13C Dipolar Couplings Research Articles

Efficient 15N-13C Polarization Transfer by Third-Spin-Assisted Pulsed Cross-Polarization Magic-Angle-Spinning NMR for Protein Structure Determination.

We introduce a pulsed third-spin-assisted recoupling experiment that produces high-intensity long-range 15N-13C cross peaks using low radiofrequency (rf) energy. This Proton-Enhanced Rotor-echo Short-Pulse IRradiATION Cross-Polarization (PERSPIRATIONCP) pulse sequence operates with the same principle as the Proton-Assisted Insensitive-Nuclei Cross-Polarization (PAINCP) experiment but uses only a fraction of the rf energy by replacing continuous-wave 13C and 15N irradiation with rotor-echo 90° pulses. Using formyl-Met-Leu-Phe (f-MLF) and β1 immunoglobulin binding domain of protein G (GB1) as model proteins, we demonstrate experimentally how PERSPIRATIONCP polarization transfer depends on the CP contact time, rf power, pulse flip angle, and 13C carrier frequency and compare the PERSPIRATIONCP performance with the performances of PAINCP, RESPIRATIONCP, and SPECIFICCP for measuring 15N-13C cross peaks. PERSPIRATIONCP achieves long-range 15N-13C transfer and yields higher cross peak-intensities than that of the other techniques. Numerical simulations reproduce the experimental trends and moreover indicate that PERSPIRATIONCP relies on 15N-1H and 13C-1H dipolar couplings rather than 15N-13C dipolar coupling for polarization transfer. Therefore, PERSPIRATIONCP is an rf-efficient and higher-sensitivity alternative to PAINCP for measuring long-range 15N-13C correlations, which are essential for protein resonance assignment and structure determination. Using cross peaks from two PERSPIRATIONCP 15N-13C correlation spectra as the sole distance restraints, supplemented with (φ, ψ) torsion angles obtained from chemical shifts, we calculated the GB1 structure and obtained a backbone root-mean-square deviation of 2.0 Å from the high-resolution structure of the protein. Therefore, this rf-efficient PERSPIRATIONCP method is useful for obtaining many long-range distance restraints for protein structure determination.

Synthesis, Structure, and Local Molecular Dynamics for Crystalline Rotors Based on Hecogenin/Botogenin Steroidal Frameworks

The synthesis and solid-state characterization of a series of cyclic/acyclic molecular rotors derived from naturally occurring steroidal 12-oxosapogenins are described. The bridged molecular rotors with rigid steroidal frameworks were obtained by employing ring-closing metathesis (RCM) as a key step. The X-ray diffraction technique was employed for determination and refinement of the crystal and molecular structure of selected models giving good quality single crystals. In the case of the bridged hecogenin molecular rotor 11E for which poor quality crystals were obtained, an NMR crystallography approach was used for fine refinement of the structure. Solid state NMR spectroscopic techniques were applied for the study of local molecular dynamics of the featured acyclic/cyclic molecular rotors. Analysis of 13C principal components of chemical shift tensors and chemical shift anisotropy (CSA) as well as heteronuclear 1H–13C dipolar couplings (DC) unambiguously proved that aromatic rings located in the space within the rigid steroidal framework both for cyclic and acyclic rotors are under kHz exchange regime. Experimental results were confirmed by theoretical calculations of rotation barrier on the density functional theory level. Small distinctions in the values of CSA and DC for the rotors under investigation are explained on the basis of differences in their molecular structures.

A cross-polarization based rotating-frame separated-local-field NMR experiment under ultrafast MAS conditions

