PubMed Health⌕ Search

Biomedical subjects

Ramadurai Ramachandran

Publications and source records attributed to Ramadurai Ramachandran.

17 recordsLinked to original sources
PubMed2005

A novel cGUUAg tetraloop structure with a conserved yYNMGg-type backbone conformation from cloverleaf 1 of bovine enterovirus 1 RNA.

The 5'-terminal cloverleaf (CL)-like RNA structures are essential for the initiation of positive- and negative-strand RNA synthesis of entero- and rhinoviruses. SLD is the cognate RNA ligand of the viral proteinase 3C (3C(pro)), which is an indispensable component of the viral replication initiation complex. The structure of an 18mer RNA representing the apical stem and the cGUUAg D-loop of SLD from the first 5'-CL of BEV1 was determined in solution to a root-mean-square deviation (r.m.s.d.) (all heavy atoms) of 0.59 A (PDB 1Z30). The first (antiG) and last (synA) nucleotide of the D-loop forms a novel 'pseudo base pair' without direct hydrogen bonds. The backbone conformation and the base-stacking pattern of the cGUUAg-loop, however, are highly similar to that of the coxsackieviral uCACGg D-loop (PDB 1RFR) and of the stable cUUCGg tetraloop (PDB 1F7Y) but surprisingly dissimilar to the structure of a cGUAAg stable tetraloop (PDB 1MSY), even though the cGUUAg BEV D-loop and the cGUAAg tetraloop differ by 1 nt only. Together with the presented binding data, these findings provide independent experimental evidence for our model [O. Ohlenschlager, J. Wohnert, E. Bucci, S. Seitz, S. Hafner, R. Ramachandran, R. Zell and M. Gorlach (2004) Structure, 12, 237-248] that the proteinase 3C(pro) recognizes structure rather than sequence.

3C Viral Proteases↗
PubMed2005

TEDOR with adiabatic inversion pulses: Resonance assignments of 13C/15N labelled RNAs.

We have examined via numerical simulations the performance characteristics of different 15N RF pulse schemes employed in the transferred echo double resonance (TEDOR) experimental protocol for generating 13C-15N dipolar chemical shift correlation spectra of isotopically labelled biological systems at moderate MAS frequencies (omega(r) approximately 10 kHz). With an 15N field strength of approximately 30-35 kHz that is typically available in 5 mm triple resonance MAS NMR probes, it is shown that a robust TEDOR sequence with significant tolerance to experimental imperfections sa as H1 inhomogeneity and resonance offsets can be effectively implemented using adiabatic heteronuclear dipolar recoupling pulse schemes. TEDOR-based 15N-13C and 15N-13C-13C chemical shift correlation experiments were carried out for obtaining 13C and 15N resonance assignments of an RNA composed of 97 (CUG) repeats which has been implicated in the neuromuscular disease myotonic dystrophy.

Carbon Isotopes↗
PubMed2005

Characterisation of hydrogen bonding networks in RNAs via magic angle spinning solid state NMR spectroscopy.

It is demonstrated that the spatial proximity of (1)H nuclei in hydrogen bonded base-pairs in RNAs can be conveniently mapped via magic angle spinning solid state NMR experiments involving proton spin diffusion driven chemical shift correlation of low gamma nuclei such as the imino and amino nitrogens of nucleic acid bases. As different canonical and non-canonical base-pairing schemes encountered in nucleic acids are characterised by topologically different networks of proton dipolar couplings, different base-pairing schemes lead to characteristic cross-peak intensity patterns in such correlation spectra. The method was employed in a study of a 100 kDa RNA composed of 97 CUG repeats, or (CUG)(97) that has been implicated in the neuromuscular disease myotonic dystrophy. (15)N-(15)N chemical shift correlation studies confirm the presence of Watson-Crick GC base pairs in (CUG)(97).

Base Pairing↗
PubMed2004

NMR solution structure of a parallel LNA quadruplex.

The solution structure of a locked nucleic acid (LNA) quadruplex, formed by the oligomer d(TGGGT), containing only conformationally restricted LNA residues is reported. NMR and CD spectroscopy, as well as molecular dynamics and mechanic calculations, has been used to characterize the complex. The molecule adopts a parallel stranded conformation with a 4-fold rotational symmetry, showing a right-handed helicity and the guanine residues in an almost planar conformation with three well-defined G-tetrads. The thermal stability of Q-LNA has been found to be comparable with that of [r(UGGGU)]4, while a T(m) increment of 20 degrees C with respect to the corresponding DNA quadruplex structure [d(TGGGT)]4 has been observed. The structural features of the LNA quadruplex reported here may open new perspectives for the biological application of LNAs as novel versatile tools to design aptamer or catalyst oligonucleotides.

Base Sequence↗
PubMed2004

Identification of NH...N hydrogen bonds by magic angle spinning solid state NMR in a double-stranded RNA associated with myotonic dystrophy.

