Investigation of the dynamics of an elastin-mimetic polypeptide using solid-state NMR

Elastin is the main structural protein that provides elasticity to various tissues and organs in vertebrates. Molecular motions are believed to play a significant role in its elasticity. We have used solid-state NMR spectroscopy to characterize the dynamics of an elastin-mimetic protein as a function of hydration to better understand the origin of elastin elasticity. Poly(Lys-25), [(VPGVG)(4)(VPGKG)](39), has a repeat sequence common to natural elastin. (13)C cross-polarization and direct polarization spectra at various hydration levels indicate that water enhances the protein motion in a non-uniform manner. Below 20% hydration, the backbone motion increases only slightly whereas above 30% hydration, both the backbone and the side-chains undergo large-amplitude motions. The motional amplitudes are extracted from (13)C-(1)H and (1)H-(1)H dipolar couplings using 2D isotropic-anisotropic correlation experiments. The root mean square fluctuation angles are found to be 11-18 degrees in the dry protein and 16-21 degrees in the 20% hydrated protein. Dramatically, the amplitudes increase to near isotropic at 30% hydration. Field-dependent (1)H rotating-frame spin-lattice relaxation times (T(1rho)) indicate that significant motions occur on the microsecond time-scale (1.2-2.3 micros). The large-amplitude and low-frequency motion of poly(Lys-25) at relatively mild hydration indicates that the conformational entropy of the protein in the relaxed state contributes significantly to the elasticity.     

Design of an N-methylated peptide inhibitor of alpha-synuclein aggregation guided by solid-state NMR

Many neurodegenerative diseases are associated with the aggregation of misfolded proteins into amyloid oligomers or fibrils that are deposited as pathological lesions within areas of the brain. An attractive therapeutic strategy for preventing or ameliorating amyloid formation is to identify agents that inhibit the onset or propagation of protein aggregation. Here we demonstrate how solid-state nuclear magnetic resonance (ssNMR) may be used to identify key residues within amyloidogenic protein sequences that may be targeted to inhibit the aggregation of the host protein. For alpha-synuclein, the major protein component of Lewy bodies associated with Parkinson's disease, we have used a combination of ssNMR and biochemical data to identify the key region for self-aggregation of the protein as residues 77-82 (VAQKTV). We used our new structural information to design a peptide derived from residues 77 to 82 of alpha-synuclein with an N-methyl group at the C-terminal residue, which was able to disrupt the aggregation of alpha-synuclein. Thus, we have shown how structural data obtained from ssNMR can guide the design of modified peptides for use as amyloid inhibitors, as a primary step toward developing therapeutic compounds for prevention and/or treatment of amyloid diseases.     

Structure of (NH4)3GaF6 investigated by multinuclear magic-angle spinning NMR spectroscopy in comparison with rietveld refinement

The structure of ammonium gallium cryolite (NH(4))(3)GaF(6) was investigated by (19)F and (69,71)Ga magic-angle spinning (MAS) NMR in comparison with X-ray powder diffraction followed by Rietveld refinement. In agreement with previous thermodynamic measurements, NMR experiments on (NH(4))(3)GaF(6) support the model of rigid GaF(6) octahedra. At high spinning speeds (30 kHz), the scalar coupling between the six equivalent (19)F nuclei and (69,71)Ga can be directly observed in the powder spectra. The coupling constants are J(19)F(69)Ga = 197 Hz and J(19)F(71)Ga = 264 Hz. To explain the (71)Ga spectra recorded at 3 kHz a small distribution of quadrupolar frequencies has to be included. The spread of the spinning sidebands hints to a largest nu(Q) value of 28 kHz for (71)Ga. This can be explained by the occurrence of highly symmetric GaF(6) octahedra, which are tilted against the surrounding atoms. In addition, the incomplete motional excitation does not average out the quadrupolar effects. NMR findings are in discrepancy to those of Rietveld refinement. As result it appears that X-ray diffraction is not sensitive enough to deliver proper results.     