Rotating-frame separated-local-field solid-state NMR experiments measure highly resolved heteronuclear dipolar couplings which, in turn, provide valuable interatomic distances for structural and dynamic studies of molecules in the solid-state. Though many different rotating-frame SLF sequences have been put forth, recent advances in ultrafast MAS technology have considerably simplified pulse sequence requirements due to the suppression of proton–proton dipolar interactions. In this study we revisit a simple two-dimensional 1H–13C dipolar coupling/chemical shift correlation experiment using 13C detected cross-polarization with a variable contact time (CPVC) and systematically study the conditions for its optimal performance at 60kHz MAS. In addition, we demonstrate the feasibility of a proton-detected version of the CPVC experiment. The theoretical analysis of the CPVC pulse sequence under different Hartmann–Hahn matching conditions confirms that it performs optimally under the ZQ (w1H−w1C=±wr) condition for polarization transfer. The limits of the cross polarization process are explored and precisely defined as a function of offset and Hartmann–Hahn mismatch via spin dynamics simulation and experiments on a powder sample of uniformly 13C-labeled L-isoleucine. Our results show that the performance of the CPVC sequence and subsequent determination of 1H–13C dipolar couplings are insensitive to 1H/13C frequency offset frequency when high RF fields are used on both RF channels. Conversely, the CPVC sequence is quite sensitive to the Hartmann–Hahn mismatch, particularly for systems with weak heteronuclear dipolar couplings. We demonstrate the use of the CPVC based SLF experiment as a tool to identify different carbon groups, and hope to motivate the exploration of more sophisticated 1H detected avenues for ultrafast MAS.

Molecular Structure of RADA16-I Designer Self-Assembling Peptide Nanofibers

The designer self-assembling peptide RADA16-I forms nanofiber matrices which have shown great promise for regenerative medicine and three-dimensional cell culture. The RADA16-I amino acid sequence has a β-strand-promoting alternating hydrophobic/charged motif, but arrangement of β-strands into the nanofiber structure has not been previously determined. Here we present a structural model of RADA16-I nanofibers, based on solid-state NMR measurements on samples with different schemes for (13)C isotopic labeling. NMR peak positions and line widths indicate an ordered structure composed of β-strands. The NMR data show that the nanofibers are composed of two stacked β-sheets stabilized by a hydrophobic core formed by alanine side chains, consistent with previous proposals. However, the previously proposed antiparallel β-sheet structure is ruled out by measured (13)C-(13)C dipolar couplings. Instead, neighboring β-strands within β-sheets are parallel, with a registry shift that allows cross-strand staggering of oppositely charged arginine and aspartate side chains. The resulting structural model is compared to nanofiber dimensions observed via images taken by transmission electron microscopy and atomic force microscopy. Multiple NMR peaks for each alanine side chain were observed and could be attributed to multiple configurations of side chain packing within a single scheme for intermolecular packing.

The Alzheimer's Amyloid-β(1–42) Peptide Forms Off-Pathway Oligomers and Fibrils That Are Distinguished Structurally by Intermolecular Organization

Increasing evidence suggests that soluble aggregates of amyloid-β (Aβ) initiate the neurotoxicity that eventually leads to dementia in Alzheimer's disease. Knowledge on soluble aggregate structures will enhance our understanding of the relationship between structures and toxicities. Our group has reported a stable and homogeneous preparation of Aβ(1–42) oligomers that has been characterized by various biophysical techniques. Here, we have further analyzed this species by solid state nuclear magnetic resonance (NMR) spectroscopy and compared NMR results to similar observations on amyloid fibrils. NMR experiments on Aβ(1–42) oligomers reveal chemical shifts of labeled residues that are indicative of β-strand secondary structure. Results from two-dimensional dipolar-assisted rotational resonance experiments indicate proximities between I31 aliphatic and F19 aromatic carbons. An isotope dilution experiment further indicates that these contacts between F19 and I31 are intermolecular, contrary to models of Aβ oligomers proposed previously by others. For Aβ(1–42) fibrils, we observed similar NMR lineshapes and inter-side-chain contacts, indicating similar secondary and quaternary structures. The most prominent structural differences between Aβ(1–42) oligomers and fibrils were observed through measurements of intermolecular 13C–13C dipolar couplings observed in PITHIRDS-CT experiments. PITHIRDS-CT data indicate that, unlike fibrils, oligomers are not characterized by in-register parallel β-sheets. Structural similarities and differences between Aβ(1–42) oligomers and fibrils suggest that folded β-strand peptide conformations form early in the course of self-assembly and that oligomers and fibrils differ primarily in schemes of intermolecular organization. Distinct intermolecular arrangements between Aβ(1–42) oligomers and fibrils may explain why this oligomeric state appears off-pathway for monomer self-assembly to fibrils.

