Amide Proton Exchange Research Articles

Using NMR-detected hydrogen-deuterium exchange to quantify protein stability in cosolutes, under crowded conditions in vitro and in cells

We review the use of nuclear magnetic resonance (NMR) spectroscopy to assess the exchange of amide protons for deuterons (HDX) in efforts to understand how high concentration of cosolutes, especially macromolecules, affect the equilibrium thermodynamics of protein stability. HDX NMR is the only method that can routinely provide such data at the level of individual amino acids. We begin by discussing the properties of the protein systems required to yield equilibrium thermodynamic data and then review publications using osmolytes, sugars, denaturants, synthetic polymers, proteins, cytoplasm and in cells.

NMR sample optimization and backbone assignment of a stabilized neurotensin receptor

G protein-coupled receptors (GPCRs) are involved in a multitude of cellular signaling cascades and consequently are a prominent target for pharmaceutical drugs. In the past decades, a growing number of high-resolution structures of GPCRs has been solved, providing unprecedented insights into their mode of action. However, knowledge on the dynamical nature of GPCRs is equally important for a better functional understanding, which can be obtained by NMR spectroscopy. Here, we employed a combination of size exclusion chromatography, thermal stability measurements and 2D-NMR experiments for the NMR sample optimization of the stabilized neurotensin receptor type 1 (NTR1) variant HTGH4 bound to the agonist neurotensin. We identified the short-chain lipid di-heptanoyl-glycero-phosphocholine (DH7PC) as a promising membrane mimetic for high resolution NMR experiments and obtained a partial NMR backbone resonance assignment. However, internal membrane-incorporated parts of the protein were not visible due to lacking amide proton back-exchange. Nevertheless, NMR and hydrogen deuterium exchange (HDX) mass spectrometry experiments could be used to probe structural changes at the orthosteric ligand binding site in the agonist and antagonist bound states. To enhance amide proton exchange we partially unfolded HTGH4 and observed additional NMR signals in the transmembrane region. However, this procedure led to a higher sample heterogeneity, suggesting that other strategies need to be applied to obtain high-quality NMR spectra of the entire protein. In summary, the herein reported NMR characterization is an essential step toward a more complete resonance assignment of NTR1 and for probing its structural and dynamical features in different functional states.

Metal interactions of α-synuclein probed by NMR amide-proton exchange.

The aberrant aggregation of α-synuclein (αS), a disordered protein primarily expressed in neuronal cells, is strongly associated with the underlying mechanisms of Parkinson's disease. It is now established that αS has a weak affinity for metal ions and that these interactions alter its conformational properties by generally promoting self-assembly into amyloids. Here, we characterised the nature of the conformational changes associated with metal binding by αS using nuclear magnetic resonance (NMR) to measure the exchange of the backbone amide protons at a residue specific resolution. We complemented these experiments with 15N relaxation and chemical shift perturbations to obtain a comprehensive map of the interaction between αS and divalent (Ca2+, Cu2+, Mn2+, and Zn2+) and monovalent (Cu+) metal ions. The data identified specific effects that the individual cations exert on the conformational properties of αS. In particular, binding to calcium and zinc generated a reduction of the protection factors in the C-terminal region of the protein, whereas both Cu(II) and Cu(I) did not alter the amide proton exchange along the αS sequence. Changes in the R2/R1 ratios from 15N relaxation experiments were, however, detected as a result of the interaction between αS and Cu+ or Zn2+, indicating that binding to these metals induces conformational perturbations in distinctive regions of the protein. Collectively our data suggest that multiple mechanisms of enhanced αS aggregation are associated with the binding of the analysed metals.

Pure shift amide detection in conventional and TROSY-type experiments of 13C,15N-labeled proteins

Large coupling networks in uniformly 13C,15N-labeled biomolecules induce broad multiplets that even in flexible proteins are frequently not recognized as such. The reason is that given multiplets typically consist of a large number of individual resonances that result in a single broad line, in which individual components are no longer resolved. We here introduce a real-time pure shift acquisition scheme for the detection of amide protons which is based on 13C-BIRDr,X. As a result the full homo- and heteronuclear coupling network can be suppressed at low power leading to real singlets at substantially improved resolution and uncompromised sensitivity. The method is tested on a small globular and an intrinsically disordered protein (IDP) where the average spectral resolution is increased by a factor of ~ 2 and higher. Equally important, the approach works without saturation of water magnetization for solvent suppression and exchanging amide protons are not affected by saturation transfer.

