Chemical Exchange Rates Research Articles

Hydrophobic Side Chain Dynamics of a Glutamate Receptor Ligand Binding Domain

Ionotropic glutamate receptors are ligand-gated ion channels that mediate much of the fast excitatory neurotransmission in the central nervous system. The extracellular ligand binding core (S1S2) of the GluR2 subtype of ionotropic glutamate receptors can be produced as a soluble protein with properties essentially identical to the corresponding domain in the intact, membrane-bound protein. Using a variety of biophysical techniques, much has been learned about the structure and dynamics of S1S2 and the relationship between its ligand-induced conformational changes and the function of the receptor. It is clear that dynamic processes are essential to the function of ionotropic glutamate receptors. We have isotopically labeled side chain methyls of GluR2 S1S2 and used NMR spectroscopy to study their dynamics on the ps-ns and mus-ms time scales. Increased disorder is seen in regions that are part of the key dimer interface in the intact protein. When glutamate is bound, the degree of ps-ns motion is less than that observed with other ligands, suggesting that the physiological agonist binds to a preformed binding site. At the slower time scales, the degree of S1S2 flexibility induced by ligand binding is greatest for willardiine partial agonists, least for antagonists, and intermediate for full agonists. Notable differences among bound ligands are in the region of the protein that forms a hinge between two lobes that close upon agonist binding, and along the beta-sheet in Lobe 2. These motions provide clues as to the functional properties of partial agonists and to the conformational changes associated with lobe closure and channel activation.

Towards p-type conductivity inSnO2 nanocrystals through Li doping

This paper examines electrical transport properties and Li doping inSnO2 synthesized by the sol–gel method. Solid-state 7Li-NMR lineshapes reveal that Li ions occupy two distinct sites with differing dynamicmobilities. The chemical exchange rate between the two sites is, however, too slowfor detection on the NMR timescale. Compressed nanoparticulate films of thisdoped semiconductor exhibit a positive Seebeck coefficient implying a p-typeconductivity. A variable-temperature direct current conductivity, over a 25–350 °C temperature range, follows an Efros–Shklovskii variable range hopping (ES-VRH) conduction mechanism(ln(ρ) versusT−1/2) at temperaturesbelow 100 °C with a crossover to 2D Mott variable range hopping (M-VRH)(ln(ρ) versusT−1/3) conduction attemperatures above 250 °C. In a transition region between these two limiting behaviors, the dc resistivityexhibits an anomalous temperature-independent plateau. We suggest that itsorigin may lie in a carrier inversion phenomenon wherein the majority carriersswitch from holes to electrons due to Li ion expulsion from the crystalline core andcreation of oxygen vacancies generated by loss of oxygen at elevated temperatures.

Structural tightening and interdomain communication in the catalytic cycle of phosphoglycerate kinase.

Changes in amide-NH chemical shift and hydrogen exchange rates as phosphoglycerate kinase progresses through its catalytic cycle have been measured to assess whether they correlate with changes in hydrogen bonding within the protein. Four representative states were compared: the free enzyme, a product complex containing 3-phosphoglyceric acid (3PG), a substrate complex containing ADP and a transition-state analogue (TSA) complex containing a 3PG-AlF(4)(-)-ADP moiety. There are an overall increases in amide protection from hydrogen exchange when the protein binds the substrate and product ligands and an additional increase when the TSA complex is formed. This is consistent with stabilisation of the protein structure by ligand binding. However, there is no correlation between the chemical shift changes and the protection factor changes, indicating that the protection factor changes are not associated with an overall shortening of hydrogen bonds in the protected ground state, but rather can be ascribed to the properties of the high-energy, exchange-competent state. Therefore, an overall structural tightening mechanism is not supported by the data. Instead, we observed that some cooperativity is exhibited in the N-domain, such that within this domain the changes induced upon forming the TSA complex are an intensification of those induced by binding 3PG. Furthermore, chemical shift changes induced by 3PG binding extend through the interdomain region to the C-domain beta-sheet, highlighting a network of hydrogen bonds between the domains that suggests interdomain communication. Interdomain communication is also indicated by amide protection in one domain being significantly altered by binding of substrate to the other, even where no associated change in the structure of the substrate-free domain is indicated by chemical shifts. Hence, the communication between domains is also manifested in the accessibility of higher-energy, exchange-competent states. Overall, the data that are consistent with structural tightening relate to defined regions and are close to the 3PG binding site and in the hinge regions of 3-phosphoglycerate kinase.

