Amide Proton Exchange Rates Research Articles

In vivo pH mapping with omega plot-based quantitative chemical exchange saturation transfer MRI.

Chemical exchange saturation transfer (CEST) MRI is promising for detecting dilute metabolites and microenvironment properties, which has been increasingly adopted in imaging disorders such as acute stroke and cancer. However, in vivo CEST MRI quantification remains challenging because routine asymmetry analysis (MTRasym ) or Lorentzian decoupling measures a combined effect of the labile proton concentration and its exchange rate. Therefore, our study aimed to quantify amide proton concentration and exchange rate independently in a cardiac arrest-induced global ischemia rat model. The amide proton CEST (APT) effect was decoupled from tissue water, macromolecular magnetization transfer, nuclear Overhauser enhancement, guanidinium, and amine protons using the image downsampling expedited adaptive least-squares (IDEAL) fitting algorithm on Z-spectra obtained under multiple RF saturation power levels, before and after global ischemia. Omega plot analysis was applied to determine amide proton concentration and exchange rate simultaneously. Global ischemia induces a significant APT signal drop from intact tissue. Using the modified omega plot analysis, we found that the amide proton exchange rate decreased from 29.6 ± 5.6 to 12.1 ± 1.3s-1 (P < 0.001), whereas the amide proton concentration showed little change (0.241 ± 0.035% vs. 0.202 ± 0.034%, P=0.074) following global ischemia. Our study determined the labile proton concentration and exchange rate underlying the in vivo APT MRI. The significant change in the exchange rate, but not the concentration of amide proton demonstrated that the pH effect dominates the APT contrast during tissue ischemia.

Amide proton transfer imaging in stroke.

Amide proton transfer (APT) imaging, a variant of chemical exchange saturation transfer MRI, has shown promise in detecting ischemic tissue acidosis following impaired aerobic metabolism in animal models and in human stroke patients due to the sensitivity of the amide proton exchange rate to changes in pH within the physiological range. Recent studies have demonstrated the possibility of using APT-MRI to detect acidosis of the ischemic penumbra, enabling the assessment of stroke severity and risk of progression, monitoring of treatment progress, and prognostication of clinical outcome. This paper reviews current APT imaging methods actively used in ischemic stroke research and explores the clinical aspects of ischemic stroke and future applications for these methods.

Influence of phosphate concentration on amine, amide, and hydroxyl CEST contrast.

To evaluate the influence of phosphate on amine, amide, and hydroxyl CEST contrast using Bloch-McConnell simulations applied to physical phantom data. Phantom solutions of 4 representative metabolites with exchangeable protons-glycine (α-amine protons), Cr (η-amine protons), egg white protein (amide protons), and glucose (hydroxyl protons)-were prepared at different pH levels (5.6 to 8.9) and phosphate concentrations (5 to 80 mM). CEST images of the phantom were collected with CEST-EPI sequence at 3 tesla. The CEST data were then fitted to full Bloch-McConnell equation simulations to estimate the exchange rate constants. With the fitted parameters, simulations were performed to evaluate the intracellular and extracellular contributions of CEST signals in normal brain tissue and brain tumors, as well as in dynamic glucose-enhanced experiments. The exchange rates of α-amine and hydroxyl protons were found to be highly dependent on both pH and phosphate concentrations, whereas the exchange rates of η-amine and amide protons were pH-dependent, albeit not catalyzed by phosphate. With phosphate being predominantly intracellular, CEST contrast of α-amine exhibited a higher sensitivity to changes in the extracellular microenvironment. Simulations of dynamic glucose-enhanced signals demonstrated that the contrast between normal and tumor tissue was mostly due to the extracellular CEST effect. The proton exchange rates in some metabolites can be greatly catalyzed by the presence of phosphate at physiological concentrations, which substantially alters the CEST contrast. Catalytic agents should be considered as confounding factors in future CEST-MRI research. This new dimension may also benefit the development of novel phosphate-sensitive imaging methods.

Sequence-Dependent Correlated Segments in the Intrinsically Disordered Region of ChiZ.

