Dissecting functional interactions in coagulation protein complexes by use of NMR spectroscopy

The blood coagulation cascade can be considered as a system of well-orchestrated protein activation reactions involving and leading to the formation of large macromolecular assemblies. NMR investigations performed during the last six years have focused on the structural, motional and binding properties of some protein domains and interfaces critical for the formation of these protein complexes, outlining sophisticated intermolecular adaptations. The studied protein domains are either single molecules or covalently-linked heterodimers of the epidermal growth factor (EGF) homology domains, calcium-binding EGF domains and gamma-carboxyglutamic(Gla)-containing domains responsible for calcium-dependent binding to cell membranes. The characterized binding interfaces have included those between thrombin and fibrinogen, between thrombin and thrombomodulin, between factor VIIIa and the cell membrane, between tissue factor and factor VIIa, and most recently between factor Va and prothrombin. The obtained results indicate that the regulation of blood coagulation by protein and low molecular weight cofactors may involve a significant degree of protein folding transitions with changes in molecular and conformational motions coupled to enzymatic activities. This new level of complexity of the molecular processes controlling coagulation may lead to novel strategies for the development of more effective therapeutic anticoagulants.     

Structure,dynamics and interactions in polymer systems as studied by NMR spectroscopy

The possibilities of NMR spectroscopy in studies of interactions in polymer systems are demonstrated on the example of two types of macromolecular complexes: (i) By measuring ¹H NMR high resolution line intensities, the formation of ordered associated structures of syndiotactic (s) poly(methyl methacrylate)(PMMA) in mixed solvents was quantitatively characterized. The obtained results permit us to assume that the mechanism by which the solvent affects self-association of s-PMMA involves specific interactions of the solvent molecules with PMMA units. Solid state high resolution ¹³C NMR spectra of associated s-PMMA gels were also measured and compared with the spectra of a solid s-PMMA sample. (ii) By using ¹³C solid state NMR spectroscopy, the differences in the structure of the amorphous and crystalline phases in pure poly(ethylene oxide) and its complexes with p-dichlorobenzene or p-nitrophenol were characterized. Prounounced differences also in the dynamic structure of the crystalline phase in these systems are indicated by the relaxation times T₁(C), T_1ρ(C) and T_1ρ(H).     

General framework for studying the dynamics of folded and nonfolded proteins by NMR relaxation spectroscopy and MD simulation

A general framework is presented for the interpretation of NMR relaxation data of proteins. The method, termed isotropic reorientational eigenmode dynamics (iRED), relies on a principal component analysis of the isotropically averaged covariance matrix of the lattice functions of the spin interactions responsible for spin relaxation. The covariance matrix, which is evaluated using a molecular dynamics (MD) simulation, is diagonalized yielding reorientational eigenmodes and amplitudes that reveal detailed information about correlated protein dynamics. The eigenvalue distribution allows one to quantitatively assess whether overall and internal motions are statistically separable. To each eigenmode belongs a correlation time that can be adjusted to optimally reproduce experimental relaxation parameters. A key feature of the method is that it does not require separability of overall tumbling and internal motions, which makes it applicable to a wide range of systems, such as folded, partially folded, and unfolded biomolecular systems and other macromolecules in solution. The approach was applied to NMR relaxation data of ubiquitin collected at multiple magnetic fields in the native form and in the partially folded A-state using MD trajectories with lengths of 6 and 70 ns. The relaxation data of native ubiquitin are well reproduced after adjustment of the correlation times of the 10 largest eigenmodes. For this state, a high degree of separability between internal and overall motions is present as is reflected in large amplitude and collectivity gaps between internal and overall reorientational modes. In contrast, no such separability exists for the A-state. Residual overall tumbling motion involving the N-terminal beta-sheet and the central helix is observed for two of the largest modes only. By adjusting the correlation times of the 10 largest modes, a high degree of consistency between the experimental relaxation data and the iRED model is reached for this highly flexible biomolecule.     

