Cloning, expression and purification of the DNA binding domain of RFX protein.

The RFX DNA binding domain (DBD) is a novel highly conserved motif belonging to a large number of dimer DNA binding proteins which have diverse regulatory functions in eukaryotic organisms. To characterize this novel motif, a 78mer polypeptide corresponding to the DBD of human hRFX (hrfX1/DBD), a prototypical member of the RFX family has been cloned and overproduced in Escherichia coli. A purification procedure using cation exchange chromatography has also been developed.

Conformational dynamism in d-(GAATTCCGTTATT) containing the complementary Myb responsive element d-CCGTTA: NMR and MD investigations.

Conformational features of the DNA segment d-(GAATTCCGTTATT) containing the complementary Myb responsive element CCGTTA has been studied by NMR and molecular dynamics calculations with a view to see the role of 3D structure in specific DNA recognition. From the low field imino proton NMR spectra, the DNA sequence is seen to exist as a duplex with pyrimidine mismatches in the centre. The 2D NMR spectra however show that teh molecule exhibits substantial dynamism even at 1 degree C. Several extra cross peaks, more than the expected number, are seen in particular regions in all the spectra. These observations indicate that the duplex undergoes slow transitions between base-paired and unbase-paired states due to mismatches in the centre. Hence, to characterise those transitions a restrained verlet dynamics has been performed for 50ps using X-PLOR force field. Structural intermediates at regular intervals have been analysed, and we see that the dynamism in the molecule results in substantial fluctuations in the different torsion angles. The mismatch sites are seen to exhibit the highest degree of fluctuations, with the bases stacking in and looping out of the duplex. The sugar geometry is seen to be fairly steady around the S domain for most of the residues.

Two-dimensional NMR studies on 1-10 fragment of adrenocorticotropic hormone.

Various two-dimensional NMR techniques have been used to obtain complete resonance assignments of the protons in the 1-10 fragment of adrenocorticotropic hormone (ACTH). 1H-1H coupling constants among the backbone protons and the chemical shift values measured in aqueous and in dimethyl sulphoxide solutions indicated preference for extended but different conformations in the two solvents.

Refined procedures for accurate determination of solution structures of nucleic acids by two dimensional nuclear magnetic resonance spectroscopy.

New procedures have been described for accurate determination of solution structures of nucleic acids. These are two fold; new two dimensional nuclear magnetic resonance techniques and better approaches for interpretation of nuclear magnetic resonance data for structure determination purposes. The significant development in two dimensional nuclear magnetic resonance techniques for this purpose are ω1 -scaling and recording of pure phase spectra. Use of ω1-scaled correlated and nuclear Overhauser effect spectra for estimation of interproton distances and 1H-1H coupling constants has been described. Computer simulation procedures for exact determination of structure have been described. Experimental spectra demonstrating the application of new procedures have been presented.

Recognition schemes for protein-nucleic acid interactions.

The molecular forces involved in protein-nucleic acid interaction are electrostatic, stacking and hydrogen-bonding. These interactions have a certain amount of specificity due to the directional nature of such interactions and the spatial contributions of the steric effects of different substituent groups. Quantum chemical calculations on these interactions have been reported which clearly bring out such features. While the binding energies for electrostatic interactions are an order of magnitude higher, the differences in interaction energies for structures stabilised by hydrogen-bonding and stacking are relatively small. Thus, the molecular interactions alone cannot explain the highly specific nature of binding observed in certain segments of proteins and nucleic acids. It is therefore logical to assume that the sequence dependent three dimensional structures of these molecules help to place the functional groups in the correct geometry for a favourable interaction between the two molecules. We have carried out 2D-FT nuclear magnetic resonance studies on the oligonucleotide d- GGATCCGGATCC. This oligonucleotide sequence has two binding sites for the restriction enzyme Bam H1. Our studies indicate that the conformation of this DNA fragment is predominantly B-type except near the binding sites where the ribose ring prefers a 3E conformation. This interesting finding raises the general question about the presence of specificity in the inherent backbone structures of proteins and nucleic acids as opposed to specific intermolecular interactions which may induce conformational changes to facilitate such binding.