Binding of the three-repeat domain of tau to phospholipid membranes induces an aggregated-like state of the protein

In patients with Alzheimer's disease, the microtubule-associated protein tau is found aggregated into paired helical filaments (PHFs) in neurofibrillary deposits. In solution, tau is intrinsically unstructured. However, the tubulin binding domain consisting of three or four 31–32 amino acid repeat regions exhibits both helical and β-structure propensity and makes up the proteolysis resistant core of PHFs. Here, we studied the structure and dynamics of the three-repeat domain of tau (i.e. K19) when bound to membranes consisting of a phosphatidylcholine and phosphatidylserine mixture or phosphatidylserine alone. Tau K19 binds to phospholipid vesicles with submicromolar affinity as measured by fluorescence spectroscopy. The interaction is driven by electrostatic forces between the positively charged protein and the phospholipid head groups. The structure of the membrane-bound state of K19 was studied using CD spectroscopy and solid-state magic-angle spinning NMR spectroscopy. To this end, the protein was selectively 13C-labeled at all valine and leucine residues. Isotropic chemical shift values of tau K19 were consistent with a β-structure. In addition, motionally averaged 1H–13C dipolar couplings indicated a high rigidity of the protein backbone. The structure formation of K19 was also shown to depend on the charge density of the membrane. Phosphatidylserine membranes induced a gain in the α-helix structure along with an immersion of K19 into the phospholipid bilayer as indicated by a reduction of the lipid chain 2H NMR order parameter. Our results provide structural insights into the membrane-bound state of tau K19 and support a potential role of phospholipid membranes in mediating the physiological and pathological functions of tau.

Amyloid-Derived Peptide Forms Self-Assembled Monolayers on Gold Nanoparticle with a Curvature-Dependent β-Sheet Structure

Using a combination of Fourier transform infrared (FTIR) spectroscopy and solid-state nuclear magnetic resonance (SSNMR) techniques, the secondary structure of peptides anchored on gold nanoparticles of different sizes is investigated. The structure of the well-studied CALNN-capped nanoparticles is compared to the structure of nanoparticles capped with a new cysteine-terminated peptide, CFGAILSS. The design of that peptide is derived from the minimal amyloidogenic sequence FGAIL of the human islet polypeptide amylin. We demonstrate that CFGAILSS forms extended fibrils in solution. When constrained at a nanoparticle surface, CFGAILSS adopts a secondary structure markedly different from CALNN. Taking into account the surface selection rules, the FTIR spectra of CFGAILSS-capped gold nanoparticles indicate the formation of β-sheets which are more prominent for 25 nm diameter nanoparticles than for 5 nm nanoparticles. No intermolecular (13)C-(13)C dipolar coupling is detected with rotational resonance SSNMR for CALNN-capped nanoparticles, while CALNN is in a random coil configuration. Coupling is detected for CFGAILSS-capped gold nanoparticles, however, consistent with an intermolecular (13)C-(13)C distance of 5.0 ± 0.3 Å, in agreement with intermolecular hydrogen bonding in a parallel β-sheet structure.

1H assisted 13C/ 15N heteronuclear correlation spectroscopy in oriented sample solid-state NMR of single crystal and magnetically aligned samples

1H-irradiation under mismatched Hartmann–Hahn conditions provides an alternative mechanism for carrying out 15N/ 13C transfers in triple-resonance heteronuclear correlation spectroscopy (HETCOR) on stationary samples of single crystals and aligned samples of biopolymers, which improve the efficiency especially when the direct 15N– 13C dipolar couplings are small. In many cases, the sensitivity is improved by taking advantage of the 13C α labeled sites in peptides and proteins with 13C detection. The similarities between experimental and simulated spectra demonstrate the validity of the recoupling mechanism and identify the potential for applying these experiments to virus particles or membrane proteins in phospholipid bilayers; however, further development is needed in order to derive quantitative distance and angular constraints from these measurements.

