A PGSE diffusion and electrophoretic NMR study of Cs+ and Na+ dynamics in aqueous crown ether systems

Multinuclear pulsed gradient spin-echo (PGSE) NMR diffusion and linewidth measurements were used to probe binding and transport in aqueous Na+-15-crown-5, Na+-18-crown-6, Cs+-15-crown-5 and Cs+-18-crown-6 systems. Since direct PGSE observation of many alkali cations is precluded by either low inherent sensitivity or rapid relaxation (or both), the feasibility of proton-detected electrophoretic NMR (ENMR) measurements to complement PGSE data was investigated. ENMR measurements were performed on aqueous Cs+-, Li+-, Na+-, K+-, and Rb+- 18-crown-6 systems. The data analysis is based on a two-site binding model and its corresponding association constants. Cs+ was found to bind considerably more tightly to 18-crown-6 (K=8 M-1) than to 15-crown-5 (K approximately 2 M-1), whereas Na+ had almost equal affinity (K approximately 4.5 M-1) for 15-crown-5 and 18-crown-6. The difficulties encountered in analysing the NMR parameters, methodological limitations and the implied need for more complicated binding models are discussed.     

Mass spectrometry assisted assignment of NMR resonances in 15N labeled proteins

Application of nuclear magnetic resonance (NMR) methods for the structural characterization to larger and more complex protein systems can be facilitated through the development of new methods for resonance assignment. Here, a novel approach that relies on integration of NMR and mass spectrometry (MS) methods is explored. The approach relies on the fact that both NMR and MS are able to monitor rates of exchange of amide protons for water deuterons. Correlating the rates can connect cross-peak positions from NMR data with fragment masses from MS data to support sequential assignment. The example provided is for a small model protein, ubiquitin, but the potential for application to large, more difficult to express proteins is clear.     

Quantification of protein-ligand interactions by mass spectrometry,titration, and H/D exchange: PLIMSTEX

Protein-ligand binding and the concomitant conformational change in the protein are of crucial importance in biophysics and drug design. We report a novel method to quantify protein-ligand interactions in solution by mass spectrometry, titration, and H/D exchange (PLIMSTEX). The approach can determine the conformational change, binding stoichiometry, and affinity in protein-ligand interactions including those that involve small molecules, metal ions, and peptides. Binding constants obtained by PLIMSTEX for four model protein-ligand systems agree with K values measured by conventional methods. At higher protein concentration, the method can be used to determine quickly the binding stoichiometry and possibly the purity of proteins. Taking advantage of concentrating the protein on-column and desalting, we are able to use different concentrations of proteins, buffer systems, salts, and pH in the exchange protocol. High picomole quantities of proteins are sufficient, offering significantly better sensitivity than that of NMR and X-ray crystallography. Automation could make PLIMSTEX a high throughput method for library screening, drug discovery, and proteomics.     

Automated Analysis and Simulation of NMR Spectra: Applications to Organo Phosphorus Chemistry

Abstract

Analysis of molecular structures in solution- and liquid-state is based on modern NMR methods. A novel set of computer programs was developed which allows for a more convenient automated analysis of NMR parameters and subsequent simulation of NMR spectra. Programs and techniques used are demonstrated for model systems like:     

Solution NMR approaches for establishing specificity of weak heterodimerization of membrane proteins

Solution NMR provides a powerful approach for detecting complex formation involving weak to moderate intermolecular affinity. However, solution NMR has only rarely been used to detect complex formation between two membrane proteins in model membranes. The impact of specific binding on the NMR spectrum of a membrane protein can be difficult to distinguish from spectral changes that are induced by nonspecific binding and/or by changes that arise from forced cohabitation of the two proteins in a single model membrane assembly. This is particularly the case when solubility limits make it impossible to complete a titration to the point of near saturation of complex formation. In this work experiments are presented that provide the basis for establishing whether specific complex formation occurs between two membrane proteins under conditions where binding is not of high avidity. Application of these methods led to the conclusion that the membrane protein CD147 (also known as EMMPRIN or basigin) forms a specific heterodimeric complex in the membrane with the 99-residue transmembrane C-terminal fragment of the amyloid precursor protein (C99 or APP-βCTF), the latter being the immediate precursor of the amyloid-β polypeptides that are closely linked to the etiology of Alzheimer's disease.     