Use of selective Trp side chain labeling to characterize protein-protein and protein-ligand interactions by NMR spectroscopy

Recent studies on amino acid occurrence in protein binding sites suggest that only a reduced number of residues are responsible for most interaction energy in protein-protein and protein-ligand interactions. Above all, tryptophan (Trp) seems to be the most frequent residue in protein's hot spots. Here we report a novel, efficient, and cost-effective method to selectively incorporate specific isotope labels into the side chains of Trp residues in recombinant proteins. We show that the method proposed allows selective NMR observation of Trp side chains that enables studies of ligand binding, protein-protein interactions, hydrogen binding, protein folding, and side chain dynamics. Examples with the protein BIR3 will be given.     

The influence of electronic modifications on rotational barriers of bis‐NHC‐complexes as observed by dynamic NMR spectroscopy

There has been much debate about the σ‐donor and π‐acceptor properties of N‐heterocyclic carbenes (NHCs). While a lot of synthetic modifications have been performed with the goal of optimizing properties of the catalyst to tune reactivity in various transformations (e.g. metathesis), direct methods to characterize σ‐donor and π‐acceptor properties are still few. We believe that dynamic NMR spectroscopy can improve understanding of this aspect. Thus, we investigated the intramolecular dynamics of metathesis precatalysts bearing two NHCs. We chose four systems with one identical NHC ligand (N,N′‐Bis(2,4,6‐trimethylphenyl)‐imidazolinylidene (SIMes) in all four cases) and NHC_ewg ligands bearing four different electron‐withdrawing groups (ewg). Both rotational barriers of the respective Ru‐NHC‐bonds change significantly when the electron density of one of the NHCs (NHC_ewg) is modified. Although it is certainly not possible to fully dissect σ‐donor and π‐acceptor portions of the bonding situations in the respective Ru‐NHC‐bond via dynamic NMR spectroscopy, our studies nevertheless show that the analysis of the rotation around the Ru‐SIMes‐bond can be used as a spectroscopic parameter complementary to cyclic voltammetry. Surprisingly, we observed that the rotation around the Ru‐NHC_ewg‐bond shows the same trend as the initiation rate of a ring‐closing metathesis of the four investigated bis‐NHC‐complexes. Copyright © 2013 John Wiley & Sons, Ltd.     

Molecular dynamics of discotic charge-transfer complexes, dielectric spectroscopy and 2H NMR studie

Using a combination of solid state ²H NMR spectroscopy on selectively deuteriated samples and dielectric spectroscopy, the molecular dynamics of discotic charge-transfer (CT) complexes were investigated. These complexes show particular thermodynamic and flow properties. Considered were mixtures of low molar mass donors and acceptors, low molar mass donors with main chain acceptor polymers and covalently linked donor-acceptor twins with different lengths of the spacer. A main result is that correlated rotational motions of discotic molecules or groups about the columnar axis are observed in all systems except for the twin with the short spacer. This type of motion seems to be a general feature of columnar phases. The non-discotic acceptor which is incorporated in the columns participates in this motion. The twin possessing a long spacer displays at high temperatures an additional process: it performs a diffusion process between the columns. A further result is that broad biphasic regions exist in CT mixtures at the transition from the discotic to the isotropic state.     

Separation of polyelectrolyte chain dynamics and dynamics of counterion attachment by EPR spectroscopy

Rotational dynamics and local enrichment of counterions close to polyelectrolyte chains were studied by EPR spectroscopy in solvents of different viscosity. The results confirm previous findings (D. Hinderberger, G. Jeschke, and H. W. Spiess, Macromolecules 2002, 35, 9698) that electrostatic attachment of counterions to the chains is dynamic with lifetimes of contact ion pairs shorter than 1 ns. While in low-viscosity solvents linewidths for a dianionic nitroxide probe and their dependence on polyelectrolyte concentration are dominated by the gradient of local concentration in the vicinity of the chain, they are more strongly influenced by changes in rotational dynamics in a glycerol/water mixture. The slowdown of dynamics at higher viscosity strongly depends on polyelectrolyte concentration, suggesting that the lifetime of the attached state increases. The linewidths of trianionic triarylmethyl probes and of the center line of the nitroxide probes are dominated by local counterion enrichment both at low and high viscosity. Comparison of these linewidths and of the extent to which the lineshapes are non-Lorentzian indicates build-up of larger concentration gradients at higher viscosity.     

