Measuring Protein-Ligand Binding by Hyperpolarized Ultrafast NMR.

Protein-ligand interactions can be detected by observing changes in the transverse relaxation rates of the ligand upon binding. The ultrafast NMR technique, which correlates the chemical shift with the transverse relaxation rate, allows for the simultaneous acquisition of R2 for carbon spins at different positions. In combination with dissolution dynamic nuclear polarization (D-DNP), where the signal intensity is enhanced by thousands of times, the R2 values of several carbon signals from unlabeled benzylamine are observable within a single scan. The hyperpolarized ultrafast chemical shift-R2 correlated experiment separates chemical shift encoding from the readout phase in the NMR pulse sequence, which allows it to beat the fundamental limit on the spectral resolution otherwise imposed by the sampling theorem. Applications enabled by the ability to measure multiple relaxation rates in a single scan include the study of structural properties of protein-ligand interactions.

R2 Relaxometry of SABRE-Hyperpolarized Substrates at a Low Magnetic Field.

Nuclear magnetic resonance (NMR) relaxometry at a low magnetic field, in the milli-Tesla range or less, is enabled by signal enhancements through hyperpolarization. The parahydrogen-based method of signal amplification by reversible exchange (SABRE) provides large signals in a dilute liquid for the measurement of R2 relaxation using a single-scan Carr-Purcell-Meiboom-Gill (CPMG) experiment. A comparison of relaxation rates obtained at high and low fields indicates that an otherwise dominant contribution from chemical exchange is excluded in this low-field range. The SABRE process itself is based on exchange between the free and polarization transfer catalyst-bound forms of the substrate. At a high magnetic field of 9.4 T, typical conditions for producing hyperpolarization including 5 mM 5-fluoropyridine-3-carboximidamide as a substrate and 0.5 mM chloro(1,5-cyclooctadiene)[4,5-dimethyl-1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene]iridium(I) as a polarization transfer catalyst precursor resulted in an R2 relaxation rate as high as 3.38 s-1. This relaxation was reduced to 1.19 s-1 at 0.85 mT. A quantitative analysis of relaxation rates and line shapes indicates that milli-Tesla or lower magnetic fields are required to eliminate the exchange contribution. At this magnetic field strength, R2 relaxation rates are indicative primarily of molecular properties. R2 relaxometry may be used for investigating molecular interactions and dynamics. The SABRE hyperpolarization, which provides signal enhancements without requiring a high magnetic field or large instrumentation, is ideally suited to enable these applications.

Analysis of Large Data Sets in a Physical Chemistry Laboratory NMR Experiment Using Python.

We describe an update to an experiment demonstrating low-field NMR spectroscopy in the undergraduate physical chemistry laboratory. A Python-based data processing and analysis protocol is developed for this experiment. The Python language is used in fillable worksheets in the notebook software JupyterLab, providing an interactive means for students to work with the measured data step by step. The protocol teaches methods for the analysis of large data sets in science or engineering, a topic that is absent from traditional chemistry curricula. Python is among the most widely used modern tools for data analysis. In addition, its open-source nature reduces the barriers for adoption in an educational laboratory.

Screening of Protein-Ligand Binding Using a SABRE Hyperpolarized Reporter.

Hyperpolarization through signal amplification by reversible exchange (SABRE) provides a facile means to enhance nuclear magnetic resonance (NMR) signals of small molecules containing an N-heterocycle or other binding site for a polarization transfer catalyst. A purpose-designed reporter ligand, which is capable of binding both to a target protein and to the catalyst, makes the sensitivity enhancement by this technique compatible with the measurement of a range of biomolecular interactions. The 1H polarization of the reporter ligand 4-amidinopyridine, which is targeting trypsin, is used to screen ligands that are not themselves hyperpolarizable by SABRE. The respective protein-ligand dissociation constants (KD) are determined by an observed change in the R2 relaxation rate of the reporter. A calculation of expected signal changes indicates that the accessible ligand KD values extend over several orders of magnitude, while the concentrations of target proteins and ligands can be reduced considering the sensitivity gains from hyperpolarization. In general, the design of a single, weakly binding ligand for a target protein enables the use of SABRE hyperpolarization for ligand screening or other biophysical studies involving macromolecular interactions.

