Proton exchange as a relaxation mechanism for T1 in the rotating frame in native and immobilized protein solutions.

T1 relaxation in the rotating frame (T1rho) is a sensitive magnetic resonance imaging (MRI) contrast for acute brain insults. Biophysical mechanisms affecting T1rho relaxation rate (R1rho) and R1rho dispersion (dependency of R1rho on the spin-lock field) were studied in protein solutions by varying their chemical environment and pH in native, heat-denatured, and glutaraldehyde (GA) cross-linked samples. Low pH strongly reduced R1rho in heat-denatured phantoms displaying proton resonances from a number of side-chain chemical groups in high-resolution 1H NMR spectra. At pH of 5.5, R1rho dispersion was completely absent. In contrast, in the GA-treated phantoms with very few NMR visible side chain groups, acidic pH showed virtually no effect on R1rho. The present data point to a crucial role of proton exchange on R1rho and R1rho dispersion in immobilized protein solution mimicking tissue relaxation properties.     

Heparin dodecasaccharide binding to platelet factor-4 and growth-related protein-alpha. Induction of a partially folded state and implications for heparin-induced thrombocytopenia.

alpha-Chemokines are known heparin-binding proteins. Here, a heparin dodecasaccharide (H12) was purified and used in NMR studies to investigate binding to growth-related protein-alpha (Gro-alpha) and to platelet factor-4-M2 (PF4-M2), an N-terminal chimera of PF4. Pulsed field gradient NMR was used to derive diffusion coefficients as the protein (monomer):H12 ratio was varied. In the absence of H12, both PF4-M2 and Gro-alpha give diffusion coefficients consistent with the presence of mostly dimers. As the PF4-M2:H12 ratio is increased from 1:6 to 2:1, the diffusion coefficient increases, indicating dissociation to the monomer state. On addition of H12 to either protein, (15)N/(1)H heteronuclear single quantum coherence NMR data demonstrate loss of (1)H resonance dispersion and intensity, particularly at protein:H12 ratios of 2:1 to 4:1, indicating significant perturbation to native structures. For Gro-alpha in particular, (1)H resonance dispersion appears random coil-like. At these same ratios, circular dichroism (CD) data show general retention of secondary structure elements with a slight shift to additional helix formation. Random coil NMR resonance dispersion suggests a shift to a less compact, partially folded, and/or more flexible state. Further addition of H12 causes resonance intensity and dispersion to return making NMR spectra appear native-like. At low PF4-M2:H12 ratios, loss of resonance intensity for residues proximal to Arg-20 and Arg-22 in three-dimensional NMR HCCH-TOCSY spectra suggests that the Arg-20-Arg-22 loop either interacts most strongly with H12 and/or that binding at this site is heterogeneous. This domain was previously shown to be crucial to heparin binding. Of particular interest to the biology of PF4-heparin complex formation, heparin-induced thrombocytopenia antibody binding occurs at about the same PF4-M2:H12 ratio as does this transition to a partially folded PF4-M2 state, strongly suggesting that heparin-induced thrombocytopenia antibody recognizes a less folded, lower aggregate state of the protein.     

Abp1p and Fyn SH3 domains fold through similar low-populated intermediate states

Src homology 3 (SH3) domains are small modules that are thought to fold via a two-state mechanism, without the accumulation of significant populations of intermediate states. Relaxation dispersion NMR studies of the folding of G48V and G48M mutants of the Fyn SH3 domain have established that, at least for these modules, folding proceeds through the formation of a transient on-pathway intermediate with an equilibrium population of 1-2% that can be readily detected [Korzhnev, D. M., et al. (2004) Nature 430, 586-590]. To investigate the generality of this result, we present an (15)N relaxation dispersion NMR study of a pair of additional SH3 domains, including a G48V mutant of a stabilized Abp1p SH3 domain that shares 36% sequence identity with the Fyn SH3 module, and a A39V/N53P/V55L mutant Fyn SH3 domain. A transient folding intermediate is detected for both of the proteins studied here, and the dispersion data are well fit to a folding model of the form F <--> I <--> U, where F, I, and U correspond to folded, intermediate, and unfolded states, respectively. The temperature dependencies of the folding/unfolding rate constants were obtained so that the thermodynamic properties of each of F, I, and U could be established. The detection of I states in folding pathways of all SH3 domains examined to date via relaxation dispersion NMR spectroscopy indicates that such intermediates may well be a conserved feature in the folding of such domains in general but that their transient nature along with their low population makes detection difficult using more well-established approaches to the study of folding.     