Nuclear Magnetic Resonance and X-ray Crystallography to Improve Struvite Determination

Ammonium and phosphate removal from industrial streams is important for many chemical processes, especially wastewater and sewerage treatment. Struvite precipitation is a popular choice and has been well characterized in model systems. However, struvite is a member of a family of similar phosphates of various valences and degrees of hydration. This paper shows that the ammonium phosphate precipitation from complex waste of decaying urine is identical to the product in model and other organic systems, i.e., struvite. We have shown that titration assays are possible for struvite, but they are difficult and hence we have also used nuclear magnetic resonance (NMR) and X-ray crystallography. NMR shows the high purity of struvite. Surprisingly, the crystals from this system are large and generate improved structural determination by X-ray crystallography compared to struvite prepared in solution or discovered in other natural systems. These large crystals allow facile separation of struvite, compared to more conventional methods using fluidized bed reactors which generate irregular concretions with difficulty.     

Observation of ligand binding to cytochrome P450 BM-3 by means of solid-state NMR spectroscopy

A broad understanding of the binding modes of ligands and inhibitors to cytochrome P450 is vital for the development of new drugs. We investigated ligand binding in a site-specific fashion on cytochrome P450 BM-3 from Bacillus megaterium, a 119 kDa paramagnetic enzyme, using solid-state magic angle spinning nuclear magnetic resonance methods. Selective labeling and longitudinal relaxation effects were utilized to identify the peaks in a site-specific fashion and to provide evidence for binding. Well-resolved one-dimensional and two-dimensional NMR spectra of cytochrome P450 BM-3 reveal shifts upon binding of its substrate, N-palmitoylglycine. These data are consistent with the crystallographic result that a biochemically important amino acid residue, Phe87, moves upon ligation. This experimental scheme provides a tool for probing ligand binding for complex systems.     

Use of 13C NMR spectrometric data to produce a predictive model of estrogen receptor binding activity

We have developed a spectroscopic data-activity relationship (SDAR) model based on 13C NMR spectral data for 30 estrogenic chemicals whose relative binding affinities (RBA) are available for the alpha (ERalpha) and beta (ERbeta) estrogen receptors. The SDAR models segregated the 30 compounds into strong and medium binding affinities. The SDAR model gave a leave-one-out (LOO) cross-validation of 90%. Two compounds that were classified incorrectly in the SDAR model were in the transition zone between classifications. Real and predicted 13C NMR chemical shifts were used with test compounds to evaluate the predictive behavior of the SDAR model. The 13C NMR SDAR model using predicted 13C NMR data for the test compounds provides a rapid, reliable, and simple way to screen whether a compound binds to the estrogen receptors.     

NMR studies of sorption and adsorption phenomena in colloidal systems

NMR methods provide chemically selective tools, particularly suitable to detect the molecular environment of molecular species in micro-heterogeneous materials. They are consequently applied to solve many questions in colloid science. The present review covers NMR studies of molecular adsorption onto particle surfaces as well as sorption into colloidal particles. Various methods ranging from liquid or solid state spectral analysis over spin relaxation to pulsed field gradient diffusion NMR have been employed in this field, monitoring either the chemical environment or the restricted dynamics of adsorbed or encapsulated guest molecules. Adsorption systems include surfactant layers, stabilizing ligands, small molecules, polymer layers or polyelectrolyte multilayers at the surface of various types of particles. Sorption into colloidal particles and detection of their position in specific compartments of the colloid are particularly relevant in systems employed as colloidal carriers, such as micelles, vesicles, or hollow polymeric capsules. With guest molecules considered as model compounds for drugs these studies have large relevance for the development of nanoparticle drug delivery systems.     

