Maayan Gal

Real-time multidimensional NMR follows RNA folding with second resolution

Conformational transitions and structural rearrangements are central to the function of many RNAs yet remain poorly understood. We have used ultrafast multidimensional NMR techniques to monitor the adenine-induced folding of an adenine-sensing riboswitch in real time, with nucleotide-resolved resolution. By following changes in 2D spectra at rates of approximately 0.5 Hz, we identify distinct steps associated with the ligand-induced folding of the riboswitch. Following recognition of the ligand, long range loop-loop interactions form and are then progressively stabilized before the formation of a fully stable complex over approximately 2–3 minutes. The application of these ultrafast multidimensional NMR methods provides the opportunity to determine the structure of RNA folding intermediates and conformational trajectories.

Native-unlike Long-lived Intermediates along the Folding Pathway of the Amyloidogenic Protein β2-Microglobulin Revealed by Real-time Two-dimensional NMR

β2-microglobulin (β2m), the light chain of class I major histocompatibility complex, is responsible for the dialysis-related amyloidosis and, in patients undergoing long term dialysis, the full-length and chemically unmodified β2m converts into amyloid fibrils. The protein, belonging to the immunoglobulin superfamily, in common to other members of this family, experiences during its folding a long-lived intermediate associated to the trans-to-cis isomerization of Pro-32 that has been addressed as the precursor of the amyloid fibril formation. In this respect, previous studies on the W60G β2m mutant, showing that the lack of Trp-60 prevents fibril formation in mild aggregating condition, prompted us to reinvestigate the refolding kinetics of wild type and W60G β2m at atomic resolution by real-time NMR. The analysis, conducted at ambient temperature by the band selective flip angle short transient real-time two-dimensional NMR techniques and probing the β2m states every 15 s, revealed a more complex folding energy landscape than previously reported for wild type β2m, involving more than a single intermediate species, and shedding new light into the fibrillogenic pathway. Moreover, a significant difference in the kinetic scheme previously characterized by optical spectroscopic methods was discovered for the W60G β2m mutant.

An improved ultrafast 2D NMR experiment: Towards atom-resolved real-time studies of protein kinetics at multi-Hz rates

Multidimensional NMR spectroscopy is a well-established technique for the characterization of structure and fast-time-scale dynamics of highly populated ground states of biological macromolecules. The investigation of short-lived excited states that are important for molecular folding, misfolding and function, however, remains a challenge for modern biomolecular NMR techniques. Off-equilibrium real-time kinetic NMR methods allow direct observation of conformational or chemical changes by following peak positions and intensities in a series of spectra recorded during a kinetic event. Because standard multidimensional NMR methods required to yield sufficient atom-resolution are intrinsically time-consuming, many interesting phenomena are excluded from real-time NMR analysis. Recently, spatially encoded ultrafast 2D NMR techniques have been proposed that allow one to acquire a 2D NMR experiment within a single transient. In addition, when combined with the SOFAST technique, such ultrafast experiments can be repeated at high rates. One of the problems detected for such ultrafast protein NMR experiments is related to the heteronuclear decoupling during detection with interferences between the pulses and the oscillatory magnetic field gradients arising in this scheme. Here we present a method for improved ultrafast data acquisition yielding higher signal to noise and sharper lines in single-scan 2D NMR spectra. In combination with a fast-mixing device, the recording of 1H–15N correlation spectra with repetition rates of up to a few Hertz becomes feasible, enabling real-time studies of protein kinetics occurring on time scales down to a few seconds.

Multidimensional NMR spectroscopy in a single scan.

Multidimensional NMR has become one of the most widespread spectroscopic tools available to study diverse structural and functional aspects of organic and biomolecules. A main feature of multidimensional NMR is the relatively long acquisition times that these experiments demand. For decades, scientists have been working on a variety of alternatives that would enable NMR to overcome this limitation, and deliver its data in shorter acquisition times. Counting among these methodologies is the so‐called ultrafast (UF) NMR approach, which in principle allows one to collect arbitrary multidimensional correlations in a single sub‐second transient. By contrast to conventional acquisitions, a main feature of UF NMR is a spatiotemporal manipulation of the spins that imprints the chemical shift and/or J‐coupling evolutions being sought, into a spatial pattern. Subsequent gradient‐based manipulations enable the reading out of this information and its multidimensional correlation into patterns that are identical to those afforded by conventional techniques. The current review focuses on the fundamental principles of this spatiotemporal UF NMR manipulation, and on a few of the methodological extensions that this form of spectroscopy has undergone during the years. Copyright

A capacitively coupled temperature‐jump arrangement for high‐resolution biomolecular NMR

A simple design for performing rapid temperature jumps within a high‐resolution nuclear magnetic resonance (NMR) setting is presented and exemplified. The design is based on mounting, around a conventional NMR glass tube, an inductive radiofrequency (RF) irradiation coil that is suitably tuned by a resonant circuit and is driven by one of the NMRs console high‐power RF amplifiers. The electric fields generated by this coils thin metal strips can lead to a fast and efficient heating of the sample, amounting to temperature jumps of ≈ 20 °C in well within a second—particularly in the presence of lossy dielectric media like those provided by physiological buffers. Moreover, when wound around a 4‐mm NMR tube, the resulting device fits a conventional 5‐mm inverse probe and is wholly compatible with the field homogeneities and sensitivities expected for high‐resolution biomolecular NMR conditions. The performance characteristics of this new system were tested using saline solutions, as well as on a lyotropic liquid crystal capable of undergoing nematic → isotropic transitions in the neighborhood of ambient temperature. These settings were then incorporated into the performance of a new kind of single‐scan 2D NMR spectroscopy acquisition, correlating the anisotropic and isotropic patterns elicited by solutes dissolved in such liquid‐crystalline systems, before and after a sudden temperature jump occurring during an intervening mixing period. Copyright