Reducing the overlap problem in the proton NMR spectra of oligosaccharides by application of pseudo-four-dimensional homonuclear HOHAHA-HOHAHA-COSY.

A new homonuclear NMR experiment is described for the assignment of the proton NMR spectra of oligosaccharides, namely HOHAHA-HOHAHA-COSY (where HOHAHA is homonuclear Hartmann-Hahn spectroscopy and COSY is correlated spectroscopy). While this technique may formally be thought of as a four-dimensional NMR experiment, by use of selective pulses it is demonstrated that the analogous pseudo-four-dimensional experiment is a valuable improvement over conventional three-dimensional HOHAHA-COSY, in that the degree of resonance overlap is markedly reduced by the dispersion of resonances into a fourth effective dimension. The technique is demonstrated by application to the biantennary nonasaccharide Gal beta 1-4-GlcNAc beta 1-2Man alpha 1-6(Gal beta 1-4GlcNAc beta 1-2Man alpha 1-3)Man-beta 1-4GlcNAc beta 1-4GlcNAc.     

Atropisomeric 3-aryl-2-oxo-4-oxazolidinones and some thione analogues--enantiodifferentiation and ligand competition in applying the dirhodium method

The atropisomeric 2-oxo-4-oxazolidinones 1Z bind weakly to the rhodium atoms in the complex Rh(II)2 [(R)-(+)-MTPA]4 (Rh*, MTPA-H = methoxytrifluoromethylphenylacetic acid identical with Mosher's acid), presumably via the C-2 carbonyl oxygen atom. There are some 1H and 13C NMR signals in these compounds which show small dispersion effects suitable for enantiodifferentiation. In contrast, the thiocarbonyl sulfur atoms in 2Z and 3Z bind strongly so that significant complexation shifts (Delta delta) and diastereomeric dispersion effects (Delta nu) can be observed, and chiral discrimination and the determination of enantiomeric ratios of these thiocarbonyl compounds is easy. So, it is shown that--as expected--C=S is a much better binding site when competing with C=O. In compounds of Series 2 a "syn-methyl effect" was discovered which describes the dependence of dispersion effects of syn-oriented methyl groups 6 on the nature of the substituents Z. A mechanism of combined steric and electronic interaction influencing the conformational equilibria inside the adducts is proposed. Determination of absolute configurations by correlation fails, at least on the basis of the data available.     

Non-degradative dissolution and acetylation of ball-milled plant cell walls: high-resolution solution-state NMR

Two solvent systems for fully dissolving, and optionally derivatizing, finely ground plant cell wall material at room temperature are described: dimethylsulfoxide (DMSO) and tetrabutylammonium fluoride (TBAF) or N-methylimidazole (NMI). In situ acetylation produces acetylated cell walls (Ac-CWs) that are fully soluble in chloroform. Lignin structures tested remain fully intact. The dispersion of 13C-1H correlations afforded by two-dimensional (2D) nuclear magnetic resonance (NMR) experiments reveals the major lignin units, allowing the whole lignin fraction to be analyzed by high-resolution solution-state NMR methods for the first time. Non-degradative cell wall dissolution offers the potential to analyze polysaccharide components, and improve current cell wall analytical methods by using standard homogeneous solution-state chemistry.     