PFG NMR investigations of TPA-TMA-silica mixtures

Pulsed-field gradient (PFG) NMR studies of tetrapropylammonium (TPA)-tetramethylammonium (TMA)-silica mixtures are presented, and the effect of TMA as a foreign ion on the TPA-silica nanoparticle interactions before and after heating has been studied. Dynamic light scattering (DLS) results suggest that silica nanoparticles in these TPA-TMA systems grow via a ripening mechanism for the first 24 h of heating. PFG NMR of mixtures before heating show that TMA can effectively displace TPA from the nanoparticle surface. The binding isotherms of TPA at room temperature obtained via PFG NMR can be described by Langmuir isotherms, and indicate a decrease in the adsorbed amount of TPA upon addition of TMA. PFG NMR also shows a systematic increase in the self-diffusion coefficient of TPA in both the mixed TPA-TMA systems and pure TPA systems with heating time, indicating an increased amount of TPA in solution upon heating. By contrast, a much smaller amount of TMA is observed to desorb from the nanoparticles upon heating. These results point to the desorption of TPA from the nanoparticles being a kinetically controlled process. The apparent desorption rate constants were calculated from fitting the desorbed amount of TPA with time via a pseudosecond-order kinetic model. This analysis show the rate of TPA desorption in TPA-TMA mixtures increases with increasing TMA content, whereas for pure TPA mixtures the rate of TPA desorption is much less sensitive to the TPA concentration.     

How to compare diffusion processes assessed by single-particle tracking and pulsed field gradient nuclear magnetic resonance

Heterogeneous diffusion processes occur in many different fields such as transport in living cells or diffusion in porous media. A characterization of the transport parameters of such processes can be achieved by ensemble-based methods, such as pulsed field gradient nuclear magnetic resonance (PFG NMR), or by trajectory-based methods obtained from single-particle tracking (SPT) experiments. In this paper, we study the general relationship between both methods and its application to heterogeneous systems. We derive analytical expressions for the distribution of diffusivities from SPT and further relate it to NMR spin-echo diffusion attenuation functions. To exemplify the applicability of this approach, we employ a well-established two-region exchange model, which has widely been used in the context of PFG NMR studies of multiphase systems subjected to interphase molecular exchange processes. This type of systems, which can also describe a layered liquid with layer-dependent self-diffusion coefficients, has also recently gained attention in SPT experiments. We reformulate the results of the two-region exchange model in terms of SPT-observables and compare its predictions to that obtained using the exact transformation which we derived.     

SEAL by NMR: Glyco‐Based Selenium‐Labeled Affinity Ligands Detected by NMR Spectroscopy

We report a method for the screening of interactions between proteins and selenium‐labeled carbohydrate ligands. SEAL by NMR is demonstrated with selenoglycosides binding to lectins where the selenium nucleus serves as an NMR‐active handle and reports on binding through ⁷⁷Se NMR spectroscopy. In terms of overall sensitivity, this nucleus is comparable to ¹³C NMR, while the NMR spectral width is ten times larger, yielding little overlap in ⁷⁷Se NMR spectroscopy, even for similar compounds. The studied ligands are singly selenated bioisosteres of methyl glycosides for which straightforward preparation methods are at hand and libraries can readily be generated. The strength of the approach lies in its simplicity, sensitivity to binding events, the tolerance to additives and the possibility of having several ligands in the assay. This study extends the increasing potential of selenium in structure biology and medicinal chemistry. We anticipate that SEAL by NMR will be a beneficial tool for the development of selenium‐based bioactive compounds, such as glycomimetic drug candidates.     

Experimental and theoretical evaluation of multisite cadmium(II) exchange in designed three-stranded coiled-coil peptides

