herbicidal agents. 6
The role of the redox-active protein, thioredoxin (Trx), as a deglutathionylating enzyme in the glutathione-mediated regulation of the triosephosphate isomerase (TIM) activity has been elucidated. The structural interactions between Trx (11 kDa) and TIM (58 kDa) were monitored by NMR experiments that recorded chemical shift perturbations. Structural models of the complex were derived from docking calculations. Enzymatic studies provided proof for the deglutathionylating function of Trx. This study assumes importance since glutathionylation of metabolic proteins under conditions of redox stress serves as a mechanism for the regulation of their activity. 7
Ammonia channeling in the Plasmodium falciparum GMP synthetase (a two-domain type GMPS) was studied using a combination of biochemical
and NMR spectroscopic methods. Steady-state competition assays were carried out to quantitate the fraction of 14N ammonia (from glutamine
hydrolysis) and 15N ammonia (from bulk medium) incorporated into GMP. One-dimensional 15N-editd NMR spectroscopic studies revealed that
ammonia generated from glutamine hydrolysis does not equilibrate with the external medium, suggesting that it is channeled from the GATase
domain to the active site in the ATPPase domain. External ammonia can access the XMP-AMP intermediate in the ATPPase domain, suggesting
that alternate paths do exist for ammonia entry into the active site.
The solution structure of the GATase subunit of the Methanocaldococus jannaschii GMPS and the sites of its interaction with Mg2+ and the ATTPase subunit have been determined. 9, 10
The other major area of interest in our laboratory has been to understand the structure and function of peptide toxins. The main focus has been on
the "conotoxins" from marine cone snails. Structure and activity studies have been carried out on two related delta-conotoxins, viz., d-Am2766 and d-Am2735.
Other classes of conopeptides have also been characterised. 14, 16
We have been successful in cloning and expressing disulfide rich peptides. One such peptide is the three-disulfide scorpion toxin BTK-2, which was first identified in the venom of the red eastern Indian scorpion, Mesobuthus tamulus was cloned and expressed in bacterial cultures. Using biosynthetically derived carbon-13 and nitrogen-15 enriched samples of BTK-2, the three-dimensional solution structure of BTK-2 was determined. The biosynthetically derived peptide exhibited the same activity on the human voltage gated potassium channel, Kv1.1, as that exhibited by the natural peptide. 16, 17
We are currently in the process of producing other disulfide rich toxins of animal origin for structural characterization.