NMR spectra of oligosaccharides at ultra-high field (900 MHz) have better resolution than expected due to favourable molecular tumbling

Nuclear magnetic resonance (NMR) remains the most promising technique for acquiring atomic-resolution information in complex carbohydrates. Significant obstacles to the acquisition of such data are the poor chemical-shift dispersion and artifacts resultant from their degenerate chemical structures. The recent development of ultra-high-field NMR (at 900 MHz and beyond) gives new potential to overcome these problems, as we demonstrate on a hexasaccharide of the highly repetitive glycosaminoglycan hyaluronan. At 900 MHz, the expected increase in spectral dispersion due to higher resonance frequencies and reduction in strong coupling-associated distortions are observed. In addition, the fortuitous molecular tumbling rate of oligosaccharides results in longer T2-values that further significantly enhances resolution, an effect not available to proteins. Combined, the resolution enhancement can be as much as twofold relative to 600 MHz, allowing all 1H-resonances in the hexasaccharide to be unambiguously assigned using standard natural-abundance experiments. The use of ultra-high-field spectrometers is clearly advantageous and promises a new and exciting era in carbohydrate structural biology.     

An introduction to NMR-based approaches for measuring protein dynamics

Proteins are inherently flexible at ambient temperature. At equilibrium, they are characterized by a set of conformations that undergo continuous exchange within a hierarchy of spatial and temporal scales ranging from nanometers to micrometers and femtoseconds to hours. Dynamic properties of proteins are essential for describing the structural bases of their biological functions including catalysis, binding, regulation and cellular structure. Nuclear magnetic resonance (NMR) spectroscopy represents a powerful technique for measuring these essential features of proteins. Here we provide an introduction to NMR-based approaches for studying protein dynamics, highlighting eight distinct methods with recent examples, contextualized within a common experimental and analytical framework. The selected methods are (1) Real-time NMR, (2) Exchange spectroscopy, (3) Lineshape analysis, (4) CPMG relaxation dispersion, (5) Rotating frame relaxation dispersion, (6) Nuclear spin relaxation, (7) Residual dipolar coupling, (8) Paramagnetic relaxation enhancement. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.     

The folding pathway of an FF domain: characterization of an on-pathway intermediate state under folding conditions by (15)N, (13)C(alpha) and (13)C-methyl relaxation dispersion and (1)H/(2)H-exchange NMR spectroscopy

The FF domain from the human protein HYPA/FBP11 folds via a low-energy on-pathway intermediate (I). Elucidation of the structure of such folding intermediates and denatured states under conditions that favour folding are difficult tasks. Here, we investigated the millisecond time-scale equilibrium folding transition of the 71-residue four-helix bundle wild-type protein by (15)N, (13)C(alpha) and methyl(13)C Carr-Purcell-Meiboom-Gill (CPMG) NMR relaxation dispersion experiments and by (1)H/(2)H-exchange measurements. The relaxation data for the wild-type protein fitted a simple two-site exchange process between the folded state (F) and I. Destabilization of F in mutants A17G and Q19G allowed the detection of the unfolded state U by (15)N CPMG relaxation dispersion. The dispersion data for these mutants fitted a three-site exchange scheme, U<-->I<-->F, with I populated higher than U. The kinetics and thermodynamics of the folding reaction were obtained via temperature and urea-dependent relaxation dispersion experiments, along with structural information on I from backbone (15)N, (13)C(alpha) and side-chain methyl (13)C chemical shifts, with further information from protection factors for the backbone amide groups from (1)H/(2)H-exchange. Notably, helices H1-H3 are at least partially formed in I, while helix H4 is largely disordered. Chemical shift differences for the methyl (13)C nuclei suggest a paucity of stable, native-like hydrophobic interactions in I. These data are consistent with Phi-analysis of the rate-limiting transition state between I and F. The combination of relaxation dispersion and Phi data can elucidate whole experimental folding pathways.     