How sequences of intrinsically disordered proteins (IDPs) code for their conformational dynamics is poorly understood. Here, we combined NMR spectroscopy, small-angle X-ray scattering (SAXS), and molecular dynamics (MD) simulations to characterize the conformations and dynamics of ChiZ1-64. MD simulations, first validated by SAXS and secondary chemical shift data, found scant α-helices or β-strands but a considerable propensity for polyproline II (PPII) torsion angles. Importantly, several blocks of residues (e.g., 11–29) emerge as “correlated segments”, identified by their frequent formation of PPII stretches, salt bridges, cation-π interactions, and sidechain-backbone hydrogen bonds. NMR relaxation experiments showed non-uniform transverse relaxation rates (R2s) and nuclear Overhauser enhancements (NOEs) along the sequence (e.g., high R2s and NOEs for residues 11–14 and 23–28). MD simulations further revealed that the extent of segmental correlation is sequence-dependent; segments where internal interactions are more prevalent manifest elevated “collective” motions on the 5–10 ns timescale and suppressed local motions on the sub-ns timescale. Amide proton exchange rates provides corroboration, with residues in the most correlated segment exhibiting the highest protection factors. We propose the correlated segment as a defining feature for the conformations and dynamics of IDPs.

Experimental and Computational Characterization of the Conformational Ensemble and Interaction Motifs of Chiz N-Terminal Intrinsically Disordered Region

Cell division of Mycobacterium tuberculosis is crucial for the pathogenesis of tuberculosis. This process is mediated by the divisome, a multi-component, dynamic complex anchored by transmembrane proteins. One such protein is ChiZ, which contains a single transmembrane helix preceded by a 64-residue N-terminal intrinsically disordered region (IDR). We have characterized the conformational ensemble and membrane interactions of the ChiZ IDR by a variety of experimental techniques, including small-angle X-ray scattering (SAXS) and solution and solid-state NMR. 2D 1H-13C INEPT and PRE experiments suggest that the N-terminal IDR of ChiZ remains dynamic in the presence of liposomes, and interactions with the lipid membrane are mediated by arginine residues. To help interpret these data with atomistic details, we have carried out multiple molecular dynamics (MD) simulations in water using several force fields tailored for disordered proteins. The best agreement with SAXS, chemical shift, and NMR relaxation data was obtained by AMBER14SB/Tip4pD, followed closely by AMBER03ws/Tip4p2005s. In the MD simulations, the IDR transiently populated helices, especially in the N-terminal half, including residues 11-15, 19-22, and 28-32. This result explains the lower amide proton exchange rates in the N-terminal half of the IDR observed by solution NMR. Simulations in the presence of a lipid bilayer, in line with the solid-state NMR data, showed multiple arginine residues interacting with the membrane, where any one arginine may engage 2 or 3 lipid headgroups at once. Ongoing work aims to determine the dominant conformations of the full-length ChiZ in the membrane environment.

APT-weighted MRI: Techniques, current neuro applications, and challenging issues.

Amide proton transfer-weighted (APTw) imaging is a molecular MRI technique that generates image contrast based predominantly on the amide protons in mobile cellular proteins and peptides that are endogenous in tissue. This technique, the most studied type of chemical exchange saturation transfer imaging, has been used successfully for imaging of protein content and pH, the latter being possible due to the strong dependence of the amide proton exchange rate on pH. In this article we briefly review the basic principles and recent technical advances of APTw imaging, which is showing promise clinically, especially for characterizing brain tumors and distinguishing recurrent tumor from treatment effects. Early applications of this approach to stroke, Alzheimer's disease, Parkinson's disease, multiple sclerosis, and traumatic brain injury are also illustrated. Finally, we outline the technical challenges for clinical APT-based imaging and discuss several controversies regarding the origin of APTw imaging signals in vivo. Level of Evidence: 3 Technical Efficacy Stage: 3 J. Magn. Reson. Imaging 2019;50:347-364.

Exchangeable deuterons introduce artifacts in amide 15N CEST experiments used to study protein conformational exchange.

