Chemical Exchange Saturation Transfer NMR Research Articles

Characterization of Mannosyl Dioxanium Ions in Solution Using Chemical Exchange Saturation Transfer NMR Spectroscopy.

The stereoselective introduction of the glycosidic bond remains one of the main challenges in carbohydrate synthesis. Characterizing the reactive intermediates of this reaction is key to develop stereoselective glycosylation reactions. Herein we report the characterization of low‐populated, rapidly equilibrating mannosyl dioxanium ions that arise from participation of a C‐3 acyl group using chemical exchange saturation transfer (CEST) NMR spectroscopy. Dioxanium ion structure and equilibration kinetics were measured under relevant glycosylation conditions and highly α‐selective couplings were observed suggesting glycosylation took place via this elusive intermediate.

Characterization of Mannosyl Dioxanium Ions in Solution Using Chemical Exchange Saturation Transfer NMR Spectroscopy

AbstractThe stereoselective introduction of the glycosidic bond remains one of the main challenges in carbohydrate synthesis. Characterizing the reactive intermediates of this reaction is key to develop stereoselective glycosylation reactions. Herein we report the characterization of low‐populated, rapidly equilibrating mannosyl dioxanium ions that arise from participation of a C‐3 acyl group using chemical exchange saturation transfer (CEST) NMR spectroscopy. Dioxanium ion structure and equilibration kinetics were measured under relevant glycosylation conditions and highly α‐selective couplings were observed suggesting glycosylation took place via this elusive intermediate.

Recent developments in deuterium solid-state NMR for the detection of slow motions in proteins

Slow timescale dynamics in proteins are essential for a variety of biological functions spanning ligand binding, enzymatic catalysis, protein folding and misfolding regulations, as well as protein–protein and protein–nucleic acid interactions. In this review, we focus on the experimental and theoretical developments of 2H static NMR methods applicable for studies of microsecond to millisecond motional modes in proteins, particularly rotating frame relaxation dispersion (R1ρ), quadrupolar Carr–Purcell–Meiboom–Gill (QCPMG) relaxation dispersion, and quadrupolar chemical exchange saturation transfer NMR experiments (Q-CEST). With applications chosen from amyloid-β fibrils, we show the complementarity of these approaches for elucidating the complexities of conformational ensembles in disordered domains in the non-crystalline solid state, with the employment of selective deuterium labels. Combined with recent advances in relaxation dispersion backbone measurements for 15N/13C/1H nuclei, these techniques provide powerful tools for studies of biologically relevant timescale dynamics in disordered domains in the solid state.

A CEST NMR experiment to obtain glycine 1Hα chemical shifts in 'invisible' minor states of proteins.

Chemical exchange saturation transfer (CEST) experiments are routinely used to study protein conformational exchange between a 'visible' major state and 'invisible' minor states because they can detect minor states with lifetimes varying from ~ 3 to ~ 100ms populated to just ~ 0.5%. Consequently several 1H, 15N and 13C CEST experiments have been developed to study exchange and obtain minor state chemical shifts at almost all backbone and sidechain sites in proteins. Conspicuously missing from this extensive set of CEST experiments is a 1H CEST experiment to study exchange at glycine (Gly) 1Hα sites as the existing 1H CEST experiments that have been designed to study dynamics in amide 1H-15N spin systems and methyl 13CH3 groups with three equivalent protons while suppressing 1H-1H NOE induced dips are not suitable for studying exchange in methylene 13CH2 groups with inequivalent protons. Here a Gly 1Hα CEST experiment to obtain the minor state Gly 1Hα chemical shifts is presented. The utility of this experiment is demonstrated on the L99A cavity mutant of T4 Lysozyme (T4L L99A) that undergoes conformational exchange between two compact conformers. The CEST derived minor state Gly 1Hα chemical shifts of T4L L99A are in agreement with those obtained previously using CPMG techniques. The experimental strategy presented here can also be used to obtain methylene proton minor state chemical shifts from protein sidechain and nucleic acid backbone sites.

Multiquantum Chemical Exchange Saturation Transfer NMR to Quantify Symmetrical Exchange: Application to Rotational Dynamics of the Guanidinium Group in Arginine Side Chains.