Dynamic Nuclear Polarization Enhanced Nuclear Spin Optical Rotation.

Nuclear spin optical rotation (NSOR) has been investigated as a magneto-optical effect, which holds the potential for applications, including hybrid optical-nuclear magnetic resonance (NMR) spectroscopy and gradientless imaging. The intrinsic nature of NSOR renders its detection relatively insensitive, which has prevented it moving from a proof of concept to a method supporting chemical characterizations. In this work, the dissolution dynamic nuclear polarization technique is introduced to provide nuclear spin polarization, increasing the signal-to-noise ratio by several thousand times. NSOR signals of 1 H and 19 F nuclei are observed in a single scan for diluted compounds, which has made this effect suitable for the determination of electronic transitions from a specific nucleus in a large molecule.

Application of Relaxation Dispersion of Hyperpolarized 13 C Spins to Protein-Ligand Binding.

Nuclear spin relaxation dispersion parameters are proposed as indicators of the binding mode of a ligand to a protein. Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) provided a 13 C signal enhancement between 3000-6000 for the ligand 4-(trifluoromethyl) benzene-1-carboximidamide binding to trypsin. The measurement of 13 C R2 relaxation dispersion was enabled without isotope enrichment, using a series of single-scan Carr-Purcell-Meiboom-Gill experiments with variable refocusing delays. The magnitude in dispersion for the spins of the ligand is correlated to the position with respect to the salt bridge between protein and the amidine group of the ligand, indicating the ligand binding orientation. Hyperpolarized relaxation dispersion is an alternative to chemical shift or NOE measurements for determining ligand binding modes.

Amplification of Nuclear Overhauser Effect Signals by Hyperpolarization for Screening of Ligand Binding to Immobilized Target Proteins.

Immobilization of a target protein enhances the cross-relaxation rates for transfer of nuclear spin polarization but reduces the accessible target concentration. Hyperpolarization of ligand spins by dissolution dynamic nuclear polarization (D-DNP) is shown to increase sensitivity for observing the intraligand nuclear Overhauser effect (NOE). This effect, also known as the transferred NOE (trNOE), can be used for detection of binding and for obtaining binding-related structural information. The measurement of hyperpolarized trNOE signals is demonstrated for the ligand 4'-hydroxyazobenzene-2-carboxylic acid interacting with avidin protein immobilized on polystyrene beads. In a sample containing 63.5 µM ligands and 0.83 µM accessible protein binding sites, the signal enhancement provided by D-DNP leads to single-scan detection of the NOE buildup, despite that this signal peaks at only 2% of the total ligand signal. These buildup curves allow the confirmation of binding through a change in the sign of the NOE and the quantitative determination of cross-relaxation rates. The combination of the D-DNP technique and protein immobilization may facilitate the identification of intraligand NOEs in ligand screening for drug discovery. The same method may be applied to in vivo characterization of ligand interactions with cell surface proteins.

Characterization of Membrane Protein-Lipid Interactions in Unfolded OmpX with Enhanced Time Resolution by Hyperpolarized NMR.

Proton nuclear spins of dodecyl phosphocholine molecules below the critical micelle concentration are hyperpolarized by using dissolution dynamic nuclear polarization (D-DNP). NMR signal enhancements of 1210±400 and 1610±550 are obtained at 9.4 T, for choline methyls in the head group of the lipid and for the tail-end methyl group, respectively. This polarization is transferred to the unfolded protein through the nuclear Overhauser effect, after dilution to a final denaturant concentration of 0.8 M urea. As a result, the amide and aromatic side-chain signals of the protein are increased up to sixfold. Selective inversion pulses applied either on the head-group or tail-group of the lipid are used to identify the source of the transferred polarization. The normalized cross-relaxation rates of σN,tail =-1.8±0.1 s-1 M-1 and σN,head =-0.5±0.3 s-1 M-1 are obtained, showing a larger polarization transfer from the tail groups. These cross-relaxation rates are determined at a low urea concentration, which constitutes refolding conditions for the protein. The sensitivity enhancement by D-DNP permits to access these conditions with a measurement time on the order of seconds, and may further open the possibility to investigate structural changes in membrane proteins during folding.

