Amide Proton Exchange Research Articles

New 13C-detected experiments for the assignment of intrinsically disordered proteins

NMR assignment of intrinsically disordered proteins (IDPs) by conventional HN-detected methods is hampered by the small dispersion of the amide protons chemical shifts and exchange broadening of amide proton signals. Therefore several alternative assignment strategies have been proposed in the last years. Attempting to seize that dispersion of (13)C' and (15)N chemical shifts holds even in IDPs, we recently proposed two (13)C-detected experiments to directly correlate the chemical shifts of two consecutive (13)C'-(15)N groups in proteins, i.e. without mediation of other nuclei. Main drawback of these experiments is the interruption of the connection at prolines. Here we present new (13)C-detected experiments to correlate consecutive (13)C'-(15)N groups in IDPs, hacacoNcaNCO and hacaCOncaNCO, that overcome this limitation. Moreover, the experiments provide recognition of glycine residues, thereby facilitating the assignment process.

Proton-detected 2D radio frequency driven recoupling solid-state NMR studies on micelle-associated cytochrome-b5

Solid-state NMR spectroscopy is increasingly used in the high-resolution structural studies of membrane-associated proteins and peptides. Most such studies necessitate isotopically labeled (13C, 15N and 2H) proteins/peptides, which is a limiting factor for some of the exciting membrane-bound proteins and aggregating peptides. In this study, we report the use of a proton-based slow magic angle spinning (MAS) solid-state NMR experiment that exploits the unaveraged 1H–1H dipolar couplings from a membrane-bound protein. We have shown that the difference in the buildup rates of cross-peak intensities against the mixing time – obtained from 2D 1H–1H radio frequency-driven recoupling (RFDR) and nuclear Overhauser effect spectroscopy (NOESY) experiments on a 16.7-kDa micelle-associated full-length rabbit cytochrome-b5 (cytb5) – can provide insights into protein dynamics and could be useful to measure 1H–1H dipolar couplings. The experimental buildup curves compare well with theoretical simulations and are used to extract relaxation parameters. Our results show that due to fast exchange of amide protons with water in the soluble heme-containing domain of cyb5, coherent 1H–1H dipolar interactions are averaged out for these protons while alpha and side chain protons show residual dipolar couplings that can be obtained from 1H–1H RFDR experiments. The appearance of resonances with distinct chemical shift values in 1H–1H RFDR spectra enabled the identification of residues (mostly from the transmembrane region) of cytb5 that interact with micelles.

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.

Remaining structures at the N- and C-terminal regions of alpha-synuclein accurately elucidated by amide-proton exchange NMR with fitting

Alpha-synuclein is analyzed in physiological conditions by CLEANEX-PM methodology, in which the amide-proton exchange can be monitored at millisecond scale. The relationship between kex and [OH]− is confirmed as a linear correlation with slope 1, indicating EX2 regime. There are significant residual structures at the N- and C-terminal regions. The structure at the C-terminal region is more stable than that of the N-terminal region. The middle part including NAC region is not completely protected. The data acquired at various pH and mixing time conditions followed by linear fitting give accurate information about residual structures.

NMR Spectroscopic Studies of Intrinsically Disordered Proteins at Near‐Physiological Conditions

When approaching physiological conditions, solvent exchange of amide protons in intrinsically disordered proteins (IDPs) is so pronounced that it becomes a key feature to be considered in NMR experiment design. 13C NMR experiments recover information that is not accessible through amide proton detection, and solvent exchange can be used to increase sensitivity.

Amide proton exchange of a dynamic loop in cell extracts

Intrinsic rates of exchange are essential parameters for obtaining protein stabilities from amide (1) H exchange data. To understand the influence of the intracellular environment on stability, one must know the effect of the cytoplasm on these rates. We probed exchange rates in buffer and in Escherichia coli lysates for the dynamic loop in the small globular protein chymotrypsin inhibitor 2 using a modified form of the nuclear magnetic resonance experiment, SOLEXSY. No significant changes were observed, even in 100 g dry weight L(-1) lysate. Our results suggest that intrinsic rates from studies conducted in buffers are applicable to studies conducted under cellular conditions.