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.

High-Resolution Hydrogen-Deuterium Protection Factors from Sparse Mass Spectrometry Data Validated by Nuclear Magnetic Resonance Measurements.

Experimental measurement of time-dependent spontaneous exchange of amide protons with deuterium of the solvent provides information on the structure and dynamical structural variation in proteins. Two experimental techniques are used to probe the exchange: NMR, which relies on different magnetic properties of hydrogen and deuterium, and MS, which exploits the change in mass due to deuteration. NMR provides residue-specific information, that is, the rate of exchange or, analogously, the protection factor (i.e., the unitless ratio between the rate of exchange for a completely unstructured state and the observed rate). MS provides information that is specific to peptides obtained by proteolytic digestion. The spatial resolution of HDX-MS measurements depends on the proteolytic pattern of the protein, the fragmentation method used, and the overlap between peptides. Different computational approaches have been proposed to extract residue-specific information from peptide-level HDX-MS measurements. Here, we demonstrate the advantages of a method recently proposed that exploits self-consistency and classifies the possible sets of protection factors into a finite number of alternative solutions compatible with experimental data. The degeneracy of the solutions can be reduced (or completely removed) by exploiting the additional information encoded in the shape of the isotopic envelopes. We show how sparse and noisy MS data can provide high-resolution protection factors that correlate with NMR measurements probing the same protein under the same conditions.

Quantitative imaging of apoptosis following oncolytic virotherapy by magnetic resonance fingerprinting aided by deep learning.

Oncolytic virotherapy is a promising anticancer strategy, however, non-invasive imaging methods that detect intratumoral viral spread and an individual’s response to therapy are either slow, lack specificity or require the use of radioactive or metal-based contrast agents, hindering their clinical translation. Here, we describe a fast chemical-exchange saturation transfer magnetic resonance fingerprinting (CEST-MRF) approach that selectively labels the exchangeable amide protons of endogenous proteins and the exchangeable macromolecule protons of lipids, without requiring an exogenous contrast agent. A deep neural network, previously trained with simulated MR-fingerprints, then generates, using CEST-MRF image data, quantitative maps of intratumoral pH, and protein and lipid concentrations. In a mouse model of glioblastoma multiforme, the maps enabled the detection of the early apoptotic response to oncolytic virotherapy, characterized by decreased cytosolic pH and reduced protein synthesis. Imaging of a healthy human volunteer yields molecular parameters in good agreement with literature values, demonstrating the clinical potential of artificial intelligence-based CEST-MRF, which may be further applicable to the imaging of a wide range of pathologies, including stroke, cancer, and neurological disorders.

A Brief History and Future Prospects of CEST MRI in Clinical Non-Brain Tumor Imaging.

Chemical exchange saturation transfer (CEST) MRI is a promising molecular imaging tool which allows the specific detection of metabolites that contain exchangeable amide, amine, and hydroxyl protons. Decades of development have progressed CEST imaging from an initial concept to a clinical imaging tool that is used to assess tumor metabolism. The first translation efforts involved brain imaging, but this has now progressed to imaging other body tissues. In this review, we summarize studies using CEST MRI to image a range of tumor types, including breast cancer, pelvic tumors, digestive tumors, and lung cancer. Approximately two thirds of the published studies involved breast or pelvic tumors which are sites that are less affected by body motion. Most studies conclude that CEST shows good potential for the differentiation of malignant from benign lesions with a number of reports now extending to compare different histological classifications along with the effects of anti-cancer treatments. Despite CEST being a unique ‘label-free’ approach with a higher sensitivity than MR spectroscopy, there are still some obstacles for implementing its clinical use. Future research is now focused on overcoming these challenges. Vigorous ongoing development and further clinical trials are expected to see CEST technology become more widely implemented as a mainstream imaging technology.