Conformational dynamics of yeast calmodulin in the Ca(2+)-bound state probed using NMR relaxation dispersion

Most calmodulin (CaM) in apo and Ca(2+)-bound states show a dumb-bell-like structure, involving the N- and C-terminal domains, connected with a flexible linker. However, Ca(2+)-bound yeast calmodulin (yCaM) takes on a unique globular structure; the target-binding site of this protein is autoinhibited. We applied NMR relaxation dispersion experiments to yCaM in the Ca(2+)-bound state. The amide (15)N and (1)H(N) relaxation dispersion profiles indicated the presence of conformational dynamics for specific residues at the interface between the N- and C-terminal domains. We conclude that these conformational dynamics were derived from the mobility of the C-terminal domain.     

Proton-coupled 15N NMR spectra of neutral and protonated ethenoadenosine and ethenocytidine.

The 15N chemical shifts and 15N, 1H spin coupling constants were determined in the title compounds using the INEPT pulse sequence and assigned with the aid of selective proton decoupling. The delta/15N/ and J/N, H/ values are discussed in terms of involvement of the imidazole ring created by ethenobridging in the electronic structure of the whole molecule. Both spectral parameters indicate that the diligant nitrogen in this ring is the primary site of protonation in these modified nucleosides. It is concluded that 15N NMR of nucleoside bases can be largely a complementary method to 1H and 13C NMR studies and, in addition, can serve as a direct probe for studies of nitrogen environment in oligomeric fragments of nucleic acids even at moderately strong magnetic fields due to the higher spectral dispersion compared with 1H and 13C NMR spectra.     

920 MHz ultra-high field NMR approaches to structural glycobiology

Although NMR spectroscopy has great potential to provide us with detailed structural information on oligosaccharides and glycoconjugates, the carbohydrate NMR analyses have been hampered by the severe spectral overlapping and the insufficiency of the conformational restraints. Recently, ultra-high field NMR spectrometers have become available for applications to structural analyses of biological macromolecules. Here we demonstrate that ultra-high fields offer not only increases in sensitivity and chemical shift dispersion but also potential benefits for providing unique information on chemical exchange and relaxation, by displaying NMR spectral data of oligosaccharide, glycoprotein, and glycolipid systems recorded at a 21.6 T magnetic field (corresponding to 920 MHz (1)H observation frequency). The ultra-high field NMR spectroscopy combined with sugar library and stable-isotope labeling approaches will open new horizons in structural glycobiology.     

Solid-state NMR investigation of indomethacin-cyclodextrin complexes in PEG 6000 carrier

Various solid dispersions of alpha-, beta- and gamma-cyclodextrin (CD) in PEG 6000 with and without the addition of 5% w/w indomethacin were prepared by the melting method using the original components. The samples were investigated by solid-state (13)C NMR, and the interactions between the drug and the cyclodextrins were evaluated. The indomethacin-gamma-CD phase with tetragonal symmetry found in a previous X-ray study gave chemical shifts which suggested that this phase is a complex between indomethacin and gamma-CD. Evidence of an indomethacin-beta-CD complex were found. A distribution of the chemical shifts for beta-CD was attributed to the possible formation of different types of complexes between indomethacin and beta-CD. No complex formation was found in the alpha-CD system. The degree of relative crystallinity of the samples in the gamma-CD system was measured by (1)H NMR, X-ray powder diffraction (XRD), differential scanning calorimetry (DSC), and modulated-temperature DSC (MTDSC). The results obtained by the NMR, XRD, and DSC techniques showed that the dispersions were less crystalline than the pure polymer carrier, and the dispersion containing the indomethacin-gamma-CD complex had the lowest degree of crystallinity. By the MTDSC method a deviation was found for the PEG 6000/indomethacin dispersion. This emphasizes that the different techniques give specific information on the crystallinity.     