Kinetics of 1H→ 13C NMR cross-polarization in polymorphs and solvates of the antipsychotic drug olanzapine

The 1H→ 13C NMR cross-polarization (CP) was studied under magic-angle spinning at 7.5 kHz in various crystal forms of the antipsychotic drug olanzapine: two polymorphs (metastable I and stable II) and eight solvates containing organic solvent and water molecules. The CP kinetics followed the non-classical I–I ⁎-S model, in which CP begins in a spin cluster of proximate abundant spins I * and rare spins S, then is controlled by spin diffusion of the abundant spins I from bulk to the I * spins of the spin cluster and finally is governed by spin-lattice relaxation of the abundant spins in the rotating frame. The corresponding CP kinetics parameters were determined and analyzed. It was demonstrated that the, λ and T df values (the CP time constant, the cluster composition parameter and the 1H spin-diffusion constant, respectively) were very useful to discriminate the functional groups, especially in the 3D parameter space. In order to conveniently analyze the large amount (175) of the collected CP parameters, the number of the observed variables was reduced using the principal component (PC) analysis. The 2D plot of PC2 vs. PC1 showed adequate separation of the CH 3, CH 2, CH and C cases (C stands for carbons without adjacent hydrogens). It was demonstrated that those cases were located along the PC1 axis in the order of increasing 1H– 13C dipolar couplings: C<CH 3<CH<CH 2. Our study showed the I–I *-S model at work and established ranges of its parameters for various functional groups.

Hindered Rotation of Water near C60

Cross-polarization (CP) nuclear magnetic resonance experiments show that water within about 1 nm of C60 molecules is anisotropic and undergoes rotational motion that is greatly hindered compared with motion in bulk. The experimental observation of a Hartmann−Hahn CP contact shows that the motion of water is slow enough for intermolecular 1H (water)−13C (C60) dipolar coupling to occur, suggesting that the time scale of the rotational (tumbling) motion of the water adjacent to the C60 is slower than the microsecond range. This is much slower than the picosecond range previously observed for bulk water. The dipolar coupling decreases with temperature in the range of 3−22 °C. This work provides molecular-scale evidence that a small hydrophobic particle can order surrounding liquid water molecules.

Synthesis of Diazocine-Containing Poly(arylene ether sulfone)s for Tailored Mechanical and Electrochemical Performance

A series of poly(arylene ether sulfone) copolymers containing the diazocine unit (0, 20, 50, 80, 100 mol %) were synthesized via step-growth polymerization. Copolymer compositions exhibited number-average molecular weights exceeding 30 000 g/mol, and mechanically ductile films were obtained for all compositions. The random copolymers exhibited enhanced thermal stability (Td10% = 500−550 °C) and mechanical properties (tensile stress = 45−65 MPa), and the thermomechanical performance was predictable based on copolymer composition. A secondary transition in the dynamic mechanical analysis appeared at 28 °C, which was attributed to segmental motion of the bulky diazocine and biphenyl units. X-ray single crystallography and 1H NMR spectroscopy as a function of temperature revealed that the diazocine ring existed in the boat-shaped conformation. The effective dipolar couplings of site-specific 1H−13C dipolar couplings in the aromatic rings were measured using DIPSHIFT solid state NMR spectroscopy at various temperatures. The experimental data indicated that the bulkier diazocine group experienced slower ring-flip motion than the smaller phenylene group. Cyclic voltammograms of diazocine-containing poly(arylene ether sulfone)s exhibited two reversible reduction peaks at −0.68 and −1.21 V under inert and basic conditions. UV spectra of the diazocine group in the reduced state indicated an extended π-electron structure, which supported the boat- to planar-conformational change (molecular actuation) via redox reactions.

High-Resolution Characterization of Liquid-Crystalline [60]Fullerenes Using Solid-State Nuclear Magnetic Resonance Spectroscopy

Liquid-crystalline materials containing fullerenes are valuable in the development of supramolecular switches and in solar cell technology. In this study, we characterize the liquid-crystalline and dynamic properties of fullerene-containing thermotropic compounds using solid-state natural abundance (13)C NMR experiments under stationary and magic angle spinning sample conditions. Chemical shifts spectra were measured in isotropic, liquid-crystalline nematic and smectic A and crystalline phases using one-dimensional (13)C experiments, while two-dimensional separated local-field experiments were used to measure the (1)H- (13)C dipolar couplings in mesophases. Chemical shift and dipolar coupling parameters were used to characterize the structure and dynamics of the liquid-crystalline dyads. NMR data of fullerene-containing thermotropic liquid crystals are compared to that of basic mesogenic unit and mesomorphic promoter compounds. Our NMR results suggest that the fullerene-ferrocene dyads form highly dynamic liquid-crystalline phases in which molecules rotate fast around the symmetry axis on the characteristic NMR time scale of approximately 10 (-4) s.