Backbone dynamics of proteins studied by two-dimensional heteronuclear NMR spectroscopy and molecular dynamics simulations

To investigate the backbone dynamics of proteins ¹⁵N longitudinal and transverse relaxation experiments combined with {¹H, ¹⁵N{ NOE measurements together with molecular dynamics simulations were carried out using ribonuclease T₁ and the complex of ribonuclease T₁ with 2′GMP as a model protein. The intensity decay of individual amide cross peaks in a series of (¹H, ¹⁵N)HSQC spectra with appropriate relaxation periods was fitted to a single exponential by using a simplex algorithm in order to obtain ¹⁵N T₁ and T₂ relaxation times. The relaxation times were analyzed in terms of the “model-free” approach introduced by Lipari and Szabo. In addition, a nanosecond molecular dynamics (MD ) simulation of ribonuclease T₁ and its 2′GMP complex in water was carried out. The angular reorientations of the backbone amide groups were classified with several coordinate frames following a transformation of NH vector trajectories. In this study, NH librations and backbone dihedral angle fluctuations were distinguished. The NH bond librations were found to be similar for all amides as characterized by correlation times of librational motions in a subpicosecond scale. The angular amplitudes of these motions were found to be about 10°–12° for out-of-plane displacements and 3°–5° for the in-plane displacement. The contributions from the much slower backbone dihedral angle fluctuations strongly depend on the secondary structure. The dependence of the amplitude of local motion on the residue location in the backbone is in good agreement with the results of NMR relaxation measurements and the X-ray data. The protein dynamics is characterized by a highly restricted local motion of those parts of the backbone with defined secondary structure as well as by a high flexibility in loop regions. Comparison of the MD and NMR data of the free liganded enzyme ribonuclease T₁ clearly indicates a restriction of the mobility within certain regions of the backbone upon inhibitor binding. © 1996 John Wiley & Sons, Inc.     

Virus-ligand interactions: identification and characterization of ligand binding by NMR spectroscopy

We demonstrate the detection and characterization of ligand binding to viruses via NMR. To illustrate the methodology, the interaction of an antiviral compound with human rhinovirus serotype 2 (HRV2) was investigated. Specific interaction of a capsid-binding inhibitor and native HRV2 was monitored utilizing saturation transfer difference (STD) NMR. STD NMR experiments at atomic resolution allowed those regions of the ligand that are involved in the interaction with the virus to be determined. The approach allows for (i) the fast and robust assessment of binding, (ii) the determination of the ligand binding epitope at atomic resolution without the necessity to crystallize virus-ligand complexes, and (iii) the reuse of the virus in subsequent assays. This methodology enables one to easily identify binding of drugs, peptides, and receptor or antibody fragments to the viral capsid.     