Nuclear Spin Hyperpolarization of NH2 - and CH3 -Substituted Pyridine and Pyrimidine Moieties by SABRE.

Hyperpolarization of N-heterocycles with signal amplification by reversible exchange (SABRE) induces NMR sensitivity gains for biological molecules. Substitutions with functional groups, in particular in the ortho-position of the heterocycle, however, result in low polarization using a typical Ir catalyst with a bis-mesityl N-heterocyclic carbene ligand for SABRE, presumably due to steric hindrance. With the addition of allylamine or acetonitrile as coligands to the precatalyst chloro(1,5-cyclooctadiene)[4,5-dimethyl-1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene] iridium, the 1 H signal enhancement increased in several substrates with ortho NH2 substitutions. For example, for a proton in 2,4-diaminopyrimidine, the enhancement factors increased from -7±1 to -210±20 with allylamine or to -160±10 with acetonitrile. CH3 substituted molecules yielded maximum signal enhancements of -25±7 with acetonitrile addition, which is considerably less than the corresponding NH2 substituted molecules, despite exhibiting similar steric size. With the more electron-donating NH2 substitution resulting in greater enhancement, it is concluded that steric hindrance is not the only dominant factor in determining the polarizability of the CH3 substituted compounds. The addition of allylamine increased the signal enhancement for the 290 Da trimethoprim, a molecule with a 2,4-diaminopyrimidine moiety serving as an antibacterial agent, to -70.

Intermolecular interactions determined by NOE build-up in macromolecules from hyperpolarized small molecules.

The nuclear Overhauser effect (NOE) is a primary means to characterize intermolecular interactions using modern NMR spectroscopy. Multiple experiments measured using different mixing time can be used for quantifying NOE buildup and measuring cross-relaxation rates. However, this approach using conventional multi-dimensional NMR is time consuming. Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) can generate deviations from equilibrium spin polarization by orders of magnitude, thereby enhancing signals and allowing to characterize NOE build up in real-time. Since most small molecules can be hyperpolarized using D-DNP, this method is applicable to the study of intermolecular interactions between small molecules and macromolecules. This application is demonstrated using a model system for host-guest interactions including the third generation polyamidoamine dendrimer (G3 PAMAM) and the pharmaceutical phenylbutazone (PBZ). After mixing 1H hyperpolarized PBZ with PAMAM, the NOE build up is directly observed at different sites of the dendrimer in series of one-dimensional NMR spectra. Cross-relaxation rates specific to individual source and target spins are determined from the build up curves. Further, the polarization enhancement is shown to be sufficiently large to allow identification of cross-peaks not observed in a conventional 2D-NOESY spectrum. The improved signal-to-noise ratio provided by hyperpolarization allows for characterizing the intermolecular interaction in an almost instantaneous measurement, opening an application to macromolecular and biomacromolecular NMR.

Metabolic Measurements of Nonpermeating Compounds in Live Cells Using Hyperpolarized NMR.

Hyperpolarization by dissolution dynamic nuclear polarization (D-DNP) has emerged as a technique for enhancing NMR signals by several orders of magnitude, thereby facilitating the characterization of metabolic pathways both in vivo and in vitro. Following the introduction of an externally hyperpolarized compound, real-time NMR enables the measurement of metabolic flux in the corresponding pathway. Spin relaxation however limits the maximum experimental time and prevents the use of this method with compounds exhibiting slow membrane transport rates. Here, we demonstrate that on-line electroporation can serve as a method for membrane permeabilization for use with D-DNP in cell cultures. An electroporation apparatus hyphenated with stopped-flow sample injection permits the introduction of the hyperpolarized metabolite within 3 s after the electrical pulse. In yeast cells that do not readily take up pyruvate, the addition of the electroporation pulse to the D-DNP experiment increases the signals of the downstream metabolic products CO2 and HCO3-, which otherwise are near the detection limit, by 8.2- and 8.6-fold. Modeling of the time dependence of these signals then permits the determination of the respective kinetic rate constants. The observed conversion rate from pyruvate to CO2 normalized for cell density was found to increase by a factor of 12 due to the alleviation of the membrane transport limitation. The use of electroporation therefore extends the applicability of D-DNP to in vitro studies with a wider range of metabolites and at the same time reduces the influence of membrane transport on the observed conversion rates.

