NMR studies on truncated sequences of human telomeric DNA: observation of a novel A-tetrad.

The structure of the telomeric DNA has been a subject of extensive investigation in recent years due to the realization that it has important functional roles to play in vivo and the observations that truncated telomeric sequences exhibit a great variety of 3D structures in aqueous solutions. In this context, we describe here NMR structural studies on two truncated human telomeric DNA sequences, d-AG(3)T and d-TAG(3)T in solutions containing K(+)ions. The G(3)stretches in both the oligonucleotides were seen to form parallel-stranded quadruplexes. However, the AG(3)segment as a whole, had different structural characteristics. The structure of d-AG(3)T revealed the formation of a novel A-tetrad, which was not seen in d-TAG(3)T. The A's in the tetrad had syn glycosidic conformation as opposed to the anti conformation of the G's in the G-tetrads. The A-tetrad stacked well over the adjacent G-tetrad and the twist angle at this step was smaller in d-AG(3)T than in d-TAG(3)T. These observations are expected to be significant from the point of view of structural diversity and recognition in telomeres.

Solution structure and dynamics of GCN4 cognate DNA: NMR investigations.

A 12 bp long GCN4-binding, self-complementary duplex DNA d(CATGACGTCATG)(2) has been investigated by NMR spectroscopy to study the structure and dynamics of the molecule in aqueous solution. The NMR structure of the DNA obtained using simulated annealing and iterative relaxation matrix calculations compares quite closely with the X-ray structure of ATF/CREB DNA in complex with GCN4 protein (DNA-binding domain). The DNA is also seen to be curved in the free state and this has a significant bearing on recognition by the protein. The dynamic characteristics of the molecule have been studied by (13)C relaxation measurements at natural abundance. A correlation has been observed between sequence-dependent dynamics and recognition by GCN4 protein.

EWGWS insert in Plasmodium falciparum ookinete surface enolase is involved in binding of PWWP containing peptides: Implications to mosquito midgut invasion by the parasite.

There are multiple stages in the life cycle of Plasmodium that invade host cells. Molecular machinery involved is such host-pathogen interactions constitute excellent drug targets and/or vaccine candidates. A screen using a phage display library has previously demonstrated presence of enolase on the surface of the Plasmodium ookinete. Phage-displayed peptides that bound to the ookinete contained a conserved motif (PWWP) in their sequence. Here, direct binding of these peptides with recombinant Plasmodium falciparum enolase (rPfeno) was investigated. These peptides showed specific binding to rPfeno, but failed to bind to other enolases. Plasmodium spp enolases are distinct in having an insert of five amino acids ((104)EWGWS(108)) that is not found in host enolases. The possibility of this insert being the recognition motif for the PWWP containing peptides was examined, (i) by comparing the binding of the peptides with rPfeno and a deletion variant Δ-rPfeno lacking (104)EWGWS(108), (ii) by measuring the changes in proton chemical shifts of PWWP peptides on binding to different enolases and (iii) by inter-molecular docking experiment to locate the peptide binding site. Results from these studies showed that the pentapeptide insert of Pfeno indeed constitutes the binding site for the PWWP domain containing peptide ligands. Search for sequences homologous to phage displayed peptides among peritrophic matrix proteins resulted in identification of perlecan, laminin, peritrophin and spacran. The possibility of these PWWP domain-containing proteins in the peritrophic matrix of insect gut to interact with ookinete cell surface enolase and facilitate the invasion of mosquito midgut epithelium is discussed.

NMR identification of local structural preferences in HIV-1 protease tethered heterodimer in 6 M guanidine hydrochloride.

Understanding protein folding requires complete characterization of all the states of the protein present along the folding pathways. For this purpose nuclear magnetic resonance (NMR) has proved to be a very powerful technique because of the great detail it can unravel regarding the structure and dynamics of protein molecules. We report here NMR identification of local structural preferences in human immunodeficiency virus-1 protease in the 'unfolded state'. Analyses of the chemical shifts revealed the presence of local structural preferences many of which are native-like, and there are also some non-native structural elements. Three-bond H(N)-H(alpha) coupling constants that could be measured for some of the N-terminal and C-terminal residues are consistent with the native-like beta-structure. Unusually shifted 15N and amide proton chemical shifts of residues adjacent to some prolines and tryptophans also indicate the presence of some structural elements. These conclusions are supported by amide proton temperature coefficients and nuclear Overhauser enhancement data. The locations of the residues exhibiting preferred structural propensities on the crystal structure of the protein, give useful insights into the folding mechanism of this protein.