Flexibility of Oxidized and Reduced States of the Chloroplast Regulatory Protein CP12 in Isolation and in Cell Extracts.

In the chloroplast, Calvin–Benson–Bassham enzymes are active in the reducing environment created in the light by electrons from the photosystems. In the dark, these enzymes are inhibited, mainly caused by oxidation of key regulatory cysteine residues. CP12 is a small protein that plays a role in this regulation with four cysteine residues that undergo a redox transition. Using amide-proton exchange with solvent, measured by nuclear magnetic resonance (NMR) and mass-spectrometry, we confirmed that reduced CP12 is intrinsically disordered. Using real-time NMR, we showed that the oxidation of the two disulfide bridges is simultaneous. In oxidized CP12, the C23–C31 pair is in a region that undergoes a conformational exchange in the NMR-intermediate timescale. The C66–C75 pair is in the C-terminus that folds into a stable helical turn. We confirmed that these structural states exist in a physiologically relevant environment: a cell extract from Chlamydomonas reinhardtii. Consistent with these structural equilibria, the reduction is slower for the C66–C75 pair than for the C23–C31 pair. The redox mid-potentials for the two cysteine pairs differ and are similar to those found for glyceraldehyde 3-phosphate dehydrogenase and phosphoribulokinase, consistent with the regulatory role of CP12.

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.

Distinct residual and disordered structures of alpha-synuclein analyzed by amide-proton exchange and NMR signal intensity

The residual solution structures of two alpha-synuclein mutants, A30P and A53T, observed in family members of patients with Parkinson's disease were compared with that of wild-type by NMR. The A53T substitution had been shown to accelerate fibril formation of alpha-synuclein, whereas the A30P mutation has the negative and positive effects on the formation of the fibril and spherical oligomer, respectively. The remaining structure was analyzed via amide-proton exchange and signal intensity measurements using NMR. Amide-proton exchange was used for both the calculation of kex values and ratio of kex at different temperatures. Effects of the A30P (N-terminal region) mutation were observed at the C-terminal region as a more flexible structure, suggesting that long-range interactions exist between the N- and C-terminal regions in alpha-synuclein. In addition, the N-terminal region adopted a more rigid structure in the A53T and A30P mutants than in the wild-type. It was concluded that the structural change caused by the mutations is related to the formation of a beta-hairpin at the initiation site of the N-terminal core structure. Furthermore, the signal intensity was used to estimate the rigidity of the structure. Higher signal intensities were observed for A30P at the 112, 113, and 116 C-terminal residues, suggesting that this region adopts more flexible structure. The ratio of the intensities at different temperatures indicated more flexible or rigid structures in the N-terminal region of A30P than in that of wild-type. Thus, using different approaches and temperatures is a good method to analyze residual structure in intrinsically disordered proteins.

Confronting the Invisible: Assignment of Protein 1HN Chemical Shifts in Cases of Extreme Broadening.

NMR studies of intrinsically disordered proteins (IDPs) at neutral pH values are hampered by the rapid exchange of backbone amide protons with solvent. Although exchange rates can be modulated by changes in pH, interactions between IDPs that lead to phase separation sometimes only occur at neutral pH values or higher, where backbone amide-based experiments fail. Here we describe a simple NMR experiment for measuring amide proton chemical shifts in cases where 1HN spectra cannot be obtained. The approach uses a weak 1H B1 field, searching for elusive 1HN resonance frequencies that become encoded in the intensities of cross-peaks in three-dimensional 1Hα-detect spectra. Applications to the CAPRIN1 protein in both dilute- and phase-separated states highlight the utility of the method, establishing that accurate 1HN chemical shifts can be obtained even in cases where solvent hydrogen exchange rates are on the order of 1500 s-1.