TROSY-selected ZZ-exchange experiment for characterizing slow chemical exchange in large proteins

A TROSY-selected ZZ-exchange experiment is described for measuring slow chemical exchange rates by monitoring the TROSY component of (15)N longitudinal magnetization. Application of the proposed pulse sequence to the cadherin 8 N-terminal extracelluar domain demonstrates that enhanced sensitivity is obtained, compared to a previously described TROSY-detected ZZ-exchange sequence (Sahu et al. J Am Chem Soc 129: 13232-13237, 2007), by preserving the TROSY effect during the mixing period as well as the frequency encoding periods.

Peptidyl-Prolyl Isomerase FKBP52 Controls Chemotropic Guidance of Neuronal Growth Cones via Regulation of TRPC1 Channel Opening

Immunophilins, including FK506-binding proteins (FKBPs), are protein chaperones with peptidyl-prolyl isomerase (PPIase) activity. Initially identified as pharmacological receptors for immunosuppressants to regulate immune responses via isomerase-independent mechanisms, FKBPs are most highly expressed in the nervous system, where their physiological function as isomerases remains unknown. We demonstrate that FKBP12 and FKBP52 catalyze cis/trans isomerization of regions of TRPC1 implicated in controlling channel opening. FKBP52 mediates stimulus-dependent TRPC1 gating through isomerization, which is required for chemotropic turning of neuronal growth cones to netrin-1 and myelin-associated glycoprotein and for netrin-1/DCC-dependent midline axon guidance of commissural interneurons in the developing spinal cord. By contrast, FKBP12 mediates spontaneous opening of TRPC1 through isomerization and is not required for growth cone responses to netrin-1. Our study demonstrates a novel physiological function of proline isomerases in chemotropic nerve guidance through TRPC1 gating and may have significant implication in clinical applications of immunophilin-related therapeutic drugs.

On-line mass spectrometry: membrane inlet sampling.

Significant insights into plant photosynthesis and respiration have been achieved using membrane inlet mass spectrometry (MIMS) for the analysis of stable isotope distribution of gases. The MIMS approach is based on using a gas permeable membrane to enable the entry of gas molecules into the mass spectrometer source. This is a simple yet durable approach for the analysis of volatile gases, particularly atmospheric gases. The MIMS technique strongly lends itself to the study of reaction flux where isotopic labeling is employed to differentiate two competing processes; i.e., O2 evolution versus O2 uptake reactions from PSII or terminal oxidase/rubisco reactions. Such investigations have been used for in vitro studies of whole leaves and isolated cells. The MIMS approach is also able to follow rates of isotopic exchange, which is useful for obtaining chemical exchange rates. These types of measurements have been employed for oxygen ligand exchange in PSII and to discern reaction rates of the carbonic anhydrase reactions. Recent developments have also engaged MIMS for online isotopic fractionation and for the study of reactions in inorganic systems that are capable of water splitting or H2 generation. The simplicity of the sampling approach coupled to the high sensitivity of modern instrumentation is a reason for the growing applicability of this technique for a range of problems in plant photosynthesis and respiration. This review offers some insights into the sampling approaches and the experiments that have been conducted with MIMS.

Role of loop-loop interactions in coordinating motions and enzymatic function in triosephosphate isomerase.