Real time NMR monitoring of local unfolding of HIV-1 protease tethered dimer driven by autolysis.

Structural studies in proteases have been hampered because of their inherent autolytic function. However, since autolysis is known to be mediated via protein unfolding, careful monitoring of the autolytic reaction has the potential to throw light on the folding-unfolding equilibria. In this paper we describe real time nuclear magnetic resonance investigations on the tethered dimer construct of the human immunodeficiency virus-1 protease, which have yielded insights into the relative stabilities of several residues in the protein. The residues lying along the active site (bottom, side and top of the active site) and those in helix have lower unfolding free energy values than the other parts of the protein. The residue level stability differences suggest that the protein is well suited to adjust itself in almost all the regions of its structure, as and when perturbations occur, either due to ligand binding or due to mutations.

Solution structure of d-GAATTCGAATTC by 2D NMR. A new approach to determination of sugar geometries in DNA segments.

A new approach based on the correlated spectroscopy (COSY) in 2D NMR has been described for determination of sugar geometries in oligonucleotides. Under the usual low resolution conditions employed in COSY, the intensities of cross peaks depend on the magnitudes of coupling constants. There are five vicinal coupling constants in a deoxyribose ring which are sensitive to the sugar geometry. The presence, absence and rough comparison of relative intensities of COSY cross peaks arising from such coupling constants enable one to fix the sugar conformation to a fair degree of precision. The methodology has been applied to d-GAATTCGAATTC. It is observed that ten out of the twelve nucleotide units in this sequence exhibit a rare O1'-endo geometry. The EcoRI cleavage sites (between G and A) in the dodecanucleotide show an interesting variation in the conformation with the two sugars attached to the Gs acquiring a geometry between C2'-endo and C4'-endo.

Step-wise formation of helical structure and side-chain packing in a peptide from scorpion neurotoxin support hierarchic model of protein folding.

The mechanism of protein folding has been the subject of extensive investigation during the last decade, both because of its academic challenge and because of its relation to many diseases which are known to occur due to misfolding of proteins. In this context, we report here a systematic investigation on the step-wise formation of a helical structure by the addition of hexafluoroacetone, in a 14-residue peptide derived from a part of the scorpion neurotoxin protein. The NMR and circular dichroism results indicate that the peptide has an inherent propensity for helix formation and this is limited to the internal few residues in aqueous solution. With the addition of the fluorosolvent, the helical content progressively increases and spans the whole sequence. This is accompanied by concomitant packing of the side chains. These results provide support to the so-called hierarchic model of protein folding which dictates that the local sequence determines the secondary structures in the protein and the side chains play an important role in this process.

Biophysical investigations on the myb-DNA system.

The oncogene product c-myb is a transcriptional modulator and is known to play important roles in cell growth and differentiation. It binds to DNA in a sequence specific manner and its cognate sequence motifs have been detected in the genes of proteins implying its role in a variety of regulatory functions. The protein has a DNA binding domain consisting of three imperfect repeats with highly conserved tryptophans at regular spacings in each of the repeats. We have carried out a variety of investigations on the structure and interactions of the DNA binding domain of Drosophila c-myb and its cognate DNA target sequences. The domain has been bacterially over-expressed by subcloning a segment of the gene coding for the domain in a pET 11d vector and transforming it into E. coli BL21 (DE3). Circular dichroism of the protein has revealed that the domain is largely helical in nature. Fluorescence investigations indicated that three out of the nine tryptophans are solvent exposed and the others are buried in the interior. The recombinant protein is able to distinguish between specific and non-specific DNA targets in its binding and the interaction is largely electrostatic in nature in both cases. Dynamic fluorescence quenching experiments suggested that the DNA binding sites on the protein for specific and non-specific DNA targets are physically different. Most of the conserved tryptophans are associated with the specific DNA binding site. Simulated annealing and molecular dynamic simulations in a water matrix have been used to predict an energetically favoured conformation for the protein. Calculation of surface accessibilities of the individual residues shows that nearly 60% of the residues are less than 50% accessible to the solvent. Two and three dimensional NMR experiments with isotopically labelled protein have enabled spin system identification for many residue type and the types of residues involved in hydrophobic core formation in the protein. In an attempt to see the DNA surface possibly involved in specific interaction with the protein, a three-dimensional structure of a 12 mer cognate DNA has been determined by NMR in conjunction with restrained energy minimization. The recognition sequence shows interesting structural characteristics that may have important roles in specific interaction.