Chemical exchange saturation transfer (CEST) NMR experiments have emerged as a powerful tool for characterizing dynamics in proteins. We show here that the CEST approach can be extended to systems with symmetrical exchange, where the NMR signals of all exchanging species are severely broadened. To achieve this, multiquantum CEST (MQ-CEST) is introduced, where the CEST pulse is applied to a longitudinal multispin order density element and the CEST profiles are encoded onto nonbroadened nuclei. The MQ-CEST approach is demonstrated on the restricted rotation of guanidinium groups in arginine residues within proteins. These groups and their dynamics are essential for many enzymes and for noncovalent interactions through the formation of hydrogen bonds, salt-bridges, and π-stacking interactions, and their rate of rotation is highly indicative of the extent of interactions formed. The MQ-CEST method is successfully applied to guanidinium groups in the 19 kDa L99A mutant of T4 lysozyme.

Extending the Sensitivity of CEST NMR Spectroscopy to Micro-to-Millisecond Dynamics in Nucleic Acids Using High-Power Radio-Frequency Fields.

Biomolecules undergo motions on the micro-to-millisecond timescale to adopt low-populated transient states that play important roles in folding, recognition, and catalysis. NMR techniques, such as Carr-Purcell-Meiboom-Gill (CPMG), chemical exchange saturation transfer (CEST), and R1ρ are the most commonly used methods for characterizing such transitions at atomic resolution under solution conditions. CPMG and CEST are most effective at characterizing motions on the millisecond timescale. While some implementations of the R1ρ experiment are more broadly sensitive to motions on the micro-to-millisecond timescale, they entail the use of selective irradiation schemes and inefficient 1D data acquisition methods. Herein, we show that high-power radio-frequency fields can be used in CEST experiments to extend the sensitivity to faster motions on the micro-to-millisecond timescale. Given the ease of implementing high-power fields in CEST, this should make it easier to characterize micro-to-millisecond dynamics in biomolecules.

Deuteron Quadrupolar Chemical Exchange Saturation Transfer (Q-CEST) Solid-State NMR for Static Powder Samples: Approach and Applications to Amyloid-β Fibrils.

We provide an experimental and computational framework for 2 H quadrupolar chemical exchange saturation transfer NMR experiments (Q-CEST) under static solid-state conditions for the quantification of dynamics on μs-ms timescales. Simulations using simple 2-site exchange models provide insights into the relation between spin dynamics and motions. Biological applications focus on two sites of amyloid-β fibrils in the 3-fold symmetric polymorph. The first site, the methyl group of A2 of the disordered N-terminal domain, undergoes diffusive motions and conformational exchange due to transient interactions. Earlier 2 H rotating frame relaxation and quadrupolar CPMG measurements are combined with the Q-CEST approach to characterize the multiple conformational states of the domain. The second site, the methyl group of M35, spans the water-accessible cavity inside the fibrils' core and undergoes extensive rotameric exchange. Q-CEST permits us to refine the rotameric exchange model for this site and allows the more precise determination of populations and rotameric exchange rate constants than line shape analysis.

Thiol-water proton exchange of glutathione, cysteine, and N-acetylcysteine: Implications for CEST MRI.

Amide-, amine-, and hydroxyl-water proton exchange can generate MRI contrast through chemical exchange saturation transfer (CEST). In this study, we show that thiol-water proton exchange can also generate quantifiable CEST effects under near-physiological conditions (pH = 7.2 and 37°C) through the characterization of the pH dependence of thiol proton exchange in phosphate-buffered solutions of glutathione, cysteine, and N-acetylcysteine. The spontaneous, base-catalyzed, and buffer-catalyzed exchange contributions to the thiol exchange were analyzed. The thiol-water proton exchange of glutathione and cysteine was found to be too fast to generate a CEST effect around neutral pH due to significant base catalysis. The thiol-water proton exchange of N-acetylcysteine was found to be much slower, yet still in the fast-exchange regime with significant base and buffer catalysis, resulting in a 9.5% attenuation of the water signal at pH 7.2 in a slice-selective CEST NMR experiment. Furthermore, the N-acetylcysteine thiol CEST was also detectable in human serum albumin and agarose phantoms.

Probing excited conformational states of nucleic acids by nitrogen CEST NMR spectroscopy.

Characterizing low-populated and short-lived excited conformational states has become increasingly important for understanding mechanisms of RNA function. Interconversion between RNA ground and excited conformational states often involves base pairing rearrangements that lead to changes in the hydrogen-bond network. Here, we present two 15N chemical exchange saturation transfer (CEST) NMR experiments that utilize protonated and non-protonated nitrogens, which are key hydrogen-bond donors and acceptors, for characterizing excited conformational states in RNA. We demonstrated these approaches on the B. Cereus fluoride riboswitch, where 15N CEST profiles complement 13C CEST profiles in depicting a potential pathway for ligand-dependent allosteric regulation of the excited conformational state of the fluoride riboswitch.