The enzyme triosephosphate isomerase (TIM) has been used as a model system for understanding the relationship between protein sequence, structure, and biological function. The sequence of the active site loop (loop 6) in TIM is directly correlated with a conserved motif in loop 7. Replacement of loop 7 of chicken TIM with the corresponding loop 7 sequence from an archaeal homologue caused a 10(2)-fold loss in enzymatic activity, a decrease in substrate binding affinity, and a decrease in thermal stability. Isotope exchange studies performed by one-dimensional (1)H NMR showed that the substrate-derived proton in the enzyme is more susceptible to solvent exchange for DHAP formation in the loop 7 mutant than for WT TIM. TROSY-Hahn Echo and TROSY-selected R(1rho) experiments indicate that upon mutation of loop 7, the chemical exchange rate for active site loop motion is nearly doubled and that the coordinated motion of loop 6 is reduced relative to that of the WT. Temperature dependent NMR experiments show differing activation energies for the N- and C-terminal hinges in this mutant enzyme. Together, these data suggest that interactions between loop 6 and loop 7 are necessary to provide the proper chemical context for the enzymatic reaction to occur and that the interactions play a significant role in modulating the chemical dynamics near the active site.

Slow backbone dynamics of chicken villin headpiece subdomain probed by NMR C′N cross‐correlated relaxation

We have investigated slow correlated motions of neighboring carbonyl and nitrogen nuclei in the backbone of chicken villin headpiece subdomain. Cross-correlated chemical shift modulation experiments were performed at three temperatures where the protein remains in its folded state. The results at 8 degrees C demonstrate the presence of microseconds to milliseconds timescale motions for a number of residues belonging both to helices and unstructured regions. As the temperature is raised, the motions become progressively less visible. The reduction of the contributions of slow motions into the cross-correlated relaxation rate with the rise in temperature is caused by the increase of the chemical exchange rate constants for the slow motion processes.

A study of adsorption and molecular dynamics of spin-labeled molecules on the surface of silica nanoparticles

Stable nitroxyl radicals of different structures, hydrophobicities, and electric charges were used as spin probes for studying the adsorption and molecular dynamics of the adsorbed molecules on the surface of LEVASIL silica nanoparticles. Neutral hydrophobic probes, namely, spin labeled derivatives of indole, are not adsorbed on the nanoparticles; however, the microviscosity and hydrophobicity of their environment differ from those in aqueous solutions. pH in the LEVASIL suspension is measured using a probe with an amino group. A study of the adsorption of a series of positively charged spin probes with hydrocarbon substituents of different lengths showed that the hydrophobic interactions do not contribute to their binding to the nanoparticle surface. The binding constants, the average number of negatively charged adsorption centers per nanoparticle, and the surface potential were determined from the adsorption isotherms of the radical cations. The rotational mobility parameters of the adsorbed radicals were estimated after analysis of the EPR line shape. A dependence of these parameters on the total spin probe concentration is observed. This is explained by the rapid dynamic equilibrium (chemical exchange) between the adsorbed and unbound spin probes. The chemical exchange rates are estimated. A slow increase in adsorption in the equilibrium nanoparticle suspension (with a characteristic time of ∼104 min) and a change in adsorption isotherms (accompanied by a change in the dynamical parameters of the adsorbed particles) are detected. Slow changes in these parameters are attributed to the existence of sufficiently deep energy traps (pores) where the adsorbed radicals slowly diffuse. The rotational mobility of the radicals in these pores is less than in the surface adsorption centers.

Dynamical Quorum Sensing and Synchronization in Large Populations of Chemical Oscillators

Populations of certain unicellular organisms, such as suspensions of yeast in nutrient solutions, undergo transitions to coordinated activity with increasing cell density. The collective behavior is believed to arise through communication by chemical signaling via the extracellular solution. We studied large, heterogeneous populations of discrete chemical oscillators (approximately 100,000) with well-defined kinetics to characterize two different types of density-dependent transitions to synchronized oscillatory behavior. For different chemical exchange rates between the oscillators and the surrounding solution, increasing oscillator density led to (i) the gradual synchronization of oscillatory activity, or (ii) the sudden "switching on" of synchronized oscillatory activity. We analyze the roles of oscillator density and exchange rate of signaling species in these transitions with a mathematical model of the interacting chemical oscillators.