The dynamics and orientation of a lipophilic drug within model membranes determined by 13C solid-state NMR

Methods for determining how a drug interacts with cellular membranes at the molecular level can give valuable insight into the mode of action of the drug and its absorption, distribution and metabolism profile. A procedure is described here to determine the orientation and location of the lipophilic drug trifluoperazine (TFP) intercalated into dimyristoylphosphatidylcholine (DMPC) bilayers, by using a novel combination of high-resolution solid-state nuclear magnetic resonance (SSNMR) methods to observe signals from (13)C within the drug at natural abundance. SSNMR measurements of (1)H-(13)C dipolar couplings for TFP and selective broadening of (13)C NMR peaks by paramagnetic Mn(2+) together suggest a model for the location, orientation and dynamics of the drug within lipid bilayers that offers an explanation for the lysoprotective effect of the drug at low concentrations. The experiments described are straightforward to implement and can be used for the routine analysis of drug-membrane interactions to provide useful information for drug design and structure refinement.

Local and translational dynamics in DNA–lipid assemblies monitored by solid-state and diffusion NMR

The influence of electrostatic interactions on the dynamic properties of complexes containing DNA and mixtures of cationic- (DDA) and zwitterionic (DLPC) lipids are studied by means of NMR. The systems are arranged in lamellar membrane stacks intercalated by DNA molecules. This is confirmed by 31P-NMR, where a superposition of an axially symmetric powder pattern arising from the phospholipid membrane and an asymmetric tensor due to DNA can be fitted to the experimentally observed lineshape. The local mobility and order is assessed using two solid-state NMR techniques applicable to samples with natural isotopic abundance: WIdeline SEparation (WISE) and Separated Local Field (SLF) spectroscopy. Both experiments yield highly resolved 13C spectra in the direct dimension. The indirect dimension contains information about molecular dynamics through the 1H dipolar linewidth (WISE) or the 1H– 13C dipolar coupling constant (SLF). The experiments suggest that DNA is static while it induces an increased disorder in the hydrocarbon chains as compared to the parent lipid case. DDA chain order is more affected than DLPC due to the attractive electrostatic interaction between DNA and the cationic lipid. Translational dynamics of the lipids and the water was measured with the Pulsed Field Gradient STimulated Echo (PFG STE) technique. The influence of lamellar domain size and the angular dependence of the diffusion coefficients and nuclear relaxation times on the results of the PFG STE experiments are discussed. The local water diffusion coefficient is reduced by a factor four from the value of bulk water, and increases as the DLPC content is increased. We observe two lipid components with an order of magnitude difference in diffusion coefficients in the DNA:DDA:DLPC precipitate and these are assigned to DLPC (fast) and DDA (slow). Cationic lipid (DDA) diffusion is decreasing a factor of 2 when DLPC is added to the pure DNA:DDA system, indicating DNA-induced lipid segregation within the bilayer and the transition from locally 2D to 1D diffusion of the DDA. The results show that DNA–lipid electrostatic interactions reduce the long-range lipid mobility but locally enhance the hydrocarbon chain dynamics by perturbing the preferred lipid packing.

NMR studies of membranes composed of glycolipids and phospholipids

Lipid membranes composed of monogalactosyldiacylglycerol (MGDG) and dimyristoylphosphatidylcholine (DMPC) were studied by means of NMR spectroscopy. The macroscopic phase behaviour was investigated by 31P NMR under stationary conditions, whereas microscopic properties such as segmental ordering were probed by two-dimensional 1H– 13C separated local field experiments under magic-angle spinning conditions. Our results clearly show that ordering/disordering effects occur for the headgroups as well as for the acyl chains when the sample composition is varied. In particular, the 1H– 13C dipolar couplings within the galactose headgroup of MGDG exhibited significant concentration dependence.