RNA plays a central role in biological processes and exhibits a variety of secondary and tertiary structural features that are often stabilized via hydrogen bonds. The distance between the donor and acceptor nitrogen nuclei involved in NH...N hydrogen bonds in nucleic acid base pairs is typically in the range of 2.6-2.9 A. Here, we show for the first time that such spatial proximity between 15N nitrogen nuclei can be conveniently monitored via magic angle spinning solid state NMR on a uniformly 15N-labelled RNA. The presence of NH.N hydrogen bonds is reflected as cross-peaks between the donor and acceptor nitrogen nuclei in 2D 15N dipolar chemical shift correlation spectra. The RNA selected for this experimental study was a CUG repeat expansion implicated in the neuromuscular disease myotonic dystrophy. The results presented provide direct evidence that the CUG repeat expansion adopts a double-stranded conformation.

Base Pairing↗
PubMed2004

Homonuclear chemical shift correlation in rotating solids via RNnu n symmetry-based adiabatic RF pulse schemes.

The efficacy of RN(nu) (n) symmetry-based adiabatic Zero-Quantum (ZQ) dipolar recoupling schemes for obtaining chemical shift correlation data at moderate magic angle spinning frequencies has been evaluated. RN(nu) (n) sequences generally employ basic inversion elements that correspond to a net 180 degrees rotation about the rotating frame x-axis. It is shown here via numerical simulations and experimental measurements that it is also possible to achieve efficient ZQ dipolar recoupling via RN(nu) (n) schemes employing adiabatic pulses. Such an approach was successfully used for obtaining (13)C chemical shift correlation spectra of a uniformly labelled sample of (CUG)(97) - a triplet repeat expansion RNA that has been implicated in the neuromuscular disease myotonic dystrophy. An analysis of the (13)C sugar carbon chemical shifts suggests, in agreement with our recent (15)N MAS-NMR studies, that this RNA adopts an Alpha-helical conformation.

Nuclear Magnetic Resonance, Biomolecular↗
PubMed2004

The structure of the stemloop D subdomain of coxsackievirus B3 cloverleaf RNA and its interaction with the proteinase 3C.

Stemloop D (SLD) of the 5' cloverleaf RNA is the cognate ligand of the coxsackievirus B3 (CVB3) 3C proteinase (3Cpro). Both are indispensable components of the viral replication initiation complex. SLD is a structurally autonomous subunit of the 5' cloverleaf. The SLD structure was solved by NMR spectroscopy to an rms deviation of 0.66 A (all heavy atoms). SLD contains a novel triple pyrimidine mismatch motif with a central Watson-Crick type C:U pair. SLD is capped by an apical uCACGg tetraloop adopting a structure highly similar to stable cUNCGg tetraloops. Binding of CVB3 3Cpro induces changes in NMR spectra for nucleotides adjacent to the triple pyrimidine mismatch and of the tetraloop implying them as sites of specific SLD:3Cpro interaction. The binding of 3Cpro to SLD requires the integrity of those structural elements, strongly suggesting that 3Cpro recognizes a structural motif instead of a specific sequence.

3C Viral Proteases↗
PubMed2004

RFDR with adiabatic inversion pulses: application to internuclear distance measurements.

In the context of the structural characterisation of biomolecular systems via MAS solid state NMR, the potential utility of homonuclear dipolar recoupling with adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements. The results obtained suggest that it is possible to obtain reliable estimates of internuclear distances via an analysis of the initial cross-peak intensity buildup curves generated from two-dimensional adiabatic inversion pulse driven longitudinal magnetisation exchange experiments.

Computer Simulation↗
PubMed2004

Triple resonance MAS NMR with (13C, 15N) labelled molecules: reduced dimensionality data acquisition via 13C-15N heteronuclear two-spin coherence transfer pathways.

A reduced dimensionality magic angle spinning solid-state NMR experimental protocol for obtaining chemical shift correlation spectra of dipolar coupled nuclei in uniformly ((13)C, (15)N) labelled biological systems is described and demonstrated. The method involves a mapping of the evolution frequencies of heteronuclear (13)C-(15)N zero- and double-quantum coherences. In comparison to a reduced dimensionality procedure involving the simultaneous incrementation of two single-quantum chemical shift evolution periods, the approach described here could be potentially advantageous for minimising the heat dissipated in the probe by high power (1)H decoupling in experiments requiring long t (1) acquisition times.

Carbon Isotopes↗
PubMed2004

Adiabatic TOBSY in rotating solids.

A MAS solid state NMR approach for achieving efficient scalar coupling mediated through-bond (13)C chemical shift correlations of the aliphatic carbons in uniformly labelled peptides/proteins is described. The method involves the application of a continuous train of adiabatic inversion pulses, as in the adiabatic TOCSY experiments carried out in solution state NMR studies. While rotor synchronised application of adiabatic inversion pulses leads to dipolar correlations, it is shown here via numerical simulations and experimental measurements that asynchronous application of adiabatic pulses can facilitate the mapping of through-bond connectivities. The method employs a suitable phasing scheme for generating the desired isotropic mixing Hamiltonian and requires moderate (13)C RF field strength only.

Carbon Isotopes↗
PubMed2004

Adiabatic heteronuclear decoupling in rotating solids.

