Solution NMR Spectra Research Articles

Integral Membrane Proteins in Nanodiscs Can Be Studied by Solution NMR Spectroscopy

We present a two-dimensional solution NMR spectrum of an integral membrane protein (IMP) in a nanodisc. Solution NMR relies on rapid isotropic tumbling of the analyte with correlation times in the nanosecond range. IMPs in a cellular membrane do not satisfy this condition. Previous liquid-state NMR studies on IMPs were conducted in organic solvent or artificial membrane mimicking particles like detergent micelles. Nanodiscs are relatively small (150 kDa), detergent-free model membranes that are suitable for functional reconstitution of IMPs. Nanodiscs allow solubilization of integral membrane proteins in a nearly native lipid bilayer environment. The 70 residue polypeptide CD4mut was incorporated into nanodiscs. CD4mut features one transmembrane helix. The aliphatic (1)H-(13)C HSQC spectrum of nanodiscs with inserted, ((13)C, (15)N)-labeled CD4mut exhibits reasonably dispersed protein and lipid NMR signals. Our results demonstrate that IMPs in nanodiscs are amenable to liquid-state NMR methodology.

Solution NMR mapping of water-accessible residues in the transmembrane β-barrel of OmpX

The atomic structure of OmpX, the smallest member of the bacterial outer membrane protein family, has been previously established by X-ray crystallography and NMR spectroscopy. In apparent conflict with electrophysiological studies, the lumen of its transmembrane beta-barrel appears too tightly packed with amino acid side chains to let any solute flow through. In the present study, high-resolution solution NMR spectra were obtained of OmpX kept water-soluble by either amphipol A8-35 or the detergent dihexanoylphosphatidylcholine. Hydrogen/deuterium exchange measurements performed after prolonged equilibration show that, whatever the surfactant used, some of the amide protons of the membrane-spanning region exchange much more readily than others, which likely reflects the dynamics of the barrel.

Structural studies on aryl‐substituted enaminoketones and their thio analogues. Part I. Analysis of high‐resolution 1H, 13C NMR and 13C CP MAS spectra combined with GIAO‐DFT calculations

A series of aryl-substituted enaminoketones and their thio analogues in CDCl(3) solution and in the solid state were studied by the use of high-resolution (1)H and (13)C as well as (13)C cross polarization magic angle spinning (CP MAS) NMR spectra in combination with gauge including atomic orbitals-density functional theory (GIAO-DFT) calculations performed at the B3PW91/6-311 + + G(d,p) level of theory using the B3PW91/6-311 + + G(d,p)-optimized geometries. The analysis of the (13)C NMR spectra in solution was done by using the Incredible Natural Abundance DoublE QUAntum Transfer Experiment (INADEQUATE) technique, whereas trends observed in the (13)C shielding constants, calculated for the compounds studied, were a great help in assigning most of the signals in the (13)C CP MAS NMR spectra. It was established on the basis of the experimental and theoretical NMR data that both groups of compounds exist in the form of Z-s-Z-s-E isomers in CDCl(3) solution as well as in the solid state, with the NH hydrogen atom involved in intramolecular hydrogen bonding. This conclusion is in agreement with the fact that some of the compounds studied reveal liquid-crystalline properties. Three-bond H, H and C, H coupling constants measured in solution played a crucial role in the structure elucidation.

Solute order parameters in liquid crystals from NMR spectra solved with evolutionary algorithms: Application of double Maier–Saupe Kobayashi–McMillan theory

We obtain dipolar couplings via a novel application of evolutionary algorithms solving for multiple spin-systems simultaneously and automatically from the NMR spectra of several solutes in several nematic and smectic liquid crystal solvents. The order parameters obtained from the dipolar couplings are used to test a novel Hamiltonian that includes two Maier–Saupe nematic terms plus Kobayashi–McMillan smectic A terms. It is shown that this Hamiltonian can rationalize the NMR experiments with physically reasonable smectic order parameters and Hamiltonian prefactors.