A Designed Three-Stranded β-Sheet in an α/β Hybrid Peptide

The incorporation of β-amino acid residues into the antiparallel β-strand segments of a multi-stranded β-sheet peptide is demonstrated for a 19-residue peptide, Boc-LV(β)FV(D)PGL(β)FVVL(D)PGLVL(β)FVV-OMe (BBH19). Two centrally positioned (D)Pro-Gly segments facilitate formation of a stable three-stranded β-sheet, in which β-phenylalanine ((β)Phe) residues occur at facing positions 3, 8 and 17. Structure determination in methanol solution is accomplished by using NMR-derived restraints obtained from NOEs, temperature dependence of amide NH chemical shifts, rates of H/D exchange of amide protons and vicinal coupling constants. The data are consistent with a conformationally well-defined three-stranded β-sheet structure in solution. Cross-strand interactions between (β)Phe3/(β)Phe17 and (β)Phe3/Val15 residues define orientations of these side-chains. The observation of close contact distances between the side-chains on the N- and C-terminal strands of the three-stranded β-sheet provides strong support for the designed structure. Evidence is presented for multiple side-chain conformations from an analysis of NOE data. An unusual observation of the disappearance of the Gly NH resonances upon prolonged storage in methanol is rationalised on the basis of a slow aggregation step, resulting in stacking of three-stranded β-sheet structures, which in turn influences the conformational interconversion between type I' and type II' β-turns at the two (D)Pro-Gly segments. Experimental evidence for these processes is presented. The decapeptide fragment Boc-LV(β)FV(D)PGL(β)FVV-OMe (BBH10), which has been previously characterized as a type I' β-turn nucleated hairpin, is shown to favour a type II' β-turn conformation in solution, supporting the occurrence of conformational interconversion at the turn segments in these hairpin and sheet structures.

Identification of Regions of Rabbit Muscle Pyruvate Kinase Important for Allosteric Regulation by Phenylalanine, Detected by H/D Exchange Mass Spectrometry

Mass spectrometry has been used to determine the number of exchangeable backbone amide protons and the associated rate constants that are altered when rabbit muscle pyruvate kinase (rM1-PYK) binds either the allosteric inhibitor (phenylalanine) or a nonallosteric analogue of the inhibitor. Alanine is used as the nonallosteric analogue because it binds competitively with phenylalanine but elicits a negligible allosteric inhibition, i.e., a negligible reduction in the affinity of rM1-PYK for the substrate, phosphoenolpyruvate. This experimental design is expected to distinguish changes in the protein caused by effector binding (i.e., those changes common upon the addition of alanine vs phenylalanine) from changes associated with allosteric regulation (i.e., those elicited by the addition of phenylalanine binding, but not alanine binding). High-quality peptic fragments covering 98% of the protein were identified. Changes in both the number of exchangeable protons per peptide and in the rate constant associated with exchange highlight regions of the protein with allosteric roles. The set of allosterically relevant peptides identified by this technique includes residues previously identified by mutagenesis to have roles in allosteric regulation by phenylalanine.

Amide proton exchange of a dynamic loop in cell extracts

Amide proton exchange of a dynamic loop in cell extracts

Macromolecular Crowding and Protein Stability

An understanding of cellular chemistry requires knowledge of how crowded environments affect proteins. The influence of crowding on protein stability arises from two phenomena, hard-core repulsions and soft (i.e., chemical) interactions. Most efforts to understand crowding effects on protein stability, however, focus on hard-core repulsions, which are inherently entropic and stabilizing. We assessed these phenomena by measuring the temperature dependence of NMR-detected amide proton exchange and used these data to extract the entropic and enthalpic contributions of crowding to the stability of ubiquitin. Contrary to expectations, the contribution of chemical interactions is large and in many cases dominates the contribution from hardcore repulsions. Our results show that both chemical interactions and hard-core repulsions must be considered when assessing the effects of crowding and help explain previous observations about protein stability and dynamics in cells.

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.

Iron(II) Complexes Containing Octadentate Tetraazamacrocycles as ParaCEST Magnetic Resonance Imaging Contrast Agents

Iron(II) complexes of the macrocyclic ligands 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane (TCMC) and (1S,4S,7S,10S)-1,4,7,10-tetrakis(2-hydroxypropyl)-1,4,7,10-tetraazacyclododecane (STHP) contain a highly stabilized Fe(II) center in the high-spin state, which is encapsulated by an octadentate macrocycle. The complexes are resistant to acid, metal cations, phosphate, carbonate, and oxygen in aqueous solution. [Fe(TCMC)](2+) contains exchangeable amide protons, and [Fe(STHP)](2+) contains exchangeable protons attributed to alcohol OH donors, which give chemical exchange saturation transfer (CEST) peaks at physiological pH and 37 °C at 50 and 54 ppm from bulk water, respectively. The distinct pH dependence of the CEST peak of the two complexes over the range of pH 6-8 shows that these two groups may be useful in the development of ratiometric pH sensors based on iron(II).