New “multicolor” polypeptide diamagnetic chemical exchange saturation transfer (DIACEST) contrast agents for MRI

An array of 33 prototype polypeptides was examined as putative contrast agents that can be distinguished from each other based on the chemical exchange saturation transfer (CEST) mechanism. These peptides were chosen based on predictions of the chemical exchange rates of exchangeable amide, amine, and hydroxyl protons that produce this contrast, and tested at 11.7T for their CEST suitability. Artificial colors were assigned to particular amino acid units (lysine, arginine, threonine, and serine) based on the separate resonance frequencies of these exchangeable protons. The magnitude of the CEST effect could be fine-tuned by altering the amino acid sequence, and these three exchangeable groups could be distinguished in an MR phantom based on their different chemical shifts ("colors"). These new diamagnetic CEST (DIACEST) agents possess a wide range of electrostatic charges, compositions, and protein stabilities in vivo, making them potentially suitable for a variety of biological applications such as designing MR reporter genes for imaging cells and distinguishing multiple targets within the same MR image.

Inclusion complexes of cryptophane–E with dichloromethane and chloroform: A thermodynamic and kinetic study using the 1D‐EXSY NMR method

Complexation equilibria and kinetics of exchange of chloroform and dichloromethane molecules between the cavity of cryptophane-E and bulk solution were investigated using NMR methods. Using one-dimensional magnetization transfer (1D-EXSY type sequence), chemical exchange rates were measured in different temperature ranges, limited by the equilibrium constant values of the complexes and the boiling points of the guest substances. From the kinetic data, activation energies were calculated using the Arrhenius equation. From the temperature-dependence of the association constant data, the enthalpy and entropy of complexation were estimated and compared with values for similar complexes of other cryptophanes.

Imaging pH using the chemical exchange saturation transfer (CEST) MRI: Correction of concomitant RF irradiation effects to quantify CEST MRI for chemical exchange rate and pH

Chemical exchange saturation transfer (CEST) MRI has been shown capable of detecting dilute labile protons and abnormal tissue glucose/oxygen metabolism, and thus, may serve as a complementary imaging technique to the conventional MRI methods. CEST imaging, however, is also dependent on experimental parameters such as the power, duration, and waveform of the irradiation RF pulse. As a result, its sensitivity and specificity for microenvironment properties such as pH is not optimal. In this study, the dependence of CEST contrast on experimental parameters was solved and an iterative compensation algorithm was proposed that corrects the experimentally measured CEST contrast from the concomitant RF irradiation effects. The proposed algorithm was verified with both numerical simulation and experimental measurements from a tissue-like pH phantom, and showed that pH derived from the compensated CEST imaging agrees reasonably well with pH-electrode measurements within 0.1 pH unit. In sum, our study validates the use of a correction algorithm to compensate CEST imaging from concomitant RF irradiation effects for accurate calibration of the chemical exchange rate, and demonstrates the feasibility of pH imaging with CEST MRI.

Oxygen surface exchange studies in thin film Gd-doped ceria

Electrical conductivity relaxation measurements were performed on gadolinium doped ceria thin films to evaluate chemical surface exchange rate of oxygen (Kex, cm/s) under reducing ambient. The measurements were performed under identical conditions in bulk and thin films as a function of thickness (35–440nm), temperature (943–1158K), and oxygen partial pressure (10−21–10−12atm) using a custom built small volume cell assembly. The Kex in thin films is found to be over an order lower than for bulk samples. Segregation effects in thin films likely lead to near-surface carrier depletion thereby decreasing oxygen exchange rate.