( 1Z, 3Z)-3-[Quinolin-2(1 H)-ylidene]-1-(quinolin-2-yl)prop-1-en-2-ol: An unexpected most stable tautomer of 1,3-bis(quinolin-2-yl)acetone

1H, 13C and 15N NMR spectra reveal that CDCl 3 solution of 1,3-bis(quinolin-2-yl)acetone contains only ( 1Z, 3Z)-3-[quinolin-2(1 H)-ylidene]-1-(quinolin-2-yl)prop-1-en-2-ol. The proton transfer takes place between two basic centers of the molecule, which means that the process is an identity reaction by character. The situation is completely different from that detected in chloroform solution of 1,3-bis(pyridin-2-yl)acetone where three different tautomers are in equilibrium with each other. Although the proton transfers in both ( 1Z, 3Z)-3-[quinolin-2(1 H)-ylidene]-1-(quinolin-2-yl)prop-1-en-2-ol and ( 1Z, 3Z)-3-hydroxy-1-[quinolin-2(1 H)-ylidene-4-quinolin-2-yl]but-3-en-2-one have the identity reaction character and are fast on the NMR time-scale, this process takes place in two six-membered rings condensed to each other in the former and in two six-membered rings conjugated with each other in the latter compound. In consequence, NMR spectra of solutions of these compounds differ not only qualitatively but also quantitatively. The character of the double proton transfer in the molecule of ( 1Z, 3Z)-3-[quinolin-2(1 H)-ylidene]-1-(quinolin-2-yl)prop-1-en-2-ol is also supported by comparison of the experimental and calculated (GIAO) 13C and 15N chemical shifts.

Influence of the Solvent on the Formation of New Tin(II) Methoxides Containing Thienyl Substituents: Crystal Structure and NMR Investigations

AbstractThe synthesis, structural characterization, and NMR studies of new tin(II) alkoxides with thiophene‐based substituents are presented. The reaction of ligand HO–C(C4H3S)3 (1) with bis(hexamethyldisilazyl)tin {Sn[N(SiMe3)2]2} in the ratio 2:1 in toluene yields the dinuclear compound {Sn[OC(C4H3S)3]2}2·2 toluene (2). The same reaction in tetrahydrofuran allows the isolation of two compounds: a dinuclear {Sn[OC(C4H3S)3]2}2·2 thf (3) and a mononuclear Sn[OC(C4H3S)3]2(thf) (4) molecule. The molecular structures of these tin(II) methoxides containing thienyl substituents 2–4 were investigated by single‐crystal X‐ray crystallography. The NMR spectra of solutions of {Sn[OC(C4H3S)3]2}2·2 toluene (2) or {Sn[OC(C4H3S)3]2}2·2 thf (3) and Sn[OC(C4H3S)3]2(thf) (4) are almost identical, showing only variations of the molar ratio of the dimeric and monomeric compounds.

Study of the ethanolic tributylmethylammonium aluminosilicate solutions using 27Al NMR

Alkaline solutions containing both silicate and aluminate ions are of considerable research interest inter alia because of their involvement in the synthesis of zeolites. In the present work, we use 27Al NMR to characterize alkaline aqueous and ethanolic tributylmethylammonium aluminosilicate solutions with different Al/Si ratios. Tributylmethylammonium (TBMA) used as a cation template and no alkaline metals are used for preparation of aluminosilicate solutions. Aluminum-27 NMR spectra of TBMA aluminosilicate solutions indicate that considerable changes occurred by changing the Si/Al ratio. The distribution of aluminosilicate species was affected by the presence of the methanol and the method of mixing the silicate and aluminosilicate solutions. To understand the reaction between silicate and aluminate ions in this system, the evolutions of 27Al NMR spectra with time are investigated.