Detection and Chiral Recognition of α‐Hydroxyl Acid through 1H and CEST NMR Spectroscopy Using a Ytterbium Macrocyclic Complex

AbstractChiral α‐hydroxyl acids are of great importance in chemical synthesis. Current methods for recognizing their chirality by 1H NMR are limited by their small chemical shift differences and intrinsic solubility problem in organic solvents. Herein, we developed three YbDO3A(ala)3 derivatives to recognize four different commercially available chiral α‐hydroxyl acids in aqueous solution through 1H NMR and chemical exchange saturation transfer (CEST) spectroscopy. The shift difference between chiral α‐hydroxyl acid observed by proton and CEST NMR ranged from 15–40 and 20–40 ppm, respectively. Our work demonstrates for first time, that even one chiral center on the side‐arm chain of cyclen could set the stage for rotation of the other two non‐chiral side chains into a preferred position. This is ascribed to the lower energy state of the structure. The results show that chiral YbDO3A‐like complexes can be used to discriminate chiral α‐hydroxyl acids with a distinct signal difference.

Detection and Chiral Recognition of α-Hydroxyl Acid through 1 H and CEST NMR Spectroscopy Using a Ytterbium Macrocyclic Complex.

Chiral α-hydroxyl acids are of great importance in chemical synthesis. Current methods for recognizing their chirality by 1 H NMR are limited by their small chemical shift differences and intrinsic solubility problem in organic solvents. Herein, we developed three YbDO3A(ala)3 derivatives to recognize four different commercially available chiral α-hydroxyl acids in aqueous solution through 1 H NMR and chemical exchange saturation transfer (CEST) spectroscopy. The shift difference between chiral α-hydroxyl acid observed by proton and CEST NMR ranged from 15-40 and 20-40 ppm, respectively. Our work demonstrates for first time, that even one chiral center on the side-arm chain of cyclen could set the stage for rotation of the other two non-chiral side chains into a preferred position. This is ascribed to the lower energy state of the structure. The results show that chiral YbDO3A-like complexes can be used to discriminate chiral α-hydroxyl acids with a distinct signal difference.

Equilibrium folding dynamics of meACP in water, heavy water, and low concentration of urea

Many proteins fold in apparent two-state behavior, as partially folded intermediates only transiently accumulate and easily escape detection. Besides a native form and a mainly unfolded form, we captured a partially unfolded form of an acyl carrier protein from Micromonospora echinospora (meACP) in the folding/unfolding equilibrium using chemical exchange saturation transfer NMR experiments. The C-terminal region of the partially unfolded form is mainly folded and the N-terminal is unfolded. Furthermore, to understand how the folding process of meACP is influenced by solvent environments, we compared the folding dynamics of meACP in D2O, H2O and low concentration of urea. As the environment becomes more denaturing from D2O to H2O and then to urea, the unfolded state becomes increasingly populated, and the folding rate decreases. Adding a small amount of urea, which does not change solvent viscosity, has little effects on the unfolding rates, while changing H2O to D2O reduces the unfolding rates possibly due to the increase of solvent viscosity. The quantified solvent effects on the protein folding Gibbs energy and activation energy suggest that the transition state of folding may have a similar structure to the native state of the protein.

Measuring Residual Dipolar Couplings in Excited Conformational States of Nucleic Acids by CEST NMR Spectroscopy.

Nucleic acids undergo structural transitions to access sparsely populated and transiently lived conformational states--or excited conformational states--that play important roles in diverse biological processes. Despite ever-increasing detection of these functionally essential states, 3D structure determination of excited states (ESs) of RNA remains elusive. This is largely due to challenges in obtaining high-resolution structural constraints in these ESs by conventional structural biology approaches. Here, we present nucleic-acid-optimized chemical exchange saturation transfer (CEST) NMR spectroscopy for measuring residual dipolar couplings (RDCs), which provide unique long-range angular constraints in ESs of nucleic acids. We demonstrate these approaches on a fluoride riboswitch, where one-bond (13)C-(1)H RDCs from both base and sugar moieties provide direct structural probes into an ES of the ligand-free riboswitch.