Protein molecules sample different conformations in solution and characterizing these conformations is crucial to understanding protein function. 15N CEST experiments are now routinely used to study slow conformational exchange of protein molecules between a 'visible' major state and 'invisible' minor states. These experiments have also been adapted to measure the solvent exchange rates of amide protons by exploiting the one bond deuterium isotope effect on the amide 15N chemical shifts. However at moderately high temperatures (~ 50°C) that are sometimes required to populate protein minor conformers to levels (~ 1%) that can be detected by CEST experiments solvent H/D exchange can lead to 'dips' in low B115N CEST profiles that can be wrongly assigned to the conformational exchange process being characterized. This is demonstrated in the case of ~ 18kDa T4 Lysozyme (T4L) at 50°C and the ~ 11kDa E. coli hibernation promoting factor (HPF) at 52°C. This problem is trivially solved by eliminating the exchangeable deuterons in the solvent by using either an external D2O lock or by using a small amount (~ 1-3%) of a molecule like d6-DMSO that does not contain exchangeable deuterons to lock the spectrometer.

Quantifying amide proton exchange rate and concentration in chemical exchange saturation transfer imaging of the human brain

Current chemical exchange saturation transfer (CEST) neuroimaging protocols typically acquire CEST-weighted images, and, as such, do not essentially provide quantitative proton-specific exchange rates (or brain pH) and concentrations. We developed a dictionary-free MR fingerprinting (MRF) technique to allow CEST parameter quantification with a reduced data set. This was accomplished by subgrouping proton exchange models (SPEM), taking amide proton transfer (APT) as an example, into two-pool (water and semisolid macromolecules) and three-pool (water, semisolid macromolecules, and amide protons) models. A variable radiofrequency saturation scheme was used to generate unique signal evolutions for different tissues, reflecting their CEST parameters. The proposed MRF-SPEM method was validated using Bloch-McConnell equation-based digital phantoms with known ground-truth, which showed that MRF-SPEM can achieve a high degree of accuracy and precision for absolute CEST parameter quantification and CEST phantoms. For in-vivo studies at 3 T, using the same model as in the simulations, synthetic Z-spectra were generated using rates and concentrations estimated from the MRF-SPEM reconstruction and compared with experimentally measured Z-spectra as the standard for optimization. The MRF-SPEM technique can provide rapid and quantitative human brain CEST mapping.

Measurement of Very Fast Exchange Rates of Individual Amide Protons in Proteins by NMR Spectroscopy.

NMR spectroscopy is a pivotal technique to measure hydrogen exchange rates in proteins. However, currently available NMR methods to measure backbone exchange are limited to rates of up to a few per second. To raise this limit, we have developed an approach that is capable of measuring proton exchange rates up to approximately 104 s-1 . Our method relies on the detection of signal loss due to the decorrelation of antiphase operators 2Nx Hz by exchange events that occur during a series of pi pulses on the 15 N channel. In practice, signal attenuation was monitored in a series of 2D H(CACO)N spectra, recorded with varying pi-pulse spacing, and the exchange rate was obtained by numerical fitting to the evolution of the density matrix. The method was applied to the small calcium-binding protein Calbindin D9k , where exchange rates up to 600 s-1 were measured for amides, where no signal was detectable in 15 N-1 H HSQC spectra. A temperature variation study allowed us to determine apparent activation energies in the range 47-69 kJ mol-1 for these fast exchanging amide protons, consistent with hydroxide-catalyzed exchange.

Rapid and quantitative chemical exchange saturation transfer (CEST) imaging with magnetic resonance fingerprinting (MRF).