State-to-state dissociation–recombination and chemical exchange rate coefficients in excited diatomic gas flows

In this paper, simple analytical state-to-state rate coefficients for the dissociation–recombination and chemical exchange reactions are presented on the basis of kinetic theory in nonequilibrium excited diatomic gases. They take into account the excited vibrational and electronic states of the chemical species and are expressed according to the preferential character of the chemical reactions. Evolution of these rate coefficients varying according to the translational temperature, bringing into play molecules CO and C 2, are discussed.

Oxygen nonstoichiometry and ionic transport in La2Ni(Fe)O4 + δ

Abstract The steady-state oxygen permeation fluxes through dense La2NiO4 + δ and La2Ni0.9Fe0.1O4 + δ ceramics, studied at 973–1223 K for membrane thickness range 0.6 to 2.0 mm, are limited by both bulk ambipolar conductivity and surface exchange kinetics. The permeability data, in combination with total conductivity and equilibrium p(O2)–T–δ diagrams, were used in numerical regression analysis to extract the local chemical potential gradients, defect concentrations, partial conductivities and exchange rates. Doping with iron was found to increase oxygen-ion mobility in K2NiF4-type lanthanum nickelate at 1173–1223 K, whilst activation energies remain essentially similar, 69–80 kJ/mol. At lower temperatures, the surface kinetics and ionic transport in La2Ni0.9Fe0.1O4 + δ become both slower than those in La2NiO4 + δ. Possible defect-interaction and exchange mechanisms relevant to this behavior are briefly discussed.

Measurement of spin‐lattice relaxation times and chemical exchange rates in multiple‐site systems using progressive saturation

A new method for measuring spin-lattice relaxation times and chemical exchange (CE) rate constants in multiple-site exchanging systems is described. The method, chemical exchange and T(1) measurement using progressive saturation (CUPS), was applied to determine T(1)s and analyze phosphorus exchange among phosphocreatine (PCr), ATP, and inorganic phosphate (Pi), mediated by creatine kinase (CK) and ATP synthase, using (31)P-MRS. Two-site exchange was analyzed in vitro and in the rat leg, and three-site exchange was analyzed in the rat heart. Data were fitted to a model of progressive saturation incorporating T(1) relaxation and CE. For the in vitro system at 8.45 T, we found T(1)(PCr)=2.86 s and T(1)(gamma-ATP)=1.72 s. For the rat gastrocnemius at 1.9T, we found T(1)(PCr) = 6.60 s and T(1)(gamma-ATP) = 2.06 s. For the rat heart at 9.4 T, we found T(1)(PCr)=3.35 s, T(1)(gamma-ATP)=0.69 s, and T(1)(Pi=1.83 s. All of these values were within 20% of literature values. Similarly, the determined exchange rates were in the same range as published values. Using simulations, we compared CUPS with transient saturation transfer as a method for measuring T(1)s and rates. The two methods showed similar sensitivity to noise. We conclude that CUPS is a viable alternative for measuring T(1)s and CE rates in exchanging systems.