Probing platinum azido complexes by 14N and 15N NMR spectroscopy

Metal azido complexes are of general interest due to their high energetic properties, and platinum azido complexes in particular because of their potential as photoactivatable anticancer prodrugs. However, azido ligands are difficult to probe by NMR spectroscopy due to the quadrupolar nature of (14)N and the lack of scalar (1)H coupling to enhance the sensitivity of the less abundant (15)N by using polarisation transfer. In this work, we report (14)N and (15)N NMR spectroscopic studies of cis,trans,cis-[Pt(N(3))(2)(OH)(2)(NH(3))] (1) and trans,trans,trans-[Pt(N(3))(2)(OH)(2)(X)(Y)], where X=Y=NH(3) (2); X=NH(3), Y=py (3) (py=pyridine); X=Y=py (4); and selected Pt(II) precursors. These studies provide the first (15)N NMR data for azido groups in coordination complexes. We discuss one- and three-bond J((15)N,(195)Pt) couplings for azido and am(m)ine ligands. The (14)N(α) (coordinated azido nitrogen) signal in the Pt(IV) azido complexes is extremely broad (W(1/2)≈2124 Hz for 4) in comparison to other metal azido complexes, attributable to a highly asymmetrical electric field gradient at the (14)N(α) atom. Through the use of anti-ringing pulse sequences, the (14)N NMR spectra, which show resolution of the broad (14)N(α) peak, were obtained rapidly (e.g., 1.5 h for 10 mM 4). The linewidths of the (14)N(α) signals correlate with the viscosity of the solvent. For (15) N-enriched samples, it is possible to detect azido (15)N resonances directly, which will allow photoreactions to be followed by 1D (15)N NMR spectroscopy. The T(1) relaxation times for 3 and 4 were in the range 5.7-120 s for (15)N, and 0.9-11.3 ms for (14)N. Analysis of the (1)J((15)N,(195)Pt) coupling constants suggests that an azido ligand has a moderately strong trans influence in octahedral Pt(IV) complexes, within the series 2-pic相似文献   

Determination of membrane protein structure and dynamics by magic-angle-spinning solid-state NMR spectroscopy

It is shown that molecular structure and dynamics of a uniformly labeled membrane protein can be studied under magic-angle-spinning conditions. For this purpose, dipolar recoupling experiments are combined with novel through-bond correlation schemes that probe mobile protein segments. These NMR schemes are demonstrated on a uniformly [13C,15N] variant of the 52-residue polypeptide phospholamban. When reconstituted in lipid bilayers, the NMR data are consistent with an alpha-helical trans-membrane segment and a cytoplasmic domain that exhibits a high degree of structural disorder.     

Study of acid-base interactions of mono- and diazaporphyrin indium complexes with acetic and trifluoroacetic acids by <Superscript>1</Superscript>H NMR and electronic spectroscopy

Indium complex of 13,17-dibutyl-2,3,7,8,12,18-hexamethyl-5-azaporphyrin (Cl)InMAP was synthesized, and acid-base interactions of the meso-nitrogen atoms in (Cl)InMAP and its diaza analog (Cl)InDAP with acetic and trifluoroacetic acids were studied by ¹H NMR and electronic spectroscopy. Depending on the medium, the complexes and proton-donor species HA give rise to acid solvates >N(HA)_n which are converted to final acid-base interaction products, H-complexes >NH+A^–(HA)_m or ionic associates >NH⁺A^–(HA)_l , as the acidity of the medium rises. In acetic acid solution, the acid solvates derived from more basic (Cl)InMAP exist in equilibrium with the H-associates (pK _a1 = 4.45±0.03). From (Cl)InDAP, the corresponding H-associates are formed only in the presence of H₂SO₄ (pK _a1 = 2.10±0.03). In more polar media (solutions of trifluoroacetic acid in methylene chloride), ionic associates are formed, which involve one [(Cl)InMAP, pK ₁ = 2.46±0.02] or two meso-nitrogen atoms [(Cl)InDAP, pK ₁ = 2.11±0.03, pK ₂ = 0.41±0.04).Translated from Zhurnal Obshchei Khimii, Vol. 74, No. 9, 2004, pp. 1546–1556.Original Russian Text Copyright © 2004 by Stuzhin, Ivanova, Migalova.This revised version was published online in April 2005 with a corrected cover date.     

Ortho-aryl substituted DPEphos ligands: rhodium complexes featuring C–H anagostic interactions and B–H agostic bonds

