Integrated NMR/Molecular Dynamics Determination of the Ensemble Conformation of a Thermodynamically Stable CUUG RNA Tetraloop.

Both experimental and theoretical structure determinations of RNAs have remained challenging due to the intrinsic dynamics of RNAs. We report here an integrated nuclear magnetic resonance/molecular dynamics (NMR/MD) structure determination approach to describe the dynamic structure of the CUUG tetraloop. We show that the tetraloop undergoes substantial dynamics, leading to averaging of the experimental data. These dynamics are particularly linked to the temperature-dependent presence of a hydrogen bond within the tetraloop. Interpreting the NMR data by a single structure represents the low-temperature structure well but fails to capture all conformational states occurring at a higher temperature. We integrate MD simulations, starting from structures of CUUG tetraloops within the Protein Data Bank, with an extensive set of NMR data, and provide a structural ensemble that describes the dynamic nature of the tetraloop and the experimental NMR data well. We thus show that one of the most stable and frequently found RNA tetraloops displays substantial dynamics, warranting such an integrated structural approach.

A 300-fold enhancement of imino nucleic acid resonances by hyperpolarized water provides a new window for probing RNA refolding by 1D and 2D NMR.

NMR sensitivity-enhancement methods involving hyperpolarized water could be of importance for solution-state biophysical investigations. Hyperpolarized water (HyperW) can enhance the 1H NMR signals of exchangeable sites by orders of magnitude over their thermal counterparts, while providing insight into chemical exchange and solvent accessibility at a site-resolved level. As HyperW's enhancements are achieved by exploiting fast solvent exchanges associated with minimal interscan delays, possibilities for the rapid monitoring of chemical reactions and biomolecular (re)folding are opened. HyperW NMR can also accommodate heteronuclear transfers, facilitating the rapid acquisition of 2-dimensional (2D) 15N-1H NMR correlations, and thereby combining an enhanced spectral resolution with speed and sensitivity. This work demonstrates how these qualities can come together for the study of nucleic acids. HyperW injections were used to target the guanine-sensing riboswitch aptamer domain (GSRapt) of the xpt-pbuX operon in Bacillus subtilis Unlike what had been observed in proteins, where residues benefited of HyperW NMR only if/when sufficiently exposed to water, these enhancements applied to every imino resonance throughout the RNA. The >300-fold enhancements observed in the resulting 1H NMR spectra allowed us to monitor in real time the changes that GSRapt undergoes upon binding hypoxanthine, a high-affinity interaction leading to conformational refolding on a ∼1-s timescale at 36 °C. Structural responses could be identified for several nucleotides by 1-dimensional (1D) imino 1H NMR as well as by 2D HyperW NMR spectra acquired upon simultaneous injection of hyperpolarized water and hypoxanthine. The folding landscape revealed by this HyperW strategy for GSRapt, is briefly discussed.

Refolding of Cold-Denatured Barstar Induced by Radio-Frequency Heating: A New Method to Study Protein Folding by Real-Time NMR Spectroscopy.

The C40A/C82A double mutant of barstar has been shown to undergo cold denaturation above the water freezing point. By rapidly applying radio-frequency power to lossy aqueous samples, refolding of barstar from its cold-denatured state can be followed by real-time NMR spectroscopy. Since temperature-induced unfolding and refolding is reversible for this double mutant, multiple cycling can be utilized to obtain 2D real-time NMR data. Barstar contains two proline residues that adopt a mix of cis and trans conformations in the low-temperature-unfolded state, which can potentially induce multiple folding pathways. The high time resolution real-time 2D-NMR measurements reported here show evidence for multiple folding pathways related to proline isomerization, and stable intermediates are populated. By application of advanced heating cycles and state-correlated spectroscopy, an alternative folding pathway circumventing the rate-limiting cis-trans isomerization could be observed. The kinetic data revealed intermediates on both, the slow and the fast folding pathway.

Optimal Destabilization of DNA Double Strands by Single-Nucleobase Caging.

Photolabile protecting groups are widely used to trigger oligonucleotide activity. The ON/OFF-amplitude is a critical parameter. An experimental setup has been developed to identify protecting group derivatives with superior caging properties. Bulky rests are attached to the cage moiety via Cu-catalyzed azide-alkyne cycloaddition post-synthetically on DNA. Interestingly, the decrease in melting temperature upon introducing o-nitrobenzyl-caged (NPBY-) and diethylaminocoumarin-cages (DEACM-) in DNA duplexes reaches a limiting value. NMR spectroscopy was used to characterize individual base-pair stabilities and determine experimental structures of a selected number of photocaged DNA molecules. The experimental structures agree well with structures predicted by MD simulations. Combined, the structural data indicate that once a sterically demanding group is added to generate a tri-substituted carbon, the sterically less demanding cage moiety points towards the neighboring nucleoside and the bulkier substituents remain in the major groove.

Unprecedented Carbon Signal Enhancement in Liquid-State NMR Spectroscopy.

We shall overcome: As a result of efforts to overcome the sensitivity challenge of liquid-state NMR spectroscopy, a thousand-fold signal enhancement was achieved by dynamic nuclear polarization (DNP) for 13 C signals at high magnetic field (3.4 T) and room temperature, thereby exceeding the predicted limitations of high-field liquid-state in situ DNP.

[Marchiafava-Bignami disease (Case-report)]. / Marchiafava-Bignami-betegség (Egy 57 éves nobeteg esetének ismertetése).

Marchiafava-Bignami disease (MBD) is caused by damage of the corpus callosum. There are acute, subacute and chronic forms, it occurs most frequently among alcoholic patients. A variety of neurological symptoms, epileptic seizures, and coma may be associated with the disease, but the chronic form may start with acute confusion and dementia, interhemispherial disconnection syndrome or with slow progressive changes in behavior. In 2001, only 250 cases were reported, of which 200 died, 30 cases contributed to severe dementia or bed rest, and favorable outcome occured in only 20 cases. The MBD diagnosis of our patient was based on the anamnesis and cranial MRI and the treatment consisted of administration of B vitamin complex, folic acid, memantine, piracetam and haloperidol. Reviewing the international literature currently recommended therapeutic options are thiamin and folic acid. According to some authors the immediate administration of thiamine affects the outcome of the disease, and there are case reports of beneficial effects of amantadine and steroids.

Real-time nuclear magnetic resonance spectroscopy in the study of biomolecular kinetics and dynamics.

The review describes the application of nuclear magnetic resonance (NMR) spectroscopy to study kinetics of folding, refolding and aggregation of proteins, RNA and DNA. Time-resolved NMR experiments can be conducted in a reversible or an irreversible manner. In particular, irreversible folding experiments pose large requirements for (i) signal-to-noise due to the time limitations and (ii) synchronising of the refolding steps. Thus, this contribution discusses the application of methods for signal-to-noise increases, including dynamic nuclear polarisation, hyperpolarisation and photo-CIDNP for the study of time-resolved NMR studies. Further, methods are reviewed ranging from pressure and temperature jump, light induction to rapid mixing to induce rapidly non-equilibrium conditions required to initiate folding.