Downstream processing and characterization of pullulan from a novel colour variant strain of Aureobasidium pullulans FB-1

Exopolysaccharide produced by a new novel colour variant strain of Aureobasidium pullulans FB-1 was purified by cell harvesting and precipitation of the polymer. Various organic solvents were screened for pullulan precipitation. Isolation and purification of pullulan from fermentation broth was carried out using single-step purification strategy by isopropyl alcohol precipitation. Ratio of culture supernatant to isopropyl alcohol and time of precipitation were optimized for pullulan precipitation. Maximum yield (4.47%, w/v) of polysaccharide was obtained when two volumes of ice-cold isopropyl alcohol were added to one volume of supernatant with precipitation time of 12 h. IR spectra as well as carbon-13 and proton NMR spectra in aqueous solution of intact polysaccharide obtained from A. pullulans FB-1 and commercially available pullulan (Sigma, USA) revealed solely α-(1 → 6) linked maltosyl units, in accord with the generally accepted structure of pullulan. Maximum hydrolysis (94.25%) of purified pullulan at 50 °C by pullulanase was achieved under agitation (150 rpm) after 360 min.

Intrinsically Disordered Titin PEVK as a Molecular Velcro: Salt-Bridge Dynamics and Elasticity

It is increasingly recognized that many proteins are intrinsically disordered and do not have a unique compact structure as those found in globular proteins. Titin is a giant modular protein (3-4 MDa) found in the muscle sarcomere that is comprised of both globular and disordered modules. The elastic titin PEVK segment, with tandem repeats of ∼28 residue modules, plays a major role in the passive tension of skeletal and heart tissues. We have proposed based on AFM studies of a cloned titin PEVK fragment, that salt-bridges play a central role in the elasticity of this PEVK polyelectrolyte. We have engineered a construct of 15 repeats of a single titin 28-residue PEVK module (human exon 172). The 50 kDa polyprotein shows well-resolved NMR spectra in dilute solution and in highly concentrated gels. Both chemical shifts and sequential NOE's indicate the presence of polyproline II helices. From long-range NOE's, we observed, for the first time, stable K to E salt-bridges with non-random pairings. Simulated annealing with NMR restraints yielded a manifold of plausible structures for an exon 172 trimer showing many salt-bridges. Steered molecular dynamics simulations (SMD) were done to study how the manifold of salt-bridges evolves during the stretching experiment. Repeated SMD simulations at slow velocity (0.0005 nm/ps) show force spectra consistent with experimental AFM force spectra of the polyprotein. SMD shows that salt-bridges occur even at high degrees of stretch and that these short range interactions are in integral part of the mechanical properties of PEVK. We propose that the long-range, non-stereospecific nature of electrostatic interactions provide a facile mechanism to tether and untether the flexible chains, which in turn affect elasticity as well as control the accessibility of protein-protein interaction to these nanogel-like proteins.

Evolutionary algorithms to solve complicated NMR spectra

The complexity of (1)H NMR spectra of solutes in partially ordered solvents such as liquid crystals increases rapidly with the number of spins. Spectra of simple solutes with sufficient symmetry and containing not too many spins (typically <or=8) are readily analyzed. The analysis of larger spin systems is more difficult, and often impossible. In this paper we present the application of a general automated evolutionary algorithm to solve the highly complex proton NMR spectrum of the 12-spin system pentane, a solute that interconverts rapidly among several symmetry-unrelated conformations. The interpretation of the spectral parameters that are obtained from the analysis requires the use of a model to connect relative orientational orders in symmetry-unrelated conformers.

Synthesis and structures of halides and pseudohalides of bis[2,6-bis(dimethylamino)phenyl]tin(iv)

Oxidative addition reactions of Sn{C6H3(NMe2)(2)-2,6}2 [ identical with Sn(ArN2)2] () with XeF2, HgCl2 or TeCl4, dabco.2Br2, I2, or SiMe3N3 afforded [Sn(ArN2)2F2] (), [Sn(ArN2)2Cl2] (), [Sn(ArN2)2Br2] (), [Sn(ArN2)2I2] (), or [Sn(ArN2)2{N(SiMe3)2}N3] (), respectively. Compound was also obtained from and aqueous KF. Treatment of , under phase transfer conditions using [NBnEt3]Cl in H2O, with NaN3, KOCN, or KSCN yielded [Sn(ArN2)2(N3)2] (), [Sn(ArN2)2(NCO)2] (), or [Sn(ArN2)2(NCS)2] (), respectively. Alternative pathways to are suggested. Excellent (, and ) and satisfactory () yields were recorded; each compound was characterised by C, H, N microanalysis, mass spectra and multinuclear NMR solution spectra at ambient temperature, which invariably showed equivalent NMe2(1)H and 13C signals for the two NMe2 groups. The X-ray structures of crystalline [Sn(ArN2)Cl] (), , , , and showed that only one of the 2- and 6-Me2N substituents of an (ArN2) group was in a close SnN contact. Reaction of with Li[AlMe4] or LiMe gave [Sn(ArN2)(micro-Me)2AlMe2] () or c-(SnMe2)6, respectively.

