16 research outputs found
Hadamard magnetization transfers achieve dramatic sensitivity enhancements in homonuclear multidimensional NMR correlations of labile sites in proteins, polysaccharides and nucleic acids
EXSY, TOCSY and NOESY lie at the foundation of homonuclear NMR experiments in
organic and pharmaceutical chemistry, as well as in structural biology. Limited
magnetization transfer efficiency is an intrinsic downside of these methods,
particularly when targeting rapidly exchanging species such as labile protons
ubiquitous in polysaccharides, sidechains and backbones of proteins, and in
bases and sugars of nucleic acids: the fast decoherence imparted on these
protons through solvent exchanges, greatly reduces their involvement in
homonuclear correlation experiments. We have recently discussed how these
decoherences can be visualized as an Anti-Zeno Effect, that can be harnessed to
enhance the efficiency of homonuclear transfers within Looped PROjected
SpectroscopY (L-PROSY) leading to 200-300% enhancements in NOESY and TOCSY
cross-peaks for amide groups in biomolecules. This study demonstrates that even
larger sensitivity gains per unit time, equivalent to reductions by several
hundred-folds in the duration of experiments, can be achieved by looping
inversion or using saturation procedures. In the ensuing experiments a priori
selected frequencies are encoded according to Hadamard recipes, and
subsequently resolved along the indirect dimension via linear combinations.
Magnetization-transfer (MT) processes reminiscent of those occurring in CEST
provide significant enhancements in the resulting cross-peaks, in only a
fraction of acquisition time of a normal 2D experiment. The effectiveness of
the ensuing three-way polarization transfer interplay between water, labile and
non-labile protons was corroborated experimentally for proteins,
homo-oligosaccharides and nucleic acids. In all cases, cross-peaks barely
detectable in conventional 2D NMR counterparts, were measured ca. 10-fold
faster and with 200-600% signal enhancements by the Hadamard MT counterparts
Relaxation-Assisted Magnetization Transfer Phenomena for a Sensitivity-Enhanced 2D NMR
2D NOESY and TOCSY play central roles in contemporary NMR. We have recently discussed how solvent-driven exchanges can significantly enhance the sensitivity of such methods when attempting correlations between labile and nonlabile protons. This study explores two scenarios where similar sensitivity enhancements can be achieved in the absence of solvent exchange: the first one involves biomolecular paramagnetic systems, while the other involves small organic molecules in natural abundance. It is shown that, in both cases, the effects introduced by either differential paramagnetic shift and relaxation or by polarization sharing among networks of protons can provide a similar sensitivity boost, as previously discussed for solvent exchange. The origin and potential of the resulting enhancements are analyzed, and experiments that demonstrate them in protein and natural products are exemplified. Limitations and future improvements of these approaches are also briefly discussed.ISSN:1520-6882ISSN:0003-270
Cross-Polarization Schemes for Improved Heteronuclear Transfers Involving Labile Protons in Biomolecular Solution NMR
INEPT-based experiments are widely used for ÂčHâÂčâ”N transfers, but often fail when involving labile protons due to solvent exchanges. J-based cross polarization (CP) strategies offer a more efficient alternative to perform such transfers, particularly when leveraging the HÊ·á”á”á”Êł â HáŽș exchange process to boost the ÂčHâÂčâ”N transfer process. This leveraging, however, demands the simultaneous spin-locking of both HÊ·á”á”á”Êł and HáŽș protons by a strong ÂčH RF field, while fulfilling the ÎłâBâ,â=ÎłâBâ,â Hartmann-Hahn matching condition. Given the low value of Îłâ/Îłâ, however, these demands are often incompatibleâparticularly when experiments are executed by the power-limited cryogenic probes used in contemporary high field NMR. The present manuscript discusses CP alternatives that can alleviate this limitation, and evaluates their performance on urea, amino acids, and intrinsically disordered proteins. These alternatives include new CP variants based on frequency-swept and phase-modulated pulses, designed to simultaneously fulfill the aforementioned conflicting conditions. Their performances vis-Ă -vis current options are theoretically analyzed with Liouville-space simulations, and experimentally tested with double and triple resonance transfer experiments.ISSN:1433-7851ISSN:1521-3773ISSN:0570-083
Relaxation-Assisted Magnetization Transfer Phenomena for a Sensitivity-Enhanced 2D NMR
2D NOESY and TOCSY play central roles in contemporary
NMR. We have
recently discussed how solvent-driven exchanges can significantly
enhance the sensitivity of such methods when attempting correlations
between labile and nonlabile protons. This study explores two scenarios
where similar sensitivity enhancements can be achieved in the absence
of solvent exchange: the first one involves biomolecular paramagnetic
systems, while the other involves small organic molecules in natural
abundance. It is shown that, in both cases, the effects introduced
by either differential paramagnetic shift and relaxation or by polarization
sharing among networks of protons can provide a similar sensitivity
boost, as previously discussed for solvent exchange. The origin and
potential of the resulting enhancements are analyzed, and experiments
that demonstrate them in protein and natural products are exemplified.