Identification of Intracellular and Extracellular Metabolites in Cancer Cells Using 13C Hyperpolarized Ultrafast Laplace NMR.

Ultrafast Laplace NMR (UF-LNMR), which is based on the spatial encoding of multidimensional data, enables one to carry out 2D relaxation and diffusion measurements in a single scan. Besides reducing the experiment time to a fraction, it significantly facilitates the use of nuclear spin hyperpolarization to boost experimental sensitivity, because the time-consuming polarization step does not need to be repeated. Here we demonstrate the usability of hyperpolarized UF-LNMR in the context of cell metabolism, by investigating the conversion of pyruvate to lactate in the cultures of mouse 4T1 cancer cells. We show that 13C ultrafast diffusion- T2 relaxation correlation measurements, with the sensitivity enhanced by several orders of magnitude by dissolution dynamic nuclear polarization (D-DNP), allows the determination of the extra- vs intracellular location of metabolites because of their significantly different values of diffusion coefficients and T2 relaxation times. Under the current conditions, pyruvate was located predominantly in the extracellular pool, while lactate remained primarily intracellular. Contrary to the small flip angle diffusion methods reported in the literature, the UF-LNMR method does not require several scans with varying gradient strength, and it provides a combined diffusion and T2 contrast. Furthermore, the ultrafast concept can be extended to various other multidimensional LNMR experiments, which will provide detailed information about the dynamics and exchange processes of cell metabolites.

Applications of dissolution dynamic nuclear polarization in chemistry and biochemistry.

Sensitivity of detection is one of the most limiting aspects when applying NMR spectroscopy to current problems in the molecular sciences. A number of hyperpolarization methods exist for increasing the population difference between nuclear spin Zeeman states and enhance the signal-to-noise ratio by orders of magnitude. Among these methods, dissolution dynamic nuclear polarization (D-DNP) is unique in its capability of providing high spin polarization for many types of molecules in the liquid state. Originally proposed for biomedical applications including in vivo imaging, applications in high resolution NMR spectroscopy are now emerging. These applications are the focus of the present review. Using D-DNP, a small sample aliquot is first hyperpolarized as a frozen solid at low temperature, followed by dissolution into the liquid state. D-DNP extends the capabilities of liquid state NMR spectroscopy towards shorter timescales and enables the study of nonequilibrium processes, such as the kinetics and mechanisms of reactions. It allows the determination of intermolecular interactions, in particular based on spin relaxation parameters. At the same time, a challenge in the application of this hyperpolarization method is that spin polarization is nonrenewable. Substantial effort has been devoted to develop methods for enabling rapid correlation spectroscopy, the measurement of time-dependent signals, and the extension of the observable time window. With these methods, D-DNP has the potential to open new application areas in the chemical and biochemical sciences.

Characterization of Chemical Exchange Using Relaxation Dispersion of Hyperpolarized Nuclear Spins.

Chemical exchange phenomena are ubiquitous in macromolecules, which undergo conformational change or ligand complexation. NMR relaxation dispersion (RD) spectroscopy based on a Carr-Purcell-Meiboom-Gill pulse sequence is widely applied to identify the exchange and measure the lifetime of intermediate states on the millisecond time scale. Advances in hyperpolarization methods improve the applicability of NMR spectroscopy when rapid acquisitions or low concentrations are required, through an increase in signal strength by several orders of magnitude. Here, we demonstrate the measurement of chemical exchange from a single aliquot of a ligand hyperpolarized by dissolution dynamic nuclear polarization (D-DNP). Transverse relaxation rates are measured simultaneously at different pulsing delays by dual-channel 19F NMR spectroscopy. This two-point measurement is shown to allow the determination of the exchange term in the relaxation rate expression. For the ligand 4-(trifluoromethyl)benzene-1-carboximidamide binding to the protein trypsin, the exchange term is found to be equal within error limits in neutral and acidic environments from D-DNP NMR spectroscopy, corresponding to a pre-equilibrium of trypsin deprotonation. This finding illustrates the capability for determination of binding mechanisms using D-DNP RD. Taking advantage of hyperpolarization, the ligand concentration in the exchange measurements can reach on the order of tens of µM and protein concentration can be below 1 µM, i.e., conditions typically accessible in drug discovery.