Reversible Neutral Dissociation of the N−Si Dative Bond in Hexacoordinate Hydrido Complexes of Silicon

Hexacoordinate silicon dichelates with hydrido and methyl monodentate ligands were prepared by transsilylation. The temperature-dependent 29Si NMR spectra of toluene-d8 or CDCl3 solutions provide evidence for a reversible, neutral dissociation of the dative N−Si bond, accompanied by a shift of the 29Si resonance from high field (hexacoordinate) to lower field (pentacoordinate) with increasing temperature. When a phenyl group is attached directly to silicon (replacing methyl), the N−Si dissociation is prevented, presumably due to electron withdrawal by the phenyl. Two of the silicon complexes were characterized by crystallographic analysis. Quantum chemical calculations at the RB3LYP/6-31G(d) level confirm the stability of an N−Si dissociated species (1.03 kcal mol−1 lower in energy than the hexacoordinate dichelate) and rule out the alternative possibility of an O−Si zwitterionic dissociation, thus supporting the mechanism suggested by the experimental results.

Silicon-29 NMR Study of Alkaline Aqueous and Alcoholic Tri-butylmethyl Ammonium (TBMA) Silicate Solutions

ABSTRACT Silicon-29 NMR spectroscopy was used to characterize aqueous and alcoholic alkaline solutions of tri-butylmethyl ammonium (TBMA) silicates. The effect of TBMA cation on the equilibrium of silicate oligomers in aqueous alkaline silicate solutions was investigated using 29Si NMR spectra. It was found that TBMA cation has a structure directing role and directs the silicate species to form minor amounts of silicate anion in the presence of high concentration of silicon. Silicon-29 NMR spectra of TBMA silicate solutions indicate that considerable changes occurred by changing the Si/TBMA ratio. The distribution of silicate species was affected by the presence of the alcohols, specifically methanol.

Syntheses, spectroscopies and structures of zinc complexes with malate

Reactions of zinc(II) ion with racemic malic acid (C 4H 6O 5 = H 3mal) result in the isolation of four new zinc(II) malato complexes: (NH 4)[Zn( R-H 2mal) 3] · H 2O ( 1), trans-[Zn( R-H 2mal)( S-H 2mal)(H 2O) 2] · 2H 2O ( 2), (NH 4) 2[Zn( R-Hmal)( S-Hmal)] · 2H 2O ( 3), and [Zn 2( R-Hmal)( S-Hmal)(H 2O) 4] n · 2 nH 2O ( 4). Three R-malic acids in 1 act as bidentate ligands via their alcoholic and the central carboxy groups with Zn(II) ion, leaving the terminal carboxylic acid groups free. The R- and S-malates of 2 coordinate in a bidentate manner with zinc ion in trans-form. In 3, Zn(II) ion is coordinated by R- and S-malates in a tridentate fashion via their alcoholic and two carboxy groups. Complex 4 forms a two-dimensional layered structure through the links of a new dimeric unit [Zn 2( R-Hmal)( S-Hmal)(H 2O) 4] with one of the oxygen atoms from the terminal carboxy group of malate ligand. The coordination of malates depends on pH variation, on Zn:malate ratio, and also on temperature. Tridentate chelation of malate in 3 is found between pH 4.5–9.0. The soluble monomeric species 1– 3 have been investigated using 13C NMR spectra by long-time acquisition. The solution NMR spectra indicate that zinc malate complexes dissociate in H 2O (D 2O). Obvious downfield shifts of the central carboxy carbon atoms in 1– 3 are observed compared with those of free malate, which indicate that these zinc malate complexes dissociate in aqueous solution.