To develop a fast magnetic resonance fingerprinting (MRF) method for quantitative chemical exchange saturation transfer (CEST) imaging. We implemented a CEST-MRF method to quantify the chemical exchange rate and volume fraction of the Nα -amine protons of L-arginine (L-Arg) phantoms and the amide and semi-solid exchangeable protons of in vivo rat brain tissue. L-Arg phantoms were made with different concentrations (25-100 mM) and pH (pH 4-6). The MRF acquisition schedule varied the saturation power randomly for 30 iterations (phantom: 0-6 μT; in vivo: 0-4 μT) with a total acquisition time of ≤2 min. The signal trajectories were pattern-matched to a large dictionary of signal trajectories simulated using the Bloch-McConnell equations for different combinations of exchange rate, exchangeable proton volume fraction, and water T1 and T2 relaxation times. The chemical exchange rates of the Nα -amine protons of L-Arg were significantly (P < 0.0001) correlated with the rates measured with the quantitation of exchange using saturation power method. Similarly, the L-Arg concentrations determined using MRF were significantly (P < 0.0001) correlated with the known concentrations. The pH dependence of the exchange rate was well fit (R2 = 0.9186) by a base catalyzed exchange model. The amide proton exchange rate measured in rat brain cortex (34.8 ± 11.7 Hz) was in good agreement with that measured previously with the water exchange spectroscopy method (28.6 ± 7.4 Hz). The semi-solid proton volume fraction was elevated in white (12.2 ± 1.7%) compared to gray (8.1 ± 1.1%) matter brain regions in agreement with previous magnetization transfer studies. CEST-MRF provides a method for fast, quantitative CEST imaging.

15N detection harnesses the slow relaxation property of nitrogen: Delivering enhanced resolution for intrinsically disordered proteins

Studies over the past decade have highlighted the functional significance of intrinsically disordered proteins (IDPs). Due to conformational heterogeneity and inherent dynamics, structural studies of IDPs have relied mostly on NMR spectroscopy, despite IDPs having characteristics that make them challenging to study using traditional 1H-detected biomolecular NMR techniques. Here, we develop a suite of 3D 15N-detected experiments that take advantage of the slower transverse relaxation property of 15N nuclei, the associated narrower linewidth, and the greater chemical shift dispersion compared with those of 1H and 13C resonances. The six 3D experiments described here start with aliphatic 1H magnetization to take advantage of its higher initial polarization, and are broadly applicable for backbone assignment of proteins that are disordered, dynamic, or have unfavorable amide proton exchange rates. Using these experiments, backbone resonance assignments were completed for the unstructured regulatory domain (residues 131-294) of the human transcription factor nuclear factor of activated T cells (NFATC2), which includes 28 proline residues located in functionally important serine-proline (SP) repeats. The complete assignment of the NFATC2 regulatory domain enabled us to study phosphorylation of NFAT by kinase PKA and phosphorylation-dependent binding of chaperone protein 14-3-3 to NFAT, providing mechanistic insight on how 14-3-3 regulates NFAT nuclear translocation.