Real-Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP†NMR Spectroscopy.

The kinase inhibitory domain of the cell cycle regulatory protein p27Kip1 (p27) was nuclear spin hyperpolarized using dissolution dynamic nuclear polarization (D-DNP). While intrinsically disordered in isolation, p27 adopts secondary structural motifs, including an α-helical structure, upon binding to cyclin-dependent kinase 2 (Cdk2)/cyclin A. The sensitivity gains obtained with hyperpolarization enable the real-time observation of 13 C NMR signals during p27 folding upon binding to Cdk2/cyclin A on a time scale of several seconds. Time-dependent intensity changes are dependent on the extent of folding and binding, as manifested in differential spin relaxation. The analysis of signal decay rates suggests the existence of a partially folded p27 intermediate during the timescale of the D-DNP NMR experiment.

Site specific polarization transfer from a hyperpolarized ligand of dihydrofolate reductase.

Protein-ligand interaction is often characterized using polarization transfer by the intermolecular nuclear Overhauser effect (NOE). For such NOE experiments, hyperpolarization of nuclear spins presents the opportunity to increase the spin magnetization, which is transferred, by several orders of magnitude. Here, folic acid, a ligand of dihydrofolate reductase (DHFR), was hyperpolarized on (1)H spins using dissolution dynamic nuclear polarization (D-DNP). Mixing hyperpolarized ligand with protein resulted in observable increases in protein (1)H signal predominantly in the methyl group region of the spectra. Using (13)C single quantum selection in a series of one-dimensional spectra, the carbon chemical shift ranges of the corresponding methyl groups can be elucidated. Signals observed in these hyperpolarized spectra could be confirmed using 3D isotope filtered NOESY spectra, although the hyperpolarized spectra were obtained in single scans. By further correlating the signal intensities observed in the D-DNP experiments with the occurrence of short distances in the crystal structure of the protein-ligand complex, the observed methyl proton signals could be matched to the chemical shifts of six amino acids in the active site of DHFR-folic acid binary complex. These data demonstrate that (13)C chemical shift selection of protein resonances, combined with the intrinsic selectivity towards magnetization originating from the initially hyperpolarized spins, can be used for site specific characterization of protein-ligand interactions.

Parallelized Ligand Screening Using Dissolution Dynamic Nuclear Polarization.

Protein-ligand interactions are frequently screened using nuclear magnetic resonance (NMR) spectroscopy. The dissociation constant (KD) of a ligand of interest can be determined via a spin-spin relaxation measurement of a reporter ligand in a single scan when using hyperpolarization by means of dissolution dynamic nuclear polarization (D-DNP). Despite nearly instantaneous signal acquisition, a limitation of D-DNP for the screening of protein-ligand interactions is the required polarization time on the order of tens of minutes. Here, we introduce a multiplexed NMR experiment, where a single hyperpolarized ligand sample is rapidly mixed with protein injected into two flow cells. NMR detection is achieved simultaneously on both channels, resulting in a chemical shift resolved spin relaxation measurement. Spectral resolution allows the use of reference compounds for accurate quantification of concentrations. Simultaneous use of two concentration ratios between protein and ligand broadens the range of KD that is accurately measurable in a single experiment to at least an order of magnitude. In a comparison of inhibitors for the protein trypsin, the average KD values of benzamidine and benzylamine were found to be 12.6 ± 1.4 µM and 207 ± 22 µM from three measurements, based on KD = 142 µM assumed known for the reporter ligand 4-(trifluoromethyl)benzene-1-carboximidamide. Typical confidence ranges at 95% evaluated for single experiments were (8.3 µM, 20 µM) and (151 µM, 328 µM). The multiplexed detection of two or more hyperpolarized samples increases throughput of D-DNP by the same factor, improving the applicability to most multipoint measurements that would traditionally be achieved using titrations.