A new method for detecting exchanging amide protons using chemical exchange rotation transfer

In this study, we introduce a new method for amide proton transfer imaging based on chemical exchange rotation transfer. It avoids several artifacts that plague conventional chemical exchange saturation transfer approaches by creating label and reference scans based on varying the irradiation pulse rotation angle (π and 2π radians) instead of the frequency offset (3.5 and -3.5 ppm). Specifically, conventional analysis is sensitive to confounding contributions from magnetic field (B(0)) inhomogeneities and, more problematically, inherently asymmetric macromolecular resonances. In addition, the lipid resonance at -3.5 ppm complicates the interpretation of the reference scan and decreases the resulting contrast. Finally, partial overlap of the amide signal by nearby amines and hydroxyls obscure the results. By avoiding these issues, our new method is a promising approach for imaging endogenous protein and peptide content and mapping pH.

Ca2+-binding reduces conformational flexibility of RC–LH1 core complex from thermophile Thermochromatium tepidum

The light-harvesting complex, LH1, of thermophile purple bacteria Thermochromatium tepidum consists of an array of α- and β-polypeptides which assemble the photoactive bacteriochlorophyll and closely interact with the membrane-lipids. In this study, we investigated the effect of calcium and manganese ions on the protein structure and thermostability of the reaction centre (RC)-LH1/lipid complex. The binding of Ca(2+), but not Mn(2+) is shown to shift the LH1 Q ( y ) absorption maximum from ~889 to 915nm and to significantly raise the thermostability of the RC-LH1 complex. The ATR-FTIR spectra indicate that interaction of Ca(2+) as monitored by the carboxylates' vibration of aspartate residues, but not Mn(2+) induces changes in the α-helix packing arrangement. The reduced rate of (1)H/(2)H exchange of proteins' amide protons shows that the accessibility to (2)H(2)O is significantly lowered in Ca(2+)-substituted RC-LH1/lipid complexes. In particular, exchange with the associated lipid molecules, is significantly retarded. These results suggest that the thermostability of the RC-LH1 complex is raised by the distinct interaction with calcium cations which reduces the RC-LH1/lipid dynamics, particularly, at the membrane-water interface.

Nonionic Homopolymeric Amphipols: Application to Membrane Protein Folding, Cell-Free Synthesis, and Solution Nuclear Magnetic Resonance

Nonionic amphipols (NAPols) synthesized by homotelomerization of an amphiphatic monomer are able to keep membrane proteins (MPs) stable and functional in the absence of detergent. Some of their biochemical and biophysical properties and applications have been examined, with particular attention being paid to their complementarity with the classical polyacrylate-based amphipol A8-35. Bacteriorhodopsin (BR) from Halobacterium salinarum and the cytochrome b(6)f complex from Chlamydomonas reinhardtii were found to be in their native state and highly stable following complexation with NAPols. NAPol-trapped BR was shown to undergo its complete photocycle. Because of the pH insensitivity of NAPols, solution nuclear magnetic resonance (NMR) two-dimensional (1)H-(15)N heteronuclear single-quantum coherence spectra of NAPol-trapped outer MP X from Escherichia coli (OmpX) could be recorded at pH 6.8. They present a resolution similar to that of the spectra of OmpX/A8-35 complexes recorded at pH 8.0 and give access to signals from solvent-exposed rapidy exchanging amide protons. Like A8-35, NAPols can be used to fold MPs to their native state as demonstrated here with BR and with the ghrelin G protein-coupled receptor GHS-R1a, thus extending the range of accessible folding conditions. Following NAPol-assisted folding, GHS-R1a bound four of its specific ligands, recruited arrestin-2, and activated binding of GTPγS by the G(αq) protein. Finally, cell-free synthesis of MPs, which is inhibited by A8-35 and sulfonated amphipols, was found to be very efficient in the presence of NAPols. These results open broad new perspectives on the use of amphipols for MP studies.

Mapping the Backbone Dynamics of the Alzheimer's APP Transmembrane Domain

The Amyloid Precursor Protein (APP) plays a central role in Alzheimer's disease. Its dimeric transmembrane domain (APP-TMD) is subject to several consecutive cleavage events by the gamma-secretase leading to a certain product pattern of amyloidogenic peptides (Munter et al., 2007). Here, we address the question how backbone dynamics of the alpha-helical APP-TMD is mapped.We have investigated the backbone dynamics of several APP-TMD model peptides with hydrogen-deuterium exchange (HDX) experiments coupled to mass spectroscopy. The underlying principle is the exchange of amide-protons for deuterons which is dependent on the dynamics of the alpha-helix.The investigation demonstrates that the N-terminal part of the APP transmembrane domain exhibits higher backbone dynamics than the C-terminal part. Further results show that exchanging threonines for valines along the APP-TMD sequence changes the helix backbone flexibility. To characterize the dynamics of each cleavage region along the APP-TMD a hybrid peptide set was examined. The hybrid peptides consist of a constant poly-leucine sequence and a varying 8 residue long APP-TMD-region where the last and last but two/ three residues represent cleavage sites.To this end, we could discover local differences in APP-TMD helix dynamics. The findings are discussed in terms of substrate recognition and binding and could explain the A beta product pattern in gamma-secretase proteolysis.Munter, L. M., Voigt, P., et al. (2007). “GxxxG motifs within the amyloid precursor protein transmembrane sequence are critical for the etiology of A beta 42.” EMBO Journal 26(6): 1702-1712.