Acid−Base Interactions and Secondary Structures of Poly-l-Lysine Probed by 15N and 13C Solid State NMR and Ab initio Model Calculations

The interactions of the 15N-labeled amino groups of dry solid poly-L-lysine (PLL) with various halogen and oxygen acids HX and the relation to the secondary structure have been studied using solid-state 15N and 13C CPMAS NMR spectroscopy (CP = cross polarization and MAS = magic angle spinning). For comparison, 15N NMR spectra of an aqueous solution of PLL were measured as a function of pH. In order to understand the effects of protonation and hydration on the 15N chemical shifts of the amino groups, DFT and chemical shielding calculations were performed on isolated methylamine-acid complexes and on periodic halide clusters of the type (CH3NH3(+)X(-))n. The combined experimental and computational results reveal low-field shifts of the amino nitrogens upon interaction with the oxygen acids HX = HF, H2SO4, CH3COOH, (CH3)2POOH, H3PO4, HNO3, and internal carbamic acid formed by reaction of the amino groups with gaseous CO2. Evidence is obtained that only hydrogen-bonded species of the type (Lys-NH2***H-X)n are formed in the absence of water. 15N chemical shifts are maximum when H is located in the hydrogen bond center and then decrease again upon full protonation, as found for aqueous solution at low pH. By contrast, halogen acids interact in a different way. They form internal salts of the type (Lys-NH3(+)X(-))n via the interaction of many acid-base pairs. This salt formation is possible only in the beta-sheet conformation. By contrast, the formation of hydrogen-bonded complexes can occur both in beta-sheet domains as well as in alpha-helical domains. The 15N chemical shifts of the protonated ammonium groups increase when the size of the interacting halogen anions is increased from chloride to iodide and when the number of the interacting anions is increased. Thus, the observed high-field 15N shift of ammonium groups upon hydration is the consequence of replacing interacting halogen atoms by oxygen atoms.

The structure of Rimonabant in the solid state and in solution: An experimental and theoretical study

The X-ray molecular structure of Rimonabant methanol solvate has been determined together with the 1H, 13C and 15N NMR spectra in acetone solution. B3YP/6-311++G(d,p) calculations have been performed out to determine two minimum energy conformations, on these geometries GIAO calculations were carried out to obtain the corresponding absolute shieldings that were compared with the experimental chemical shifts.

A new unprecedented method for the synthesis of 1-fluorosilatrane

We developed the simplest and the most convenient original procedure for the synthesis of fluorosilatrane. The method is shown in the following scheme: parent very viscous liquid (in contrast to other crystalline triethanolammonium halides). To this product was added an equimolar amount of tetraethoxysilane, and stirring while heating was continued with distilling off the evolved ethanol. White powder of the formed fluorosilatrane was heated in a vacuum to remove residual (EtO)4Si and EtOH (yield of the latter was 90%). The FSa formed has sublimation temperature ~300°С at the atmospheric pressure in agreement with the published data [1, 4]. Noteworthy that at the temperature above 220°С the FSa powder is transformed into threadlike crystals. IR spectrum of FSa formed (KBr pellet) contains strong adsorption bands corresponding to vibrations of Si–F (748– 785 cm ), Si–O (790–810 cm), and С–O (1075– 1150 cm) bonds. H and С NMR spectra of solution of FSa in DMSO-d6 are characterized by the following parameters: δH 2.92 and 3.69 ppm, δС 50.5 and 56.8 ppm. The IR and NMR spectra of the fluorosilatrane obtained are consistent with the published data [3, 7– 10].

A solid state and solution NMR study of the tautomerism in hydroxyquinoline carboxylic acids

Some hydroxyquinoline carboxylic acids and their conjugate acids and bases were characterized by 13C and 15N NMR spectroscopy in solution and in the solid state. Differences in 13C and, in particular, 15N chemical shift patterns allow to distinguish between individual tautomers and confirm the presence of zwitterionic species in the solid state. Solution NMR spectra in dimethyl sulfoxide (DMSO) show effects resulting as a consequence of dynamic exchange and suggest the presence of an equilibrium mixture of hydroxyquinoline carboxylic acid and zwitterionic hydroxyquinolinium carboxylate tautomers.