Structural Basis for the Procofactor to Cofactor Transition in Human Factor V

Factor V, the inactive precursor to factor Va, has a domain organization of A1-A2-B-A3-C1-C2. Factor Va is formed by the proteolytic excision of the central B domain, which resolves the molecule into a heterodimer (A1-A2/A3-C1-C2). Removal of the B domain enables the cofactor to engage factor Xa on phosphatidylserine-containing membranes, assemble prothrombinase and greatly enhance the rate of thrombin formation. Recent studies have shown a key role for a basic region (BR), which lies approximately in the center of the B domain, in enforcing procofactor properties in human factor V (hFV). Exogenously added recombinant BR fragments can bind with high affinity to a cofactor-like variant of human hFV (hFVDT), in which a large central portion of the B domain has been deleted, interfere with Xa binding and restore procofactor-like properties. Biochemical evidence suggests that BR binding results from its interaction with an acidic region (AR2) at the C terminus of the B domain and likely also an acidic sequence (AR1) at the C terminus of the A2 domain. Our recent crystal structure of hFVDT provided the first structural evidence that AR1 and AR2, ~800 residues apart in the primary structure of hFV, are positioned adjacent to each other and could plausibly form an extended surface for high affinity BR binding to reconstitute a tripartite procofactor-regulatory region (AR1/BR/AR2). However, the lack of BR in hFVDT precluded independent structural verification of this possibility. In a computational approach, we created a molecular model for the 58 residue BR peptide. The top scoring three-dimensional models of the 58 residue BR peptide showed a helix-loop arrangement, contrary to the general belief that the B domain lacks structured regions. The best scoring BR peptide model was used for ab initio docking studies using the crystal structure of hFVDT to predict possible binding sites using PIPER and ClusPro. The most highly represented and statistically probable solutions showed the BR peptide in intimate contact with juxtaposed surfaces provided by AR1 and AR2. Interestingly, the docked BR peptide contacted regions in AR1 and on the A2 domain implicated in FXa binding in the structure of Pseudonaja textilis FV bound to snake venom factor X. Computational predictions were tested using hydrogen-deuterium exchange detected by protein fragmentation and mass spectroscopy (HDX). Proteolytic fragmentation of hFVDT and fragment detection by LC-MS was optimized to cover >95% of its 1514 residues with an average redundancy of 4.27 peptides/residue. Only 4 or 5 segments of ~10-15 residue length were not covered. Addition of the BR peptide had minor effects on amide proton exchange over the bulk of the molecule. However, BR peptide binding was accompanied by reductions in amide proton exchange rates of ~7-30-fold in immediately adjacent regions of hFVDT corresponding to sequences within A2 (626-634), AR1 (658-695), AR2 (872-881) and A3 (983-995). BR peptide binding to hFVDT is accompanied by perturbations in these spatially adjacent regions covering a small fraction of the surface area at approximately the 3 o’clock position with the molecule in the standard orientation. The marked agreement between the HDX findings and the computational docking studies supports our proposal that the BR engages an extended surface contributed by AR1 and AR2 to form a tripartite procofactor-regulatory region. The interaction of BR with AR1 and a small region in A2, both implicated in binding Xa, potentially explains how the BR might restrict Xa binding to the procofactor. Destabilization of BR binding by proteolysis at the C terminus of AR2 is envisioned to result in cofactor formation by releasing the BR and revealing sites responsible for binding Xa. Our findings provide a structural explanation for the long standing puzzle of factor V activation and pave the way for further definition of mechanistic details of procofactor and cofactor function. They have implications for how interactions with TFPIα through the basic region at its C-terminus might regulate FV(a). They also reveal previously unanticipated strategies to modulate functions of hFV and hFVa for therapeutic gain. DisclosuresCamire:Pfizer: Consultancy, Patents & Royalties, Research Funding; Bayer: Consultancy; Novo Nordisk: Research Funding; sparK: Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties. Krishnaswamy:Portola: Research Funding; Janssen: Consultancy, Research Funding.

Mapping Protein-Ligand Interactions with Proteolytic Fragmentation, Hydrogen/Deuterium Exchange-Mass Spectrometry.

Biological processes are the result of noncovalent, protein-ligand interactions, where the ligands range from small organic and inorganic molecules to lipids, nucleic acids, peptides, and proteins. Amide groups within proteins constantly exchange protons with water. When immersed in heavy water (D2O), mass spectrometry (MS) can measure the change of mass associated with the hydrogen to deuterium exchange (HDX). Protein-ligand interactions modify the hydrogen exchange rates of amide protons, and the measurement of the amide exchange rates can provide rich information regarding the dynamical structure of the protein-ligand complex. This chapter describes a protocol for conducting bottom-up, continuous uptake, proteolytic fragmentation HDX-MS experiments that can help identify and map the interacting peptides of a protein-ligand interface. This tutorial outlines the fundamental theory governing hydrogen exchange; provides practical information regarding the preparation of protein samples and solutions; and describes the exchange reaction, reaction quenching, enzymatic digestion, chromatographic separation, and peptide analysis by MS. Tables list representative combinations of fluidic components used by HDX-MS researchers and summarize the available HDX-MS analysis software packages. Additionally, two HDX-MS case studies are used to illustrate protein-ligand interactions involving: (1) a continuous sequence of interacting residues and (2) a set of discontinuously numbered residues, residing spatially near each other.