Extracranial measurements of amide proton transfer using exchange-modulated point-resolved spectroscopy (EXPRESS)

Chemical exchange saturation transfer (CEST) imaging has been used experimentally in a large range of applications. However, full quantification of CEST effects in vivo using standard imaging sequences is time consuming as a large number of saturation frequency offsets, each followed by an imaging readout, are required to define a z spectrum. Furthermore, outside the brain, the presence of fat can confound the interpretation of z spectra. A novel acquisition and post-processing technique is presented in this study, named exchange-modulated point-resolved spectroscopy (EXPRESS), which aims to address these limitations and to enable spatially localised, high signal-to-noise measurements of CEST effects in vivo. Using amide proton exchange (APT) measurements in tumours, it is demonstrated that the acquisition of two-dimensional EXPRESS spectra composed of chemical shift and saturation frequency offset dimensions allows the correction of CEST data containing both fat and water signals, which is a common confounding property of tissues found outside the brain.

TmDOTA-tetraglycinate encapsulated liposomes as pH-sensitive LipoCEST agents.

Lanthanide DOTA-tetraglycinate (LnDOTA-(gly)4 −) complexes contain four magnetically equivalent amide protons that exchange with protons of bulk water. The rate of this base catalyzed exchange process has been measured using chemical exchange saturation transfer (CEST) NMR techniques as a function of solution pH for various paramagnetic LnDOTA-(gly)4 − complexes to evaluate the effects of lanthanide ion size on this process. Complexes with Tb(III), Dy(III), Tm(III) and Yb(III) were chosen because these ions induce large hyperfine shifts in all ligand protons, including the exchanging amide protons. The magnitude of the amide proton CEST exchange signal differed for the four paramagnetic complexes in order, Yb>Tm>Tb>Dy. Although the Dy(III) complex showed the largest hyperfine shift as expected, the combination of favorable chemical shift and amide proton CEST linewidth in the Tm(III) complex was deemed most favorable for future in vivo applications where tissue magnetization effects can interfere. TmDOTA-(gly)4 − at various concentrations was encapsulated in the core interior of liposomes to yield lipoCEST particles for molecular imaging. The resulting nanoparticles showed less than 1% leakage of the agent from the interior over a range of temperatures and pH. The pH versus amide proton CEST curves differed for the free versus encapsulated agents over the acidic pH regions, consistent with a lower proton permeability across the liposomal bilayer for the encapsulated agent. Nevertheless, the resulting lipoCEST nanoparticles amplify the CEST sensitivity by a factor of ∼104 compared to the free, un-encapsulated agent. Such pH sensitive nano-probes could prove useful for pH mapping of liposomes targeted to tumors.

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.

Local and Coupled Thermodynamic Stability of the Two-Domain and Bifunctional Enzyme SlyD from Escherichia coli

SlyD (sensitive to lysis D) is a protein folding helper enzyme comprising peptidylprolyl isomerase as well as chaperone activities at the respective FKBP and IF domains. Both domains coact concerning the peptidylprolyl isomerase activity on protein substrates. Using various biophysical techniques and NMR spectroscopy, the local and global thermodynamic stability of the variant (1-165) of SlyD from Escherichia coli (SlyD*) was characterized. Structurally, both domains are rather independent. The urea-induced unfolding transitions of the two domain protein monitored by 2D NMR spectroscopy and amide proton exchange experiments, however, showed that the IF domain experiences a reduced local stability under both native and unfolding conditions compared to the FKBP domain. Nevertheless, the entire protein shows highly cooperative unfolding at elevated denaturing conditions. This cooperativity is significantly reduced in a SlyD* variant missing the IF domain. The quite low local stability due to high internal fluctuations of the IF domain might be the prerequisite for the ubiquitous chaperone function of SlyD. One physiological role of the metallochaperone SlyD is divalent cations binding. Nickel binds only to the FKBP domain but extensively increases the thermodynamic stability of both SlyD domains, verifying the coupled stability of the domains.