In Situ Determination of Tacticity, Deactivation, and Kinetics in [rac-(C2H4(1-Indenyl)2)ZrMe][B(C6F5)4] and [Cp2ZrMe][B(C6F5)4]-Catalyzed Polymerization of 1-Hexene Using (13)C Hyperpolarized NMR.

The stereochemistry, kinetics, and mechanism of olefin polymerization catalyzed by a set of zirconium-based metallocenes was studied by NMR using dissolution dynamic nuclear polarization (DNP). Hyperpolarized 1-hexene was polymerized in situ with a C2 symmetric catalyst, [(EBI)ZrMe][B(C6F5)4] (EBI = rac-(C2H4(1-indenyl)2)), and a C2v symmetric catalyst, [(Cp)2ZrMe][B(C6F5)4] (Cp = cyclopentadienyl). Hyperpolarized (13)C NMR spectra were used to characterize product tacticity following initiation of the reaction. At the same time, a signal gain of 3 orders of magnitude from (13)C hyperpolarization enabled the real time observation of catalyst-polymeryl species and deactivation products, such as vinylidene and a Zr-allyl complex. The compounds appearing in the reaction provide evidence for the existence of ß-hydride elimination and formation of a dormant site via a methane-generating mechanism. The presence of a deactivating mechanism was incorporated in a model used to determine kinetic parameters of the reaction. On this basis, rate constants were measured between 0.8 and 6.7 mol % of catalyst. The concentration dependence of the rate constants obtained indicates a second-order process for polymerization concomitant with a first-order process for deactivation. The simultaneous observation of both processes in the time evolution of (13)C NMR signals over the course of several seconds underlines the utility of hyperpolarized NMR for quantifying early events in polymerization reactions.

Application of blind source separation to real-time dissolution dynamic nuclear polarization.

The use of a blind source separation (BSS) algorithm is demonstrated for the analysis of time series of nuclear magnetic resonance (NMR) spectra. This type of data is obtained commonly from experiments, where analytes are hyperpolarized using dissolution dynamic nuclear polarization (D-DNP), both in in vivo and in vitro contexts. High signal gains in D-DNP enable rapid measurement of data sets characterizing the time evolution of chemical or metabolic processes. BSS is based on an algorithm that can be applied to separate the different components contributing to the NMR signal and determine the time dependence of the signals from these components. This algorithm requires minimal prior knowledge of the data, notably, no reference spectra need to be provided, and can therefore be applied rapidly. In a time-resolved measurement of the enzymatic conversion of hyperpolarized oxaloacetate to malate, the two signal components are separated into computed source spectra that closely resemble the spectra of the individual compounds. An improvement in the signal-to-noise ratio of the computed source spectra is found compared to the original spectra, presumably resulting from the presence of each signal more than once in the time series. The reconstruction of the original spectra yields the time evolution of the contributions from the two sources, which also corresponds closely to the time evolution of integrated signal intensities from the original spectra. BSS may therefore be an approach for the efficient identification of components and estimation of kinetics in D-DNP experiments, which can be applied at a high level of automation.

Determination of Intermolecular Interactions Using Polarization Compensated Heteronuclear Overhauser Effect of Hyperpolarized Spins.

The nuclear Overhauser effect (NOE) has long been used as a selective indicator for intermolecular interactions. Due to relatively small changes of signal intensity, often on the order of several percent, quantitative NOE measurements can be challenging. Hyperpolarization of nuclear spins can dramatically increase the NOE intensity by increasing population differences, but poses its own challenge in quantifying the original polarization level. Here, we demonstrate a method for the accurate measurement of intermolecular heteronuclear cross-relaxation rates by simultaneous acquisition of signals from both nuclei. Using this method, we measure cross-relaxation rates between water protons and (19)F of trifluoroacetic acid at concentrations ranging from 23 to 72 mM. A concentration-independent value of 2.46 × 10(-4) ± 1.02 × 10(-5) s(-1) M(-1) is obtained at a temperature of 301 K and validated using a nonhyperpolarized measurement. In a broader context, accurate measurement of heteronuclear cross-relaxation rates may enable the study of intermolecular interactions including those involving macromolecules where (19)F atoms can be introduced as site-selective labels.