(13)CHD2-CEST NMR spectroscopy provides an avenue for studies of conformational exchange in high molecular weight proteins.

An NMR experiment for quantifying slow (millisecond) time-scale exchange processes involving the interconversion between visible ground state and invisible, conformationally excited state conformers is presented. The approach exploits chemical exchange saturation transfer (CEST) and makes use of (13)CHD2 methyl group probes that can be readily incorporated into otherwise highly deuterated proteins. The methodology is validated with an application to a G48A Fyn SH3 domain that exchanges between a folded conformation and a sparsely populated and transiently formed unfolded ensemble. Experiments on a number of different protein systems, including a 360kDa half-proteasome, establish that the sensitivity of this (13)CHD2 (13)C-CEST technique can be upwards of a factor of 5 times higher than for a previously published (13)CH3 (13)C-CEST approach (Bouvignies and Kay in J Biomol NMR 53:303-310, 2012), suggesting that the methodology will be powerful for studies of conformational exchange in high molecular weight proteins.

Stable isotope-labeled RNA phosphoramidites to facilitate dynamics by NMR.

Given that Ribonucleic acids (RNAs) are a central hub of various cellular processes, methods to synthesize these RNAs for biophysical studies are much needed. Here, we showcase the applicability of 6-(13)C-pyrimidine phosphoramidites to introduce isolated (13)C-(1)H spin pairs into RNAs up to 40 nucleotides long. The method allows the incorporation of 6-(13)C-uridine and -cytidine residues at any desired position within a target RNA. By site-specific positioning of the (13)C-label using RNA solid phase synthesis, these stable isotope-labeling patterns are especially well suited to resolve resonance assignment ambiguities. Of even greater importance, the labeling pattern affords accurate quantification of important functional transitions of biologically relevant RNAs (e.g., riboswitch aptamer domains, viral RNAs, or ribozymes) in the μs- to ms time regime and beyond without complications of one bond carbon scalar couplings. We outline the chemical synthesis of the 6-(13)C-pyrimidine building blocks and their use in RNA solid phase synthesis and demonstrate their utility in Carr Purcell Meiboom Gill relaxation dispersion, ZZ exchange, and chemical exchange saturation transfer NMR experiments.

Sialo-CEST: chemical exchange saturation transfer NMR of oligo- and poly-sialic acids and the assignment of their hydroxyl groups using selective- and HSQC-TOCSY

Chemical exchange saturation transfer (CEST) is an NMR method that takes advantage of proton exchange between solute and solvent molecules in dynamic equilibrium, enabling the detection of the solute NMR signals with enhanced sensitivity. Herein, we report that the hydroxyl groups in a naturally occurring polysaccharide, α-2,8 polysialic acid in aqueous solution, yield very significant CEST effects even at 37°C where the resonances of the hydroxyl groups are not directly observed. We also report the assignments of the hydroxyl groups for the polymer and its oligomeric building blocks, from monomer to hexamer. We show that the same assignments can be made by either 1H–1H TOCSY methods or 1H–13C HSQC-TOCSY methods, to alleviate spectral overlap. Finally, we report the exchange rates of the OH groups with water and show how these rates can be used to select and fine-tune CEST effects.

Probing Slow Chemical Exchange at Carbonyl Sites in Proteins by Chemical Exchange Saturation Transfer NMR Spectroscopy

Unsichtbares sichtbar machen: Ein 13CO-NMR-CEST-Experiment (CEST=chemical exchange saturation transfer) wurde zur Untersuchung „unsichtbarer“ angeregter Proteinzustände mit Lebensdauern von 5 bis 50 ms entwickelt. Die chemischen 13CO-Verschiebungen zusammen mit den 15N-CEST-Profilen zeigen, dass die A39G-FF-Domäne sich ähnlich wie das Wildtypprotein über ein kompaktes Intermediat (I) faltet (F und U=nativer bzw. ungefalteter Zustand).

Probing Slow Chemical Exchange at Carbonyl Sites in Proteins by Chemical Exchange Saturation Transfer NMR Spectroscopy

Seeing the invisible: A 13CO NMR chemical exchange saturation transfer (CEST) experiment for the study of “invisible” excited protein states with lifetimes on the order of 5–50 ms has been developed. The 13CO chemical shifts together with those obtained from fits of 15N CEST profiles establish that the A39G FF domain folds via a similar compact intermediate (I) as the wild-type protein (F and U=native and unfolded states). As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.