The synthesis of new Schrock–Osborn Rh(i) pre-catalysts with ortho-substituted DPEphos ligands, [Rh(DPEphos-R)(NBD)][BAr^F₄] [R = Me, OMe, ⁱPr; Ar^F = 3,5-(CF₃)₂C₆H₃], is described. Along with the previously reported R = H variant, variable temperature ¹H NMR spectroscopic and single-crystal X-ray diffraction studies show that these all have axial (C–H)⋯Rh anagostic interactions relative to the d⁸ pseudo square planar metal centres, that also result in corresponding downfield chemical shifts. Analysis by NBO, QTAIM and NCI methods shows these to be only very weak C–H⋯Rh bonding interactions, the magnitudes of which do not correlate with the observed chemical shifts. Instead, as informed by Scherer''s approach, it is the topological positioning of the C–H bond with regard to the metal centre that is important. For [Rh(DPEphos–ⁱPr)(NBD)][BAr^F₄] addition of H₂ results in a Rh(iii) ⁱPr–C–H activated product, [Rh(κ³,σ-P,O,P-DPEphos-ⁱPr′)(H)][BAr^F₄]. This undergoes H/D exchange with D₂ at the ⁱPr groups, reacts with CO or NBD to return Rh(i) products, and reaction with H₃B·NMe₃/tert-butylethene results in a dehydrogenative borylation to form a complex that shows both a non-classical B–H⋯Rh 3c-2e agostic bond and a C–H⋯Rh anagostic interaction at the same metal centre.

Rh(i) complexes of ortho-substituted DPEphos-R (R = H, Me, OMe, ⁱPr) ligands show anagostic interactions; for R =ⁱPr C–H activation/dehydrogenative borylation forms a product exhibiting both B–H/Rh 3c-2e agostic and C–H/Rh anagostic motifs.     

Toward the characterization of peptidoglycan structure and protein-peptidoglycan interactions by solid-state NMR spectroscopy

Solid-state NMR spectroscopy is applied to intact peptidoglycan sacculi of the Gram-negative bacterium Escherichia coli. High-quality solid-state NMR spectra allow atom-resolved investigation of the peptidoglycan structure and dynamics as well as the study of protein-peptidoglycan interactions.     

Structural transformation and guest dynamics of methanol-loaded hydroquinone clathrate observed by temperature-dependent Raman spectroscopy

The structural transformations and guest dynamics of methanol-loaded β-form hydroquinone (HQ) clathrate were investigated using temperature-dependent Raman spectroscopy. Methanol-loaded β-form HQ clathrate was obtained by recrystallization and characterized by elemental analysis, synchrotron X-ray diffraction, solid-state (13)C NMR spectroscopy, and Raman spectroscopy. Temperature-dependent Raman spectra of methanol-loaded β-form HQ clathrate were measured in the temperature range 300-412 K at increments of 4 K. Although no significant changes were evident in the temperature range 300-376 K, abrupt changes in the relative intensity and shape of the Raman bands were observed between 380 and 412 K indicating the structural transition from methanol-loaded β-form HQ clathrate to pure α-form HQ. Methanol molecules were gradually released from the β-form HQ clathrate in the range 364-380 K. Upon returning to ambient conditions, the crystal structure of the HQ sample remained identical to that of pure α-form HQ. Therefore, the temperature-induced structural transition of methanol-loaded HQ clathrate is completely irreversible and α-form HQ is more stable at ambient conditions.     

2′-O-Trifluoromethylated RNA – a powerful modification for RNA chemistry and NMR spectroscopy

New RNA modifications are needed to advance our toolbox for targeted manipulation of RNA. In particular, the development of high-performance reporter groups facilitating spectroscopic analysis of RNA structure and dynamics, and of RNA–ligand interactions has attracted considerable interest. To this end, fluorine labeling in conjunction with ¹⁹F-NMR spectroscopy has emerged as a powerful strategy. Appropriate probes for RNA previously focused on single fluorine atoms attached to the 5-position of pyrimidine nucleobases or at the ribose 2′-position. To increase NMR sensitivity, trifluoromethyl labeling approaches have been developed, with the ribose 2′-SCF₃ modification being the most prominent one. A major drawback of the 2′-SCF₃ group, however, is its strong impact on RNA base pairing stability. Interestingly, RNA containing the structurally related 2′-OCF₃ modification has not yet been reported. Therefore, we set out to overcome the synthetic challenges toward 2′-OCF₃ labeled RNA and to investigate the impact of this modification. We present the syntheses of 2′-OCF₃ adenosine and cytidine phosphoramidites and their incorporation into oligoribonucleotides by solid-phase synthesis. Importantly, it turns out that the 2′-OCF₃ group has only a slight destabilizing effect when located in double helical regions which is consistent with the preferential C3′-endo conformation of the 2′-OCF₃ ribose as reflected in the ³J (H1′–H2′) coupling constants. Furthermore, we demonstrate the exceptionally high sensitivity of the new label in ¹⁹F-NMR analysis of RNA structure equilibria and of RNA–small molecule interactions. The study is complemented by a crystal structure at 0.9 Å resolution of a 27 nt hairpin RNA containing a single 2′-OCF₃ group that well integrates into the minor groove. The new label carries high potential to outcompete currently applied fluorine labels for nucleic acid NMR spectroscopy because of its significantly advanced performance.

