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

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

Hyperpolarized Hadamard Spectroscopy Using Flow NMR

The emergence of the dissolution dynamic nuclear polarization (D-DNP) technique provides an important breakthrough to overcome inherent sensitivity limitations in nuclear magnetic resonance (NMR) experiments. In dissolution DNP, only a small amount of frozen sample is polarized, dissolved, and injected into an NMR spectrometer. Although substantially enhanced NMR signals can be obtained, the single scan nature of this technique a priori impedes the use of correlation experiments, which represent some of the most powerful applications of NMR spectroscopy. Here, an alternative method for multiscan spectroscopy from D-DNP samples utilizing a flow NMR probe is described. Multiple hyperpolarized segments of sample are sequentially injected using a purpose designed device. Hadamard spectroscopy can then be applied for obtaining chemical shift correlation information even from a small number of scans. This capability is demonstrated with a four-scan data set for obtaining the [¹³C,¹H] correlations in the test molecule 1-butanol. Because of the effects of spin–lattice relaxation and concentration gradients in the D-DNP experiment, the subtractive process for Hadamard reconstruction requires an additional step of intensity scaling. For this purpose, a reconstruction procedure was developed that uses entropy maximization and is robust with respect to noise and signal overlap. In a broader sense, the multiscan NMR as described here is amenable to various correlation NMR experiments, and increases the versatility of D-DNP in small-molecule characterization

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

Modeling of Polarization Transfer Kinetics in Protein Hydration Using Hyperpolarized Water

Water–protein interactions play a central role in protein structure, dynamics, and function. These interactions, traditionally, have been studied using nuclear magnetic resonance (NMR) by measuring chemical exchange and nuclear Overhauser effect (NOE). Polarization transferred from hyperpolarized water can result in substantial transient signal enhancements of protein resonances due to these processes. Here, we use dissolution dynamic nuclear polarization and flow-NMR for measuring the pH dependence of transferred signals to the protein trypsin. A maximum enhancement of 20 is visible in the amide proton region of the spectrum at pH 6.0, and of 47 at pH 7.5. The aliphatic region is enhanced up to 2.3 times at pH 6.0 and up to 2.5 times at pH 7.5. The time dependence of these observed signals can be modeled quantitatively using rate equations incorporating chemical exchange to amide sites and, optionally, intramolecular NOE to aliphatic protons. On the basis of these two- and three-site models, average exchange (<i>k</i>_ex) and cross-relaxation rates (σ) obtained were <i>k</i>_ex = 12 s^–1, σ = −0.33 s^–1 for pH 7.5 and <i>k</i>_ex = 1.8 s^–1, σ = −0.72 s^–1 for pH 6.0 at a temperature of 304 K. These values were validated using conventional EXSY and NOESY measurements. In general, a rapid measurement of exchange and cross-relaxation rates may be of interest for the study of structural changes of the protein occurring on the same time scale. Besides protein–water interactions, interactions with cosolvent or solutes can further be investigated using the same methods

Parallelized Ligand Screening Using Dissolution Dynamic Nuclear Polarization

Protein–ligand interactions are frequently screened using nuclear magnetic resonance (NMR) spectroscopy. The dissociation constant (<i>K</i>_D) 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 <i>K</i>_D 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 <i>K</i>_D values of benzamidine and benzylamine were found to be 12.6 ± 1.4 μM and 207 ± 22 μM from three measurements, based on <i>K</i>_D = 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

Chemical Shift Correlations from Hyperpolarized NMR Using a Single SHOT

A significant challenge in realizing the promise of the dissolution dynamic nuclear polarization technique for signal enhancement in high-resolution NMR lies in the nonrenewability of the hyperpolarized spin state. This property prevents the application of traditional two-dimensional correlation spectroscopy, which relies on regeneration of spin polarization before each successive increment of the indirect dimension. Since correlation spectroscopy is one of the most important approaches for the identification and structural characterization of molecules by NMR, it is important to find easily applicable methods that circumvent this problem. Here, we introduce the application of scaling of heteronuclear couplings by optimal tracking (SHOT) to achieve this goal. SHOT decoupling pulses have been numerically optimized on the basis of optimal control algorithms to obtain chemical shift correlations in C–H groups, either by acquiring a single one-dimensional ¹³C spectrum with ¹H off-resonance decoupling or vice versa. Vanillin, which contains a number of functional groups, was used as a test molecule, allowing the demonstration of SHOT decoupling tailored toward simplified and accurate data analysis. This strategy was demonstrated for two cases: First, a linear response to chemical shift offset in the correlated dimension was optimized. Second, a pulse with alternating linear responses in the correlated dimension was chosen as a goal to increase the sensitivity of the decoupling response to the chemical shift offset. In these measurements, error ranges of ±0.03 ppm for the indirectly determined ¹H chemical shifts and of ±0.4 ppm for the indirectly determined ¹³C chemical shifts were found. In all cases, we show that chemical shift correlations can be obtained from information contained in a single scan, which maximizes the ratio of signal to stochastic noise. Furthermore, a comprehensive discussion of the robustness of the method toward nonideal conditions is included based on experimental and simulated data. Unique features of this technique include the abilities to control the accuracy of chemical shift determination in spectral regions of interest and to acquire such chemical shift correlations rapidlythe latter being of interest for potential application in real-time spectroscopy

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 ¹⁹F 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 ¹⁹F atoms can be introduced as site-selective labels

Multinuclear Detection of Nuclear Spin Optical Rotation at Low Field

We describe the multinuclear detection of nuclear spin optical rotation (NSOR), an effect dependent on the hyperfine interaction between nuclear spins and electrons. Signals of ¹H and ¹⁹F are discriminated by frequency in a single spectrum acquired at sub-millitesla field. The simultaneously acquired optical signal along with the nuclear magnetic resonance signal allows the calculation of the relative magnitude of the NSOR constants corresponding to different nuclei within the sample molecules. This is illustrated by a larger NSOR signal measured at the ¹⁹F frequency despite a smaller corresponding spin concentration. Second, it is shown that heteronuclear <i>J</i>-coupling is observable in the NSOR signal, which can be used to retrieve chemical information. Multinuclear frequency and <i>J</i> resolution can localize optical signals in the molecule. Properties of electronic states at multiple sites in a molecule may therefore ultimately be determined by frequency-resolved NSOR spectroscopy at low field

Measurement of Kinetics and Active Site Distances in Metalloenzymes Using Paramagnetic NMR with ¹³C Hyperpolarization

Paramagnetic relaxation enhancement (PRE) conjoint with hyperpolarized NMR reveals structural information on the enzyme–product complex in an ongoing metalloenzyme-catalyzed reaction. Substrates of pseudouridine monophosphate glycosidase are hyperpolarized using the dynamic nuclear polarization (DNP) method. Time series of ¹³C NMR spectra are subsequently measured with the enzyme containing diamagnetic Mg²⁺ or paramagnetic Mn²⁺ ions in the active site. The differences of the signal evolution and line widths in the Mg²⁺ vs Mn²⁺ reactions are explained through PRE in the enzyme-bound product, which is in fast exchange with its free form. Here, a strong distance dependence of the paramagnetically enhanced relaxation rates enables the calculation of distances from product atoms to the metal center in the complexed structure. The same method can be used to add structural information to real-time characterizations of chemical processes involving compounds with naturally present or artificially introduced paramagnetic sites