Resolution enhancement in spectra of natural products dissolved in weakly orienting media with the help of 1H homonuclear dipolar decoupling during acquisition: Application to 1H– 13C dipolar couplings measurements

In weakly orienting media such as poly-γ-benzyl- l-glutamate (PBLG) a polymer that forms a chiral liquid crystal in organic solvents, the spectral resolution for embedded molecules is usually poor because of numerous 1H, 1H dipolar couplings that generally broaden proton spectra. Therefore 1H, 13C dipolar couplings are difficult or impossible to measure. Here, we incorporate Flip-Flop decoupling during detection into an HSQC experiment. Flip-Flop removes the 1H, 1H dipolar couplings and scales the chemical shifts of the protons as well as the 1H, 13C dipolar couplings during detection. A resolution gain by a factor 1.5–4.2 and improved signal intensity by an average factor of 1.6–1.7 have been obtained. This technique is demonstrated on (+)-menthol dissolved in a PBLG/CDCl 3 phase.

Probing membrane topology by high-resolution 1H– 13C heteronuclear dipolar solid-state NMR spectroscopy

Membrane topology changes introduced by the association of biologically pertinent molecules with membranes were analyzed utilizing the 1H– 13C heteronuclear dipolar solid-state NMR spectroscopy technique (SAMMY) on magnetically aligned phospholipid bilayers (bicelles). The phospholipids 1H– 13C dipolar coupling profiles lipid motions at the headgroup, glycerol backbone, and the acyl chain region. The transmembrane segment of phospholamban, the antimicrobial peptide (KIGAKI) 3 and cholesterol were incorporated into the bicelles, respectively. The lipids 1H– 13C dipolar coupling profiles exhibit different shifts in the dipolar coupling contour positions upon the addition of these molecules, demonstrating a variety of interaction mechanisms exist between the biological molecules and the membranes. The membrane topology changes revealed by the SAMMY pulse sequence provide a complete screening method for analyzing how these biologically active molecules interact with the membrane.

NMR studies of chloroform@cryptophane-A and chloroform@ bis-cryptophane inclusion complexes oriented in thermotropic liquid crystals

Oriented inclusion complexes of chloroform@cryptophane-A and chloroform@ bis-cryptophane were prepared using a nematic thermotropic liquid crystal (ZLI 1132), and the alignment and magnetic resonance properties of these host–guest systems were studied via 13C NMR of the labeled guests. Large 1H– 13C dipolar splittings for 13CHCl 3 guests indicated significantly enhanced (∼2-fold) ordering for the trapped vs. free ligands under all conditions studied, with similar ordering observed for the two complexes—despite significant differences in size and motional freedom between the hosts. For each environment, variable-temperature studies permitted the sign and magnitude of the order parameter for chloroform's C–H bond to be independently determined from the 13C chemical shift anisotropy (CSA) shifts (via the gradient method) and the restored 1H– 13C dipolar couplings. In both systems, the results are consistent with overall alignment of the complexes such that the cage principal axis lies roughly perpendicular to the LC director.

Lipid−Ethanol Interaction Studied by NMR on Bicelles

The interaction of ethanol with phospholipids was studied in bicelles at a physiologically relevant ethanol concentration of 20 mM and a lipid content of 14 wt % by high-resolution NMR. Transient association of ethanol with magnetically aligned bicelles imparts a small degree of anisotropy to the solute. This anisotropy allows detection of residual (1)H-(1)H and (1)H-(13)C dipolar couplings, which are superimposed on scalar couplings. Residual (2)H NMR quadrupole splittings of isotope-labeled ethanol were measured as well. The analysis of residual tensorial interactions yielded information on the orientation and motions of ethanol in the membrane-bound state. The fraction of phosphatidylcholine-bound ethanol was determined independently by gas chromatography and NMR. About 4% of ethanol is bound to phosphatidylcholine at a bicelle concentration of 14 wt % at 40 degrees C. Free and bound ethanol are in rapid exchange. The lifetime of ethanol association with phosphatidylcholine membranes is of the order of a few nanoseconds.

PITANSEMA, a low-power PISEMA solid-state NMR experiment

A low radio frequency power polarization inversion spin exchange at the magic angle (PISEMA) pulse sequence is described for the measurement of heteronuclear dipolar couplings from solids. The method employs a time averaged nutation concept to significantly reduce the rf power required to spin-lock low γ nuclear spins in PISEMA experiments. The efficacy of the 2D method is demonstrated on a single crystal of n-acetyl- l- 15N-valyl- l- 15N-leucine dipeptide to measure 1H– 15N dipolar couplings and a liquid crystal sample to measure 1H– 13C dipolar couplings.