An important factor that defines the toxicity of elements such as cadmium(II), mercury(II), and lead(II) with biological macromolecules is metal ion exchange dynamics. Intriguingly, little is known about the fundamental rates and mechanisms of metal ion exchange into proteins, especially helical bundles. Herein, we investigate the exchange kinetics of Cd(II) using de novo designed three-stranded coiled-coil peptides that contain metal complexing cysteine thiolates as a model for the incorporation of this ion into trimeric, parallel coiled coils. Peptides were designed containing both a single Cd(II) binding site, GrandL12AL16C [Grand = AcG-(LKALEEK)(5)-GNH(2)], GrandL26AL30C, and GrandL26AE28QL30C, as well as GrandL12AL16CL26AL30C with two Cd(II) binding sites. The binding of Cd(II) to any of these sites is of high affinity (K(A) > 3 × 10(7) M(-1)). Using (113)Cd NMR spectroscopy, Cd(II) binding to these designed peptides was monitored. While the Cd(II) binding is in extreme slow exchange regime without showing any chemical shift changes, incremental line broadening for the bound (113)Cd(II) signal is observed when excess (113)Cd(II) is titrated into the peptides. Most dramatically, for one site, L26AL30C, all (113)Cd(II) NMR signals disappear once a 1.7:1 ratio of Cd(II)/(peptide)(3) is reached. The observed processes are not compatible with a simple "free-bound" two-site exchange kinetics at any time regime. The experimental results can, however, be simulated in detail with a multisite binding model, which features additional Cd(II) binding site(s) which, once occupied, perturb the primary binding site. This model is expanded into differential equations for five-site NMR chemical exchange. The numerical integration of these equations exhibits progressive loss of the primary site NMR signal without a chemical shift change and with limited line broadening, in good agreement with the observed experimental data. The mathematical model is interpreted in molecular terms as representing binding of excess Cd(II) to surface Glu residues located at the helical interfaces. In the absence of Cd(II), the Glu residues stabilize the three-helical structure though salt bridge interactions with surface Lys residues. We hypothesize that Cd(II) interferes with these surface ion pairs, destabilizing the helical structure, and perturbing the primary Cd(II) binding site. This hypothesis is supported by the observation that the Cd(II)-excess line broadening is attenuated in GrandL26AE28QL30C, where a surface Glu(28), close to the metal binding site, was changed to Gln. The external binding site may function as an entry pathway for Cd(II) to find its internal binding site following a molecular rearrangement which may serve as a basis for our understanding of metal complexation, transport, and exchange in complex native systems containing α-helical bundles.     

Solid-state 35/37Cl NMR spectroscopy of hydrochloride salts of amino acids implicated in chloride ion transport channel selectivity: opportunities at 900 MHz

The results of a detailed systematic chlorine solid-state NMR study of several hydrochloride salts of amino acids implicated in chloride ion transport channel selectivity are reported. (35)Cl and (37)Cl NMR spectra have been obtained for stationary and/or magic-angle spinning powdered samples of the following compounds on 500 and/or 900 MHz spectrometers: DL-arginine HCl monohydrate, L-lysine HCl, L-serine HCl, L-glutamic acid HCl, L-proline HCl, L-isoleucine HCl, L-valine HCl, L-phenylalanine HCl, and glycine HCl. Spectral analyses provide information on the anisotropic properties and relative orientations of the chlorine electric field gradient and chemical shift (CS) tensors, which are intimately related to the local molecular and electronic structure. Data obtained at 900 MHz provide unique examples of the effects of CS anisotropy on the NMR spectrum of a quadrupolar nucleus. The range of chlorine quadrupolar coupling constants (C(Q)) measured, -6.42 to 2.03 MHz, demonstrates the sensitivity of this parameter to the chloride ion environment and suggests the applicability of chlorine solid-state NMR as a novel experimental tool for defining chloride binding environments in larger ion channel systems. Salts of hydrophobic amino acids are observed to tend to exhibit larger values of C(Q) than salts of hydrophilic amino acids. A simple model for rationalizing the observed trend in C(Q) is proposed. For salts for which neutron diffraction structures are available, we identify a quantum chemical method which reproduces experimental values of C(Q) with a root-mean-square deviation of 0.1 MHz and a correlation coefficient of 0.9998. On the basis of this, chlorine NMR tensors are predicted for the Cl(-) binding site in ClC channels.     

Progress in nuclear magnetic resonance studies of surfactant systems

Nuclear magnetic resonance (NMR), as a powerful technology, is widely used to characterize the physicochemical properties of surfactants in solution. As a sensitive technique to molecular environment, NMR is beyond the reach of other spectral methods in surfactant systems. Recent years, intensive investigations of surfactants by NMR were reported but not well summarized; therefore, we highlight these significant progresses, which may shed light on the challenges to understand their behavior and mechanisms in surfactant systems. The theory of various NMR methods was introduced, including chemical shifts, diffusion, relaxation, 2D nuclear Overhauser effect spectroscopy and rotating frame nuclear Overhauser effect spectroscopy. The behavior, interaction, and mechanisms among surfactants and other molecules from NMR technologies were discussed. Challenges to understand the behavior and mechanisms in surfactant systems and instrumentation limits are addressed as perspectives.     