Histidine side-chain dynamics and protonation monitored by 13C CPMG NMR relaxation dispersion

The use of ¹³C NMR relaxation dispersion experiments to monitor micro-millisecond fluctuations in the protonation states of histidine residues in proteins is investigated. To illustrate the approach, measurements on three specifically ¹³C labeled histidine residues in plastocyanin (PCu) from Anabaena variabilis (A.v.) are presented. Significant Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion is observed for ¹³C^ε1 nuclei in the histidine imidazole rings of A.v. PCu. The chemical shift changes obtained from the CPMG dispersion data are in good agreement with those obtained from the chemical shift titration experiments, and the CPMG derived exchange rates agree with those obtained previously from ¹⁵N backbone relaxation measurements. Compared to measurements of backbone nuclei, ¹³C^ε1 dispersion provides a more direct method to monitor interchanging protonation states or other kinds of conformational changes of histidine side chains or their environment. Advantages and shortcomings of using the ¹³C^ε1 dispersion experiments in combination with chemical shift titration experiments to obtain information on exchange dynamics of the histidine side chains are discussed.     

500 MHz H-NMR studies of bile salt-phosphatidylcholine mixed micelles and vesicles Evidence for differential motional restraint on bile salt and phosphatidylcholine resonances

¹H nuclear magnetic resonance (NMR) spectra at 500 MHz have been obtained for taurocholate/egg phosphatidylcholine mixtures of varying composition. The excellent chemical shift dispersion permits identification of most resonances for each component. This high-resolution character of the NMR spectra is retained until the phosphatidylcholine (PC) mole fraction exceeds 60–70% (the exact limit depends on ionic strength). ¹H linewidths have been monitored as a function of solute composition in order to evaluate trends in local molecular mobility of each component as the distribution of aggregate particles is varied, and to examine the effects of added NaCl in altering micellar size and shape. Although prior light scattering studies (Mazer, N.A., Benedek, G.B. and Carey, M.C. (1980) Biochemistry 19, 601–615) and our own work indicate a 6-fold increase in particle hydrodynamic radius from pure taurocholate micelles to 1 : 1 taurocholate/PC mixtures containing 150 mM NaCl, both lipid components retain substantial motional freedom and exhibit narrow NMR signals in this compositional region. As the solubilization limit for PC is approached (approx. 2:1 PC:taurocholate), differential behavior is observed for the two components: the motion of taurocholate becomes preferentially restricted, while polar portions of the PC remain mobile until large multilayers predominate.     

500 MHz 1H-NMR studies of bile salt-phosphatidylcholine mixed micelles and vesicles Evidence for differential motional restraint on bile salt and phosphatidylcholine resonances

¹H nuclear magnetic resonance (NMR) spectra at 500 MHz have been obtained for taurocholate/egg phosphatidylcholine mixtures of varying composition. The excellent chemical shift dispersion permits identification of most resonances for each component. This high-resolution character of the NMR spectra is retained until the phosphatidylcholine (PC) mole fraction exceeds 60–70% (the exact limit depends on ionic strength). ¹H linewidths have been monitored as a function of solute composition in order to evaluate trends in local molecular mobility of each component as the distribution of aggregate particles is varied, and to examine the effects of added NaCl in altering micellar size and shape. Although prior light scattering studies (Mazer, N.A., Benedek, G.B. and Carey, M.C. (1980) Biochemistry 19, 601–615) and our own work indicate a 6-fold increase in particle hydrodynamic radius from pure taurocholate micelles to 1 : 1 taurocholate/PC mixtures containing 150 mM NaCl, both lipid components retain substantial motional freedom and exhibit narrow NMR signals in this compositional region. As the solubilization limit for PC is approached (approx. 2:1 PC:taurocholate), differential behavior is observed for the two components: the motion of taurocholate becomes preferentially restricted, while polar portions of the PC remain mobile until large multilayers predominate.     