The new 2′-OCF₃ label for nucleic acid NMR spectroscopy carries high potential to outcompete currently applied fluorine labels because of significantly advanced performance.     

Synthesis and characterization of cyclopalladated complexes of benzylamine by IR and NMR spectroscopy studies

The chloro-bridged dimer [Pd(μ-Cl)(C₆H₄CH₂NH₂-κ²-C,N)]₂ reacts with PPh₂Et, P(p-tolyl)₃, AsPh₃, piper (piper =?C₅H₁₀N) and Py in dichloromethane at room temperature for 24 h in a one-to-two molar ratio and undergoing bridge-splitting reactions to give [PdCl(C₆H₄CH₂NH₂–κ²-C,N)L] (L =?PPh₂Et (1a), P(p-tolyl)₃ (1b), AsPh₃ (1c), piper (1d), C₆H₄CH₂NH₂ (3e) and Py (1f)). Complex 1f in THF at room temperature reacts with a stoichiometric amount of TlTfO (thallium triflate, TfO=CF₃SO₃) and Py (molar ratio 1 : 1 : 1) to afford [Pd(C₆H₄CH₂NH₂)(Py)₂]TfO (2). Infrared and NMR spectroscopies allow unambiguous characterization of these products.     

Interaction and morphology of polyethylenimine/DNA complexes as studied by solid-state NMR spectroscopy

Solid-state ¹H wide-line and ³¹P magic angle spinning NMR were applied to a series of PEI(polyethylenimine)/DNA complexes. The experimental results revealed that the higher the nitrogen/phosphorus (N/P) molar ratio is, the more phosphorus atoms of DNA are engaged in the electrostatic interaction with PEI. ¹H spin–diffusion experiments manifested that the aggregation degree of DNA in the complexes decreases greatly when N/P ratio increases from 0.5 to 3 and changes only slightly with further increase of N/P ratio, indicating that DNA disperses in the matrix of PEI on the molecular level at higher N/P ratio.     

Folding, conformational changes, and dynamics of cytochromes C probed by NMR spectroscopy

NMR spectroscopy has become a vital tool for studies of protein conformational changes and dynamics. Oxidized Fe(III)cytochromes c are a particularly attractive target for NMR analysis because their paramagnetism (S = (1)/(2)) leads to high (1)H chemical shift dispersion, even for unfolded or otherwise disordered states. In addition, analysis of shifts induced by the hyperfine interaction reveals details of the structure of the heme and its ligands for native and nonnative protein conformational states. The use of NMR spectroscopy to investigate the folding and dynamics of paramagnetic cytochromes c is reviewed here. Studies of nonnative conformations formed by denaturation and by anomalous in vivo maturation (heme attachment) are facilitated by the paramagnetic, low-spin nature of native and nonnative forms of cytochromes c. Investigation of the dynamics of folded cytochromes c also are aided by their paramagnetism. As an example of this analysis, the expression in Escherichia coli of cytochrome c(552) from Nitrosomonas europaea is reported here, along with analysis of its unusual heme hyperfine shifts. The results are suggestive of heme axial methionine fluxion in N. europaea ferricytochrome c(552). The application of NMR spectroscopy to investigate paramagnetic cytochrome c folding and dynamics has advanced our understanding of the structure and dynamics of both native and nonnative states of heme proteins.