Long-range magnetic non-equivalence of methylene protons and pK's of amide bonds in peptides

This research aims at showing that deciphering NMR patterns of peptide anisochronous methylene protons (A and B) coupled to an adjacent NH or CH(X) hydrogen may bring novel structural and dynamical information, using for this purpose four l-Histidine-containing peptides and the NMR patterns of Glycyl or β-Alanyl residues or of the Histidyl side-chain, or else of a grafted N-butyl chain. Investigations were carried out along the following lines: ionization constants of functional end groups and speciation curves of protonated species; specific anisochronies and coupling constants assigned to each state of protonation of each compound; base-catalyzed amide proton exchange rate and peptide pK's from DNMR of anisochronous protons. Vicinal coupling constants JAX and JBX were found (i) equal along the peptide backbone, suggesting either free rotation about the N–C bond, or, more probably, a torsion angle Φ=90°, and (ii) unequal in the Histidyl side-chain, suggesting an Imidazole-carboxylate interaction preventing free rotation. Peptide pK's span a wide range of values, from 13.63 to 17.48, depending on the parent residue and the characteristics of functional end groups.

H/D exchange of a 15N labelled Tau fragment as measured by a simple Relax-EXSY experiment

We present an equilibrium H/D exchange experiment to measure the exchange rates of labile amide protons in intrinsically unfolded proteins. By measuring the contribution of the H/D exchange to the apparent T1 relaxation rates in solvents of different D2O content, we can easily derive the rates of exchange for rapidly exchanging amide protons. The method does not require double isotope labelling, is sensitive, and requires limited fitting of the data. We demonstrate it on a functional fragment of Tau, and provide evidence for the hydrogen bond formation of the phosphate moiety of Ser214 with its own amide proton in the same fragment phosphorylated by the PKA kinase.

Time-resolved multidimensional NMR with non-uniform sampling

Time-resolved experiments demand high resolution both in spectral dimensions and in time of the studied kinetic process. The latter requirement traditionally prohibits applications of the multidimensional experiments, which, although capable of providing invaluable information about structure and dynamics and almost unlimited spectral resolution, require too lengthy data collection. Our work shows that the problem has a solution in using modern methods of NMR data collection and signal processing. A continuous fast pulsing three-dimensional experiment is acquired using non-uniform sampling during full time of the studied reaction. High sensitivity and time-resolution of a few minutes is achieved by simultaneous processing of the full data set with the multi-dimensional decomposition. The method is verified and illustrated in realistic simulations and by measuring deuterium exchange rates of amide protons in ubiquitin. We applied the method for characterizing kinetics of in vitro phosphorylation of two tyrosine residues in an intrinsically disordered cytosolic domain of the B cell receptor protein CD79b. Signals of many residues including tyrosines in both phosphorylated and unmodified forms of CD79b are found in a heavily crowded region of 2D 1H–15N correlation spectrum and the significantly enhanced spectral resolution provided by the 3D time-resolved approach was essential for the quantitative site-specific analysis.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-013-9811-1) contains supplementary material, which is available to authorized users.

Flexible and rigid structures in HIV-1 p17 matrix protein monitored by relaxation and amide proton exchange with NMR

The HIV-1 p17 matrix protein is a multifunctional protein that interacts with other molecules including proteins and membranes. The dynamic structure between its folded and partially unfolded states can be critical for the recognition of interacting molecules. One of the most important roles of the p17 matrix protein is its localization to the plasma membrane with the Gag polyprotein. The myristyl group attached to the N-terminus on the p17 matrix protein functions as an anchor for binding to the plasma membrane. Biochemical studies revealed that two regions are important for its function: D14–L31 and V84–V88. Here, the dynamic structures of the p17 matrix protein were studied using NMR for relaxation and amide proton exchange experiments at the physiological pH of 7.0. The results revealed that the α12-loop, which includes the 14–31 region, was relatively flexible, and that helix 4, including the 84–88 region, was the most protected helix in this protein. However, the residues in the α34-loop near helix 4 had a low order parameter and high exchange rate of amide protons, indicating high flexibility. This region is probably flexible because this loop functions as a hinge for optimizing the interactions between helices 3 and 4. The C-terminal long region of K113-Y132 adopted a disordered structure. Furthermore, the C-terminal helix 5 appeared to be slightly destabilized due to the flexible C-terminal tail based on the order parameters. Thus, the dynamic structure of the p17 matrix protein may be related to its multiple functions.