Solvation of Lithium Ion in N,N-Diethyl-N-methyl-N-(2-methoxyethyl)ammonium Bis(trifluoromethanesulfonyl)-amide Using Raman and Multinuclear NMR Spectroscopy

The solvation structure of the Li(I) species in N,N-diethyl-N-methyl-N-(2-methoxyethyl)ammonium bis(trifluoromethane-sulfonyl) amide (DEMETFSA) was studied by measuring the Raman and multinuclear NMR spectra of DEMETFSA solutions containing LiTFSA of various concentrations (0.12-1.92 mol kg(-1), [TFSA(-)]/[Li(I)] = 20.0-2.22). It was found from Raman spectra that an intense band due to the free TFSA(-) anion at around 741 cm(-1) becomes weak, and a new band appears at around 747 cm(-1) with an increase in the concentrations of LiTFSA, and that the pseudoisosbestic point is observed at around 744 cm(-1) in the range of [TFSA(-)]/[Li(I)] = 20.0-5.00. From analyses of these Raman bands, the number of TFSA(-) anions bound to the Li(+) ion was evaluated to be 1.85 +/- 0.08, and hence, the Li(I) in DEMETFSA solutions was proposed to exist as [Li(TFSA)(2)](-) in the range of [TFSA(-)]/[Li(I)] = 20.0-5.00. Furthermore, in the range of [TFSA(-)]/[Li(I)] = 2.86-2.22, the band observed at around 747 cm(-1) became more strong, and the pseudoisosbestic point disappeared. From these phenomena, it seems that the Li(I) oligomer species are formed in the higher concentration region of LiTFSA. The (19)F NMR signal of the TFSA(-) anion observed at 42.31 ppm in neat DEMETFSA was found to shift to a higher field linearly with an increase in the concentrations of LiTFSA ([LiTFSA] = 0.00-0.99 mol kg(-1), [TFSA(-)]/[Li(+)] = 20.0-3.33), while in a higher concentration range ([LiTFSA] > or = 1.26 mol kg(-1), [TFSA(-)]/[Li(+)] < or = 2.86), a slight deviation from linearity was observed. On the other hand, the (7)Li NMR signal did not show an appreciable shift with increasing LiTFSA concentrations. These results support that the Li(I) species in DEMETFSA solutions exist as [Li(TFSA)(2)](-) and the Li(I) oligomer species in the low and high concentration regions of LiTFSA, respectively.

Synthesis and characterisation of six Fe(II or III), Co(II) or Zr(IV) complexes containing the ligand [CH(SiMe 2R)P(Ph) 2NSiMe 3] − (R = Me, NEt 2) and of [Co{N(SiMe 3)C(Ph)C(H)P(Ph) 2NSiMe 3} 2

The C, N-(trimethylsilyliminodiphenylphosphoranyl)silylmethylmetal complexes [Fe( L) 2] ( 3), [Co( L) 2] ( 4), [ZrCl 3( L)]·0.83CH 2Cl 2 ( 5), [Fe( L) 3] ( 6), [Fe( L′) 2] ( 7) and [Co( L′) 2] ( 8) have been prepared from the lithium compound Li[CH(SiMe 2R)P(Ph) 2NSiMe 3] [ 1a, (R = Me) {≡ Li( L)}; 1b, (R = NEt 2) {≡ Li( L′)}] and the appropriate metal chloride (or for 7, FeCl 3). From Li[N(SiMe 3)C(Ph)C(H)P(Ph) 2NSiMe 3] [≡ Li( L″)] ( 2), prepared in situ from Li( L) ( 1a) and PhCN, and CoCl 2 there was obtained bis(3-trimethylsilylimino- diphenylphosphoranyl-2-phenyl- N-trimethylsilyl-1-azaallyl- N, N ′)cobalt(II) ( 9). These crystalline complexes 3– 9 were characterised by their mass spectra, microanalyses, high spin magnetic moments (not 5) and for 5 multinuclear NMR solution spectra. The X-ray structure of 3 showed it to be a pseudotetrahedral bis(chelate), the iron atom at the spiro junction.