Quantifying Mass Transport during Polarization in a Li Ion Battery Electrolyte by in Situ (7)Li NMR Imaging

Poor mass transport in the electrolyte of Li ion batteries causes large performance losses in high-power applications such as vehicles, and the determination of transport properties under or near operating conditions is therefore important. We demonstrate that in situ (7)Li NMR imaging in a battery electrolyte can directly capture the concentration gradients that arise when current is applied. From these, the salt diffusivity and Li(+) transport number are obtained within an electrochemical transport model. Because of the temporal, spatial, and chemical resolution it can provide, NMR imaging will be a versatile tool for evaluating electrochemical systems and methods.     

Low-field 1H NMR spectroscopy: Factors impacting signal-to-noise ratio and experimental time in the context of mixed microstructure polyisoprenes

Low-cost, high-accuracy characterization of polymeric materials is critical for satisfying societal demand for high-quality materials with ultra-specific requirements. Low-field nuclear magnetic resonance (NMR) spectroscopy presents an opportunity to replace costlier or destructive methods while utilizing nondeuterated solvents. Many factors play key roles in the ability of low-field NMR spectroscopy to accurately analyze polymer systems. Sample characteristics such as polymer concentration, composition, and molecular weight all directly affect the capability of low-field spectrometers to accurately determine polymer microstructure compositions. In addition to inherent sample properties affecting instrumental accuracy, many choices concerning instrumental parameters (including number of scans, relaxation delay, spectral width, and points per scan) must be made that impact the quality of the resulting NMR spectra. In this work, we benchmark the capability of a 60-MHz low-field NMR spectrometer for analyzing polymer materials using mixed microstructure polyisoprenes as a model polymer system of interest. The aforementioned critical sample and instrumental variables are varied, and we report on the ability to quantitatively characterize polyisoprene microstructure to within 1–2% of a higher field NMR spectrometer (400 MHz). We anticipate our findings to be generally applicable to other low-field spectrometers of similar field strength and other polymer systems.     

Affinity NMR: decoding DNA binding

We have shown that affinity NMR can be used to edit a NMR spectrum so that ligands that have affinity to DNA can be observed in the presence of other nonbinding molecules. Diffusion encoded spectroscopy (DECODES) can be used to identify the binding ligands. We were able to identify Hoechst 33342 as binding to the Drew-Dickerson dodecamer d(CGCGAATTCGCG)2 in the presence of the nonbinding molecules adenine, adenosine, and thiamine. Affinity NMR appears to be readily applicable to DNA systems for the following reasons. (1) The relaxation rate of the DNA oligonucleotides is favorable, thus the signal intensity loss due to relaxation is not severe. (2) A comparison of the patterns of the DNA cross-peaks upon binding in the two-dimensional total correlation spectroscopy and correlation spectroscopy spectrum are easily performed, and the ligand signals in the two-dimensional DECODES spectrum can be readily identified. (3) The aromatic part of the DNA spectrum is devoid of 2D cross-peaks in these correlation spectra, greatly facilitating the interpretation of the bound ligand in the DECODES spectrum.     

MAS NMR structure of a microcrystalline Cd-bacteriochlorophyll d analogue

Solid-state NMR is an emerging method to obtain structural information in molecular biology and nanotechnology for systems that are inaccessible to solution NMR or diffraction methods. While solution NMR generally converges upon families of structures in a bottom-up approach, solid NMR structure determination will have to take into account the top-down constraints that follow from the additional requirement that the entire 3D space must be packed in an orderly fashion. We used MAS NMR together with molecular modeling calculations in steps to establish a detailed model of the local crystal structure of an aggregate of uniformly 13C- and 15N-labeled Cd-chlorophyllide d, a model for the chlorosomal antennae. In this way we converge upon a space group P21 with a = 14.3 A, b = 27.3 A, c = 6.4 A, beta = 147.2 degrees and Z = 2.