Limitations and future improvements of these approaches are also briefly
discussed
Heteronuclear transfers from labile protons in biomolecular NMR: Cross polarization, revisited
INEPT- and HMQC-based pulse sequences are widely used to transfer polarization between heteronuclei, particularly in biomolecular spectroscopy: they are easy to setup and involve low power deposition. Still, these short-pulse polarization transfers schemes are challenged by fast solvent chemical exchange. An alternative to improve these heteronuclear transfers is J-driven cross polarization (J-CP), which transfers polarization by spin-locking the coupled spins under Hartmann-Hahn conditions. J-CP provides certain immunity against chemical exchange and other T2-like relaxation effects, a behavior that is here examined in depth by both Liouville-space numerical and analytical derivations describing the transfer efficiency. While superior to INEPT-based transfers, fast exchange may also slow down these J-CP transfers, hurting their efficiency. This study therefore explores the potential of repeated projective operations to improve 1Hâ15N and 1Hâ15Nâ13C J-CP transfers in the presence of fast solvent chemical exchanges. It is found that while repeating J-CP provides little 1Hâ15N transfer advantages over a prolonged CP, multiple contacts that keep both the water and the labile protons effectively spin-locked can improve 1Hâ15Nâ13C transfers in the presence of chemical exchange. The ensuing Looped, Concatenated Cross Polarization (L-CCP) compensates for single J-CP losses by relying on the 13Câs longer lifetimes, leading to a kind of âalgorithmic coolingâ that can provide high polarization for the 15N as well as carbonyl and alpha 13Cs. This can facilitate certain experiments, as demonstrated with triple resonance experiments on intrinsically disordered proteins involving labile, chemically exchanging protons
A hybrid structure determination approach to investigate the druggability of the nucleocapsid protein of SARS-CoV-2
The pandemic caused by SARS-CoV-2 has called for concerted efforts to generate new insights into the biology of betacoronaviruses to inform drug screening and development. Here, we establish a workflow to determine the RNA recognition and druggability of the nucleocapsid N-protein of SARS-CoV-2, a highly abundant protein crucial for the viral life cycle. We use a synergistic method that combines NMR spectroscopy and protein-RNA cross-linking coupled to mass spectrometry to quickly determine the RNA binding of two RNA recognition domains of the N-protein. Finally, we explore the druggability of these domains by performing an NMR fragment screening. This workflow identified small molecule chemotypes that bind to RNA binding interfaces and that have promising properties for further fragment expansion and drug development.ISSN:1362-4962ISSN:0301-561
Magnetization transfer to enhance NOE cross-peaks among labile protons: applications to iminoâimino sequential walks in SARS-CoV-2-derived RNAs
2D NOESY plays a central role in structural NMR spectroscopy. We have recently discussed methods that rely on solvent-driven exchanges to enhance NOE correlations between exchangeable and non-exchangeable protons in nucleic acids. Such methods, however, fail when trying to establish connectivities within pools of labile protons. This study introduces an alternative that also enhances NOEs between such labile sites, based on encoding aâ
priori selected peaks by selective saturations. The resulting selective magnetization transfer (SMT) experiment proves particularly useful for enhancing the iminoâimino cross-peaks in RNAs, which is a first step in the NMR resolution of these structures. The origins of these enhancements are discussed, and their potential is demonstrated on RNA fragments derived from the genome of SARS-CoV-2, recorded with better sensitivity and an order of magnitude faster than conventional 2D counterparts