Sensitivity-enhanced three-dimensional and carbon-detected two-dimensional NMR of proteins using hyperpolarized water

Signal enhancements of up to two orders of magnitude in protein NMR can be achieved by employing HDO as a vector to introduce hyperpolarization into folded or intrinsically disordered proteins. In this approach, hyperpolarized HDO produced by dissolution-dynamic nuclear polarization (D-DNP) is mixed with a protein solution waiting in a high-field NMR spectrometer, whereupon amide proton exchange and nuclear Overhauser effects (NOE) transfer hyperpolarization to the protein and enable acquisition of a signal-enhanced high-resolution spectrum. To date, the use of this strategy has been limited to 1D and 1H-15N 2D correlation experiments. Here we introduce 2D 13C-detected D-DNP, to reduce exchange-induced broadening and other relaxation penalties that can adversely affect proton-detected D-DNP experiments. We also introduce hyperpolarized 3D spectroscopy, opening the possibility of D-DNP studies of larger proteins and IDPs, where assignment and residue-specific investigation may be impeded by spectral crowding. The signal enhancements obtained depend in particular on the rates of chemical and magnetic exchange of the observed residues, thus resulting in non-uniform ‘hyperpolarization-selective’ signal enhancements. The resulting spectral sparsity, however, makes it possible to resolve and monitor individual amino acids in IDPs of over 200 residues at acquisition times of just over a minute. We apply the proposed experiments to two model systems: the compactly folded protein ubiquitin, and the intrinsically disordered protein (IDP) osteopontin (OPN).

Hydrogen-deuterium exchange mass spectrometry of membrane proteins in lipid nanodiscs.

Hydrogen deuterium exchange mass spectrometry (H/DX MS) provides a quantitative comparison of the relative rates of exchange of amide protons for solvent deuterons. In turn, the rate of amide exchange depends on a complex combination of the stability of local secondary structure, solvent accessibility, and dynamics. H/DX MS has, therefore, been widely used to probe structure and function of soluble proteins, but its application to membrane proteins was limited previously to detergent solubilized samples. The large excess of lipids from model membranes, or from membrane fractions derived from in vivo samples, presents challenges with mass spectrometry. The lipid nanodisc platform, consisting of apolipoprotein A-derived membrane scaffold proteins, provides a native like membrane environment in which to capture analyte membrane proteins with a well defined, and low, ratio of lipid to protein. Membrane proteins in lipid nanodiscs are amenable to H/DX MS, and this is expected to lead to a rapid increase in the number of membrane proteins subjected to this analysis. Here we review the few literature examples of the application of H/DX MS to membrane proteins in nanodiscs. The incremental improvements in the experimental workflow of the H/DX MS are described and potential applications of this approach to study membrane proteins are described.

Alpha protons as NMR probes in deuteratedproteins

We describe a new labeling method that allows for full protonation atthe backbone Hα position, maintaining protein side chains with a high level ofdeuteration. We refer to the method as alpha proton exchange by transamination(α-PET) since it relies on transaminase activity demonstrated here using Escherichia coli expression. We show that α-PETlabeling is particularly useful in improving structural characterization of solidproteins by introduction of an additional proton reporter, while eliminating manystrong dipolar couplings. The approach benefits from the high sensitivity associatedwith 1.3 mm samples, more abundant information including Hα resonances, and thenarrow proton linewidths encountered for highly deuterated proteins. The labelingstrategy solves amide proton exchange problems commonly encountered for membraneproteins when using perdeuteration and backexchange protocols, allowing access toalpha and all amide protons including those in exchange-protected regions. Theincorporation of Hα protons provides new insights, as the close Hα–Hα andHα–HN contacts present in β-sheets become accessible,improving the chance to determine the protein structure as compared withHN–HN contacts alone.Protonation of the Hα position higher than 90% is achieved for Ile, Leu, Phe, Tyr,Met, Val, Ala, Gln, Asn, Thr, Ser, Glu, Asp even though LAAO is only active at thisdegree for Ile, Leu, Phe, Tyr, Trp, Met. Additionally, the glycine methylene carbonis labeled preferentially with a single deuteron, allowing stereospecific assignmentof glycine alpha protons. In solution, we show that the high deuteration leveldramatically reduces R2 relaxation rates, which is beneficialfor the study of large proteins and protein dynamics. We demonstrate the methodusing two model systems, as well as a 32 kDa membrane protein, hVDAC1, showing theapplicability of the method to study membrane proteins.

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.

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.