Polymorphic phase behavior of unsaturated lysophosphatidylethanolamines: a 31P NMR and X-ray diffraction study

The polymorphic phase behavior of aqueous dispersions of 1-oleoyl-, 1-linoleoyl-, and 1-linolenoyl-sn-3-glycerophosphoethanolamine (1-C18:1c-PE, 1-C18:2c-PE, and 1-C18:3c-PE, respectively) has been investigated by 31P NMR, small-angle and wide-angle X-ray diffraction, and freeze-fracture techniques in response to changes in temperature and pH. Between -20 and 0 degrees C at pH 7, NMR and X-ray data indicate that 1-C18:1c-PE adopts a lamellar phase. Above 20 degrees C, the X-ray diffraction from 1-C18:1c-PE reveals no long-range lattice order, whereas the NMR data indicate lamellar structure to 90 degrees C. Freeze-fracture electron microscopy shows that 1-C18:1c-PE at pH 8.2 forms closed multilamellar vesicles upon dispersion and also that large unilamellar vesicles produced by extrusion techniques (LUVETs) can be made from 1-C18:1c-PE at pH 7. Such LUVETs can trap [3H]inulin and support a K+ diffusion potential for up to 4 h. At pH 8.5 and above, 1-C18:1c-PE forms optically clear, fluid dispersions with NMR and X-ray characteristics consistent with a micellar (noninverted) phase structure. Attempts to prepare LUVETs from 1-C18:1c-PE at pH 9 result in structures that can neither trap [3H]inulin nor support a membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR structure and dynamics of a designed water-soluble transmembrane domain of nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) is an important therapeutic target for a wide range of pathophysiological conditions, for which rational drug designs often require receptor structures at atomic resolution. Recent proof-of-concept studies demonstrated a water-solubilization approach to structure determination of membrane proteins by NMR (Slovic et al., PNAS, 101: 1828-1833, 2004; Ma et al., PNAS, 105: 16537-42, 2008). We report here the computational design and experimental characterization of WSA, a water-soluble protein with ~83% sequence identity to the transmembrane (TM) domain of the nAChR α1 subunit. Although the design was based on a low-resolution structural template, the resulting high-resolution NMR structure agrees remarkably well with the recent crystal structure of the TM domains of the bacterial Gloeobacter violaceus pentameric ligand-gated ion channel (GLIC), demonstrating the robustness and general applicability of the approach. NMR T(2) dispersion measurements showed that the TM2 domain of the designed protein was dynamic, undergoing conformational exchange on the NMR timescale. Photoaffinity labeling with isoflurane and propofol photolabels identified a common binding site in the immediate proximity of the anesthetic binding site found in the crystal structure of the anesthetic-GLIC complex. Our results illustrate the usefulness of high-resolution NMR analyses of water-solubilized channel proteins for the discovery of potential drug binding sites.     

A left-handed 3(1) helical conformation in the Alzheimer Abeta(12-28) peptide

We show for the first time that the secondary structure of the Alzheimer beta-peptide is in a temperature-dependent equilibrium between an extended left-handed 3(1) helix and a flexible random coil conformation. Circular dichroism spectra, recorded at 0.03 mM peptide concentration, show that the equilibrium is shifted towards increasing left-handed 3(1) helix structure towards lower temperatures. High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study the Alzheimer peptide fragment Abeta(12-28) in aqueous solution at 0 degrees C and higher temperatures. NMR translation diffusion measurements show that the observed peptide is in monomeric form. The chemical shift dispersion of the amide protons increases towards lower temperatures, in agreement with the increased population of a well-ordered secondary structure. The solvent exchange rates of the amide protons at 0 degrees C and pH 4.5 vary within at least two orders of magnitude. The lowest exchange rates (0.03-0.04 min(-1)) imply that the corresponding amide protons may be involved in hydrogen bonding with neighboring side chains.     

Solid-state NMR study and assignments of the KcsA potassium ion channel of S. lividans

The extraordinary efficiency and selectivity of potassium channels have made them ideal systems for biophysical and functional studies of ion conduction. We carried out solid-state NMR studies of the selectivity filter region of the protein. Partial site-specific assignments of the NMR signals were obtained based on high field multidimensional solid-state NMR spectra of uniformly (13)C, (15)N enriched KcsA potassium channel from Streptomyces lividans. Both backbone and sidechain atoms were assigned for residues V76-D80 and P83-L90, in and near the selectivity filter region of the protein; this region exhibits good dispersion and useful chemical shift fingerprints. This study will enable structure, dynamic and mechanistic studies of ion conduction by NMR.