Molecular Dynamics of Pyridine Adsorbed on the Silica Surface

The molecular dynamics of pyridine adsorbed onto dry high-surface-area silica gel was studied using solid-state deuterium NMR spectroscopy. Selectively and totally deuterated pyridine samples were used to identify the spectral signatures associated with each ring location and to identify the dynamical modes present in the surface adsorbed species. Loading levels corresponding to 0.8 and 0.2 monolayer coverage were studied. At 174 K and 0.8 monolayer, the adsorbed pyridine molecules are found in three motional modes: a fraction is found to be static, a larger fraction is found executing fast discontinuous rotation (180° ring flips) about the pyridine Cγ−N axis, and a very small fraction is found in fast continuous rotation (diffusion) about the pyridine Cγ−N axis, all referred to a 5 × 10-6 s time scale. No evidence of O−H−N rotation about the Si−O bond (“swinging arm” motion) is observed. The pyridine flips and rotations permit measurement of the angle between the Cγ−N axis and each of the Cα−D and Cβ−D axes are found to be 55.9 ± 0.3° and 60.4 ± 0.5°, respectively. As the temperature is lowered, the fraction of static pyridine rings increases, while the fractions of flipping and freely rotating pyridine rings decrease. At 78 K and 0.8 monolayer, pyridine ring flip motion is still observed, implying a very low potential barrier to discontinuous rotation for some of the surface population. At 221 K, the flipping rings dominate, rotating rings are a minority, and no static rings remain. Above 221 K, the chemical exchange rate among pyridine molecules becomes significant and further motional averaging is observed. At 221 K and below, the spectra show no contributions corresponding to intermediate-rate motions; the notable absence of intermediate-rate patterns is explained in terms of a previously proposed model based on a broad distribution of motional correlation times. The surface structures and dynamics with 0.2 monolayer are found to be substantially similar to those found at 0.8 monolayer. These dynamical data are interpreted in terms of the structure of the silica surface as probed by pyridine molecules.

NMR analysis of GaPO 4 crystal growth in mixtures of phosphoric and sulfuric acids

Gallium orthophosphate solutions prepared from the system: Acid-GaPO 4-H 2O, where Acid=H 3PO 4, H 2SO 4 or H 3PO 4-H 2SO 4, have been investigated by 31P and 71Ga high resolution NMR at temperatures lower than room temperature. The effect of the concentration of acids, of their composition, as well as the concentration of gallophosphate is studied to assign peaks. Although the detailed structure of isolated species remains unresolved, the picture of the chemistry, taking place in these solutions, can be qualitatively described in terms of the first sphere of complexation of the observed nuclei. Mainly three signals are observed in 71Ga NMR spectra. A relative narrow peak at around 0 ppm is due to non-bonded hexaaquaa-gallium species, while resonances at −12 to −14 ppm and broader signals at −24 to −36 ppm are attributed to gallium in direct interaction with sulfates and phosphates species, respectively. 31P NMR spectra reveal five distinct chemical shift regions. Free phosphate species resonate at 0–1 ppm, P nuclei in phosphate moiety directly bonded to one Ga resonate at −4 to −5 ppm, to two Ga at −7 to −8 ppm, to three Ga at −9 to −10 ppm, and to four Ga at −12 to −14 ppm. A correlation between the chemical shift range and the number of Ga surrounding a phosphate group obtained in 2D EXSY 31P NMR experiments confirms the assignments of the 31P NMR resonances. The interconversions between all the phosphate species are established, and the chemical exchange rate constants are determined at −21 °C. These results are discussed on the basis of a proposal concerning the main species and their respective interconversions. Among them the four-ring (GaPO) 2 cyclic dimer, is singled out to be the most reactive building unit for the crystal growth processes.

Comparative 1H NMR and molecular modeling study of hydroxy protons of β- d-Gal p-(1→4)-β- d-Glc pNAc-(1→2)-α- d-Man p-(1→O)(CH 2) 7CH 3 analogues in aqueous solution

The 1H chemical shifts, coupling constants, temperature coefficients, exchange rates, and inter-residual ROEs have been measured, in aqueous solution, for the hydroxy and amine/amide proton resonances of a set of β- d-Gal p-(1→4)-β- d-Glc pNAc-(1→2)-α- d-Man p-(1→O)(CH 2) 7CH 3 analogues. From the structural data, a few significant structural features could be ascertained, such as a preferential anti-conformation for the amide protons of the N-acetyl and N-propionyl groups. The introduction of systematic modifications at Gal 2-C and Gal 6-C resulted in alterations of the Gal 4-OH, Gal 3-OH, and GlcNAc 3-OH areas, since variations in chemical shifts and temperature coefficient were observed. In order to verify the possibility of hydrogen bonds, molecular dynamics simulations in the gas phase and explicit solvent were performed and correlated with the experimental data. A network of hydrogen bonds to solvent molecules was observed, but no strong intramolecular hydrogen bonding was observed.