In magic angle spinning solid state NMR experiments the potential of heteronuclear (1)H decoupling employing a continuous train of adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements. It is shown that, with a (1)H RF field strength of approximately 100 kHz that is typically available in MAS NMR probes, it is possible to achieve efficient adiabatic (1)H decoupling at low magic angle spinning frequencies. It is pointed out that in the presence of H (1) inhomogeneities it will be advantageous to employ adiabatic decoupling in MAS solid state NMR experiments.

Histidine↗
PubMed2003

Broadband RFDR with adiabatic inversion pulses.

With a view to obtain (13)C chemical shift correlation spectra of uniformly labelled peptides/proteins at high magnetic fields and high magic angle spinning frequencies (omega(r)/2pi </= 20 kHz), the efficacy of RFDR with adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements employing different adiabatic pulse phasing schemes, shapes and durations. It is demonstrated that homonuclear dipolar recoupling with superior performance under resonance offset and H(1) inhomogeneity effects and without strong dependence on the (13)C chemical shift differences can be achieved with adiabatic pulses. It is shown that (13)C chemical shift correlation spectra in the entire range of carbon chemical shifts can be obtained efficiently with short adiabatic inversion pulses. In situations where correlation spectra of only the aliphatic region are required, the possibility for minimising the interference between the recoupling and decoupling RF fields with long adiabatic pulses, at low recoupling power levels and without compromising the broadband RFDR characteristics, is also indicated.

Amino Acid Sequence↗
PubMed2002

Interaction of Kazal-type inhibitor domains with serine proteinases: biochemical and structural studies.

The interaction of domains of the Kazal-type inhibitor protein dipetalin with the serine proteinases thrombin and trypsin is studied. The functional studies of the recombinantly expressed domains (Dip-I+II, Dip-I and Dip-II) allow the dissection of the thrombin inhibitory properties and the identification of Dip-I as a key contributor to thrombin/dipetalin complex stability and its inhibitory potency. Furthermore, Dip-I, but not Dip-II, forms a complex with trypsin resulting in an inhibition of the trypsin activity directed towards protein substrates. The high resolution NMR structure of the Dip-I domain is determined using multi-dimensional heteronuclear NMR spectroscopy. Dip-I exhibits the canonical Kazal-type fold with a central alpha-helix and a short two-stranded antiparallel beta-sheet. Molecular regions essential for inhibitor complex formation with thrombin and trypsin are identified. A comparison with molecular complexes of other Kazal-type thrombin and trypsin inhibitors by molecular modeling shows that the N-terminal segment of Dip-I fulfills the structural prerequisites for inhibitory interactions with either proteinase and explains the capacity of this single Kazal-type domain to interact with different proteinases.

Amino Acid Sequence↗
PubMed2002

Three-dimensional structure of the S4-S5 segment of the Shaker potassium channel.

The propagation of action potentials during neuronal signal transduction in phospholipid membranes is mediated by ion channels, a diverse group of membrane proteins. The S4-S5 linker peptide (S4-S5), that connects the S4 and S5 transmembrane segments of voltage-gated potassium channels is an important region of the Shaker ion-channel protein. Despite its importance, very little is known about its structure. Here we provide evidence for an amphipathic alpha-helical conformation of a synthetic S4-S5 peptide of the voltage-gated Drosophila melanogaster Shaker potassium channel in water/trifluoroethanol and in aqueous phospholipid micelles. The three-dimensional solution structures of the S4-S5 peptide were obtained by high-resolution nuclear magnetic resonance spectroscopy and distance-geometry/simulated-annealing calculations. The detailed structural features are discussed with respect to model studies and available mutagenesis data on the mechanism and selectivity of the potassium channel.

Amino Acid Sequence↗
PubMed2002

REDOR: an assessment of the efficacy of dipolar recoupling with adiabatic inversion pulses.

A numerical assessment of the efficacy of REDOR recoupling of heteronuclear dipolar interactions employing adiabatic dephasing pulses has been carried out by considering an isolated dipolar coupled spin 1/2 I-S system. At moderate magic angle spinning frequencies in the range of 3-6 kHz and when the CSA of the dephased spins is large, it is shown that efficient broadband heteronuclear dipolar recoupling and reliable distance estimates can be achieved even under conditions where a significant fraction of the rotor period is occupied by the adiabatic pulse. The efficacy of REDOR with adiabatic inversion pulses has been demonstrated experimentally in two model 15N-13C spin systems, (13C',15N) Aib-(15N) Aib-NH2 (Aib = alpha-aminoisobutyric acid) and (1-13C,15N) glycine.

Carbon Isotopes↗
PubMed2002

Chemical shift correlation via RFDR: elimination of resonance offset effects.

It is shown that it is possible to effectively execute RFDR experiments with adiabatic inversion pulses and obtain resonance offset compensation that is superior to what can be achieved by conventional rectangular pulses. Employing 40-micros tanh/tan adiabatic pulses at a power level of approximately 38 kHz and a spinning speed of 12 kHz it is demonstrated that the range of resonance offset compensation achieved is sufficient to generate, via a single experiment, homonuclear chemical shift correlation spectra in the entire (13)C chemical shift range in peptides/proteins at the currently available field strengths.

Amino Acids↗