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.

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.

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.

Mechanism of interaction of the antileukemic drug cytosine arabinoside with aromatic peptides: role of sugar conformation and peptide backbone.

Interaction of the antileukemic drugs, cytosine-arabinoside (Ara-C) and adenosine-arabinoside (Ara-A) and a structural analogue, cytidine, with aromatic dipeptides has been studied by fluorescence and NMR spectroscopy. Ara-C and cytidine bind tryptophanyl and histidyl dipeptides but not tyrosyl dipeptides, while Ara-A does not bind to any of them. Both studies indicate association involving stacking of aromatic moieties. NMR spectra also indicate a protonation of the histidine moiety by Ara-C. In case of cytidine, the chemical shifts observed on binding to His-Phe imply that the backbone protons of the dipeptide participate in the binding. The conformation of the sugar and the base seem to play a very important role in the binding phenomenon as three similar molecules, Ara-C, Ara-A and cytidine bind in totally different ways.

Distance estimation from NOE data in macromolecular systems: a quadratic approach.

A new procedure has been suggested for the estimation of interproton distances from Nuclear Overhauser Enhancement (NOE) data in biological macromolecules. A critical assessment of the errors and advantages has been made vis a vis the commonly used initial rate approximation. Experimental data supporting the theoretical ideas have been presented.

A frequency-time domain 3D COSY-J technique for measurement of 1H-31P coupling constants in oligonucleotides.

A frequency-time domain 3D NMR technique has been developed for measurement of heteronuclear coupling constants in oligonucleotides employing a combination of COSY and J-resolved techniques. The method employs frequency-selective excitation to generate the omega 1 axis and 2D FT to generate the omega 2 and omega 3 axes. The procedure yields high resolution, especially along the omega 1 axis. The technique is demonstrated on a dinucleotide.

NMR observation of T-tetrads in a parallel stranded DNA quadruplex formed by Saccharomyces cerevisiae telomere repeats.

We report here the NMR structure of the DNA sequence d-TGGTGGC containing two repeats of Saccharomyces cerevisiae telomere DNA which is unique in that it has a single thymine in the repeat sequence and the number of Gs can vary from one to three. The structure is a novel quadruplex incor-porating T-tetrads formed by symmetrical pairing of four Ts via O4-H3 H-bonds in a plane. This is in contrast to the previous results on other telomeric sequences which contained more than one T in the repeat sequences and they were seen mostly in the flexible regions of the structures. We observed that the T4-tetrad was nicely accommodated in the center of the G-quadruplex, but it caused a small underwinding of the right handed helix. The T tetrad stacked well on the adjacent G3-tetrad, but poorly on the G5 tetrad. Likewise, T1 also formed a stable T-tetrad at the 5' end of the quadruplex. To our knowledge, this is the first report of T-tetrad formation in DNA structures. These observations are of significance from the points of view of both structural diversity and specific recognitions.

Unfolding kinetics of tryptophan side chains in the dimerization and hinge regions of HIV-I protease tethered dimer by real time NMR spectroscopy.

HIV I protease has been the target of extensive and variety of investigations in recent years because of its importance in the AIDS viral life cycle. We describe here real time NMR studies on the unfolding kinetics of two tryptophans, W6 and W42, which are located in the dimerization and hinge domains of the protein, respectively. Unfolding seems to get initiated in the dimerization domain. The kinetic data at two temperatures, 32 and 42 degrees C, can both be described by two-state models for both the tryptophans, and the final state reached at 42 degrees C does not depend on the path of unfolding. Unfolding free energy changes derived from the kinetic fitting parameters are less than 3 kJ/mol, indicating that the energy landscape is very shallow. The free energy values and the rates for the two tryptophans are different at 32 degrees C, but are nearly the same at 42 degrees C. These are interpreted in the light of the "new view" of protein folding and the relative behaviors of the two tryptophans suggest the existence of cooperative pathways in the unfolding reaction of the protein. These observations would provide valuable insights into protein function, stability, and effects of nonactive site mutations conferring drug resistance.