Research highlights

Our research interests are primarily focused on the investigation of the three-dimensional structures and properties of biological molecules, particularly proteins and nucleic acids, in atomic detail and their correlation with biological activity.

The methods we use are largely multidimensional and multinuclear NMR techniques. Besides, we use variety of methods based on optical spectroscopy, including fluorescence and circular dichroism. Recently, we started using isothermal titration calorimetry (ITC) and Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF; TofSpec 2E) in our experimental studies. Besides we also use the techniques of protein chemistry and molecular biology for biosynthetic expression of proteins and their site-directed mutagenesis.

We had been having close collaboration with the National Institutes of Health, Bethesda, Maryland. Under this collaboration, we have an on going Indo-US Research Program entitled “Conformation and Interaction of Non-standard Forms of Nucleic Acids”, with Dr. H.T. Miles as the principal investigator from NIH. We also have close links with the European Union Large Scale Facility for Nuclear Magnetic Resonance in life sciences (CERM) at Florence, Italy. This is part of the Department of Science and Technology (DST) sponsored Indo-Italian collaborative research program. Under this program, we have an ongoing research project entitled “Structure and dynamics of calcium binding proteins”.

Significant Achievements

We developed and improved NMR techniques to obtain better quality NMR data. New strategies have been developed to obtain more precise and detailed structural information at atomic level on several DNA segments.

Our major achievements include the concentration, temperature and pH dependent 3D structural characterization of several nonstandard DNA fragments, which include duplexes with mismatched base pairs, hairpins and higher order structures such as triplexes. Significant contributions include: (i) Structural evidence for the pH driven disproportionation of an oligonucleotide sample, consisting of homo-purine (R) and homo-pyrimidine (Y) strands in 1:1 molar ratio, into an intermolecular Y.R:Y DNA triplex and a single stranded R; (ii) Structural characterization of A-I, A⁺-G, A⁺-C, T-C and G-T mismatched base pairs; (iii) New insights of the interactions stabilizing base triples, which include mismatched base triples (C⁺.G-T and T.A⁺-C). This mode of recognition offers new possibilities for the targeting of mismatched sequences by oligonucleotide-directed triple-helix formation. (iv) NMR study on a complex formed when a palindromic homopyrimidine dodecamer (d-CTTCTCCTCTTC) and a homopurine hexamer (d-GAAGAG) are mixed in 1:1 molar ratio in aqueous solutions. This led to the discovery of yet another new DNA conformation where in the two strands of each oligomer form a novel triple stranded DNA structure with a palindromic symmetry. The cytosines involved in Hoogsteen base pairing remain protonated even at neutral pH and thus stabilize the triplex under physiological conditions. This study thus throws light upon the structural feasibility of C⁺.G:C base triples at neutral pH, where biologically relevant triplex formation is likely to occur. (v) NMR characterization of a parallel stranded DNA duplex at atomic resolution.

Our studies on 3D structures of four hairpin DNA’s by NMR provided a structural basis for understanding various interactions between uracil DNA glycosylase and uracil.

Our studies on the dynamics of polymerase mediated DNA slippage, in vitro, threw light upon the effects of T.A:T and G.G:C foldback triplex structures. This study for the first time demonstrates that, such slippage at the 3’-end (proximal) of T or G strand is hampered by the foldback triplex structures at the 5’ end (distal). It is proposed that the migration of putative intermediate, namely a hairpin loop, is prevented through T.A:T or G.G:C structures.

Structural Characterization of EF-hand calcium binding proteins by NMR

Our efforts in understanding the structure-function relationship of several peptides and proteins have been challenging. We have worked on the 3D structure of several proteins by multidimensional and multinuclear NMR spectroscopy. In order to understand the mechanisms of Ca²⁺ signaling in E. histolytica, we have overexpressed two calcium binding proteins (EhCaBP1(M_r ~ 15 kDa) and EhCaBP2(M_r ~ 20 kDa)) in E. coli., and isotopically ¹³C or/and ¹⁵N labelled the protein to study their 3D structures by NMR. We deposited the 3D structure of EhCaBP1 in PDB (PDB ID: 1JFK), which revealed the presence of two globular domains connected by a flexible amino acid linker. Each domain consists of a pair of helix-loop-helix motifs similar to EF-hands seen in Ca²⁺ binding proteins such as calmodulin and troponin C. Further, we structurally characterized the molten-globular apo-form of EhCaBP1 and its equilibrium folding to its completely folded holo state.