Quantitative Bayesian model‐based analysis of amide proton transfer MRI

Amide Proton Transfer (APT) reports on contrast derived from the exchange of protons between amide groups and water. Commonly, APT contrast is quantified by asymmetry analysis, providing an ensemble contrast of both amide proton concentration and exchange rate. An alternative is to sample the off-resonant spectrum and fit an exchange model, permitting the APT effect to be quantified, correcting automatically for confounding effects of spillover, field inhomogeneity, and magnetization transfer. Additionally, it should permit amide concentration and exchange rate to be independently quantified. Here, a Bayesian method is applied to this problem allowing pertinent prior information to be specified. A three-pool model was used incorporating water protons, amide protons, and magnetization transfer effect. The method is demonstrated in simulations, creatine phantoms with varying pH and in vivo (n = 7). The Bayesian model-based approach was able to quantify the APT effect accurately (root-mean-square error < 2%) even when subject to confounding field variation and magnetization transfer effect, unlike traditional asymmetry analysis. The in vivo results gave approximate APT concentration (relative to water) and exchange rate values of 3 × 10(-3) and 15 s(-1) . A degree of correlation was observed between these parameter making the latter difficult to quantify with absolute accuracy, suggesting that more optimal sampling strategies might be required.

The pH sensitivity of –NH exchange in LnDOTA–tetraamide complexes varies with amide substituent

The amide proton exchange rates in various lanthanide(III) DOTA-tetraamide complexes were investigated by CEST as a function of variable chemical structures and charges on the amide substituents. Comparisons were made between YbDOTA-(gly)(4)(-) (Yb-1), YbDOTA-(NHCH(2)PO(3))(4 (5-) (Yb-2) and YbDOTA-(NHCH(2)PO(3)Et(2))(4)(3+) (Yb-3). The general shapes of the CEST vs pH profiles were similar for the three complexes but they showed maximum CEST intensities at different pH values, pH 8.3, 8.8 and 6.9 for Yb-1, Yb-2 and Yb-3, respectively. This indicates that a more negatively charged substituent on the amide helps stabilize the partial positive charge on the amide nitrogen and consequently more base is required to catalyze proton exchange. The chemical shifts of the -NH protons in Yb-1 and Yb-2 were similar (-17 ppm) while the -NH proton in Yb-3 was at -13 ppm. This shows that the crystal field produced by the amide oxygen donor atoms in Yb-3 is substantially weaker than that in the other two complexes. In an effort to expand the useful range of pH values that might be measured using these complexes as CEST agents, the shapes of the CEST vs pH curves were also determined for two thulium(III) complexes with much larger hyperfine shifted -NH proton resonances. The ratio of CEST from -NH exchange in Tm-1 compared with CEST from -NH exchange in Tm-3 was found to be linear over an extended pH range, from 6.3 to 7.4. This demonstrates a potential advantage of using mixtures of lanthanide(III) DOTA-tetraamides for mapping tissue pH by use of ratiometric CEST imaging.

Sulfonated amphipols: Synthesis, properties, and applications

Amphipols (APols) are amphiphatic polymers that keep membrane proteins (MPs) water-soluble. The best characterized and most widely used APol to date, A8-35, comprises a polyacrylate backbone grafted with octyl- and isopropylamine side chains. The nature of its hydrophilic moieties prevents its use at the slightly acidic pH that is desirable to slow down the rate of amide proton exchange in solution NMR studies. We describe here the synthesis and properties of pH-insensitive APols obtained by replacing isopropyles with taurine. Sulfonated APols (SAPols) can be used to trap MPs in the form of small complexes, to stabilize them, and to keep them water-soluble even at low pH. [(15) N,(1) H]-transverse relaxation-optimized spectroscopy NMR spectra obtained at pH 6.8 of a bacterial outer MP folded in SAPols show that the protein is correctly folded. The spectra have a resolution similar to that achieved with A8-35 and reveal water-exposed amide and indole protons whose resonance peaks are absent at pH 8.0.