The role of myristoylation in governing Ca²⁺-binding and Ca²⁺-induced conformational changes in a neuronal calcium sensor (NCS-1), we studied yet another Ca²⁺-binding protein. ⁴⁵Ca binding and isothermal titration calorimetric (ITC) data show that myristoylation increases the degree of cooperativity in NCS-1 and thus the myristoylated NCS-1 binds Ca²⁺ more strongly (with three Ca²⁺ binding sites) than the non-myristoylated one (with two Ca²⁺ binding sites).

Energetics and mechanism of Ca²⁺ displacement by lanthanides in EF-hand calcium binding proteins by NMR

The sequential Ca²⁺ displacement, as observed during the NMR titration experiments, is interpreted in the light of ITC data and provided insight into the intra and interdomain cooperativity and Ca²⁺ specificity, for the first time.

Tracked AuTomated NMR Assignments in Proteins (TATAPRO)

We have developed a program for automated NMR assignments in proteins, which takes few seconds to complete the assignments. The algorithm, TATAPRO (Tracked AuTomated Assignments in Proteins) utilizes the protein primary sequence and peak lists from a set of triple resonance spectra, which correlate ¹H^N and ¹⁵N chemical shifts with that of ¹³C^a, ¹³C^b and ¹³C’/¹H^a. Such a spectral data is used to create a ²master_list², consisting of all possible sets of ¹H^N_i, ¹⁵N_i, ¹³C^a_i/i-1, ¹³C^b_i/i-1 and ¹³C’_i/i-1/¹H^a_i/i-1 chemical shifts. Subsequently, based on a statistical analysis of ¹³C^a and ¹³C^b chemical shifts of all the proteins deposited in the BioMagResBank, it has been shown that the 20 amino acid residues can be grouped into 9 distinct categories, each of which is assigned a unique 2-digit code. With the help of such a code, the program uses the master_list to search for neighbouring partners of a given residue along the polypeptide chain and identifies a maximum possible stretch of residues. This results in the sequence specific resonance assignment of that stretch of residues. The procedure is repeated till all the residues are assigned. The program has been tested using experimental data on a calcium binding protein (15 kDa), which has substantial internal sequence homology, and using published data on four other proteins in the molecular weight range of 18-42 kDa. In all these cases nearly complete sequence specific resonance assignments are achieved.

This program has been written in ANSI C code and can be compiled on any Unix based workstation or windows based system equipped with a C compiler. The execution time of the program is of the order of few seconds on a R10000 based solid impact workstation (SGI).

Selective ²unlabeling² of amino acids in proteins:

We have developed novel methodologies in protein engineering and isotope labeling. One of them is for sequence specific resonance assignments in proteins, using amino acid selective ²unlabeling². The strategy was based on selective unlabeling of amino acid residues in uniformly or fractionally ¹³C or/and ¹⁵N labeled proteins, which simplifies the multidimensional heteronuclear NMR spectra. This aids in sequence specific resonance assignments of both backbone and side chain nuclei. The methodology has been demonstrated by unlabeling several amino acid residues in a 15 kDa calcium binding protein from Entamoeba Histolytica (Eh-CaBP). This methodology has also been used for stereospecific NMR assignments of methyl (CH₃) groups of Val and Leu residues in fractionally ¹³C-labeled proteins. The approach is based on selective ²unlabeling² of specific amino acids in proteins while fractionally ¹³C-labeling the rest. A 2D [¹³C-¹H] HSQC spectrum recorded on such a sample is devoid of peaks belonging to the ²unlabeled² amino acid residues. In yet another attempt, this methodology has been extended to assign pseudocontact shifted peaks and to measure residual dipolar couplings in the simplified 2D [¹⁵N-¹H] HSQC spectra of EhCaBP, which was otherwise not possible to analyze without such spectral simplification. Further, this methodology has been used to measure ¹J(N_i, C^a_i), ¹J(N_i, C¢_i-1), ²J(N_i, C^a_i-1), ²J(H^N_i, C¢_i-1) and ²J(H^N_i, C^a_i) values in biosynthetically (¹³C and ¹⁵N) labeled proteins.