The morphology of gel-spun polyethylene fibers,investigated by solid-state 13C NMR

Gel-spun polyethylene fibers were analyzed at room temperature with ¹³C NMR, using both, CP-MAS and BILEV (Bloch-decay with two-level decoupling). The analysis shows the existence of three different components in the fiber sample—a crystal component, an amorphous component and a third component, named the oriented, mobile component. This latter component has a ¹³ C chemical shift that is similar to the crystalline chemical shift, but with a mobility, expressed by T₁, that is closer to the amorphous component. The chemical shift and T₁ are as follows: 34.06 ppm and 28.1 s for the crystalline part; 31.70 ppm and 0.3 s for the amorphous part; and 34.06 ppm and 1.8 s for the oriented mobile component. The percentages are 63.2% crystalline; 34.0% oriented mobile, 2.8% amorphous component. Using proton spin-diffusion measurements, it was possible to estimate the domain size of the crystalline and oriented mobile components to be 62.8 and 13.2 nm, respectively, in agreement with the results of a full-pattern x-ray study on the same sample. After melting of the fiber at 450 K and recrystallinzation on cooling, the oriented, mobile component is dramatically reduced. © 1994 John Wiley & Sons, Inc.     

Raftlike mixtures of sphingomyelin and cholesterol investigated by solid-state 2H NMR spectroscopy

Sphingomyelin is a lipid that is abundant in the nervous systems of mammals, where it is associated with putative microdomains in cellular membranes and undergoes alterations due to aging or neurodegeneration. We investigated the effect of varying the concentration of cholesterol in binary and ternary mixtures with N-palmitoylsphingomyelin (PSM) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) using deuterium nuclear magnetic resonance ((2)H NMR) spectroscopy in both macroscopically aligned and unoriented multilamellar dispersions. In our experiments, we used PSM and POPC perdeuterated on the N-acyl and sn-1 acyl chains, respectively. By measuring solid-state (2)H NMR spectra of the two lipids separately in mixtures with the same compositions as a function of cholesterol mole fraction and temperature, we obtained clear evidence for the coexistence of two liquid-crystalline domains in distinct regions of the phase diagram. According to our analysis of the first moments M1 and the observed (2)H NMR spectra, one of the domains appears to be a liquid-ordered phase. We applied a mean-torque potential model as an additional tool to calculate the average hydrocarbon thickness, the area per lipid, and structural parameters such as chain extension and thermal expansion coefficient in order to further define the two coexisting phases. Our data imply that phase separation takes place in raftlike ternary PSM/POPC/cholesterol mixtures over a broad temperature range but vanishes at cholesterol concentrations equal to or greater than a mole fraction of 0.33. Cholesterol interacts preferentially with sphingomyelin only at smaller mole fractions, above which a homogeneous liquid-ordered phase is present. The reasons for these phase separation phenomena seem to be differences in the effects of cholesterol on the configurational order of the palmitoyl chains in PSM-d31 and POPC-d31 and a difference in the affinity of cholesterol for sphingomyelin observed at low temperatures. Hydrophobic matching explains the occurrence of raftlike domains in cellular membranes at intermediate cholesterol concentrations but not saturating amounts of cholesterol.     

Structural properties of docosahexaenoyl phospholipid bilayers investigated by solid-state 2H NMR spectroscopy.

Polyunsaturated lipids in cellular membranes are known to play key roles in such diverse biological processes as vision, neuronal signaling, and apoptosis. One hypothesis is that polyunsaturated lipids are involved in second messenger functions in biological signaling. Another current hypothesis affirms that the functional role of polyunsaturated lipids relies on their ability to modulate physical properties of the lipid bilayer. The present research has employed solid-state 2H NMR spectroscopy to acquire knowledge of the molecular organization and material properties of polyunsaturated lipid bilayers. We report measurements for a homologous series of mixed-chain phosphatidylcholines containing a perdeuterated, saturated acyl chain (n:0) at the sn-1 position, adjacent to docosahexaenoic acid (DHA, 22:6omega3) at the sn-2 position. Measurements have been performed on fluid (L(alpha))-state multilamellar dispersions as a function of temperature for saturated acyl chain lengths of n = 12, 14, 16, and 18 carbons. The saturated sn-1 chains are therefore used as an intrinsic probe with site-specific resolution of the polyunsaturated bilayer structure. The 2H NMR order parameters as a function of acyl position (order profiles) have been analyzed using a mean-torque potential model for the chain segments, and the results are discussed in comparison with the homologous series of disaturated lipid bilayers. At a given absolute temperature, as the sn-1 acyl length adjacent to the sn-2 DHA chain is greater, the order of the initial chain segments increases, whereas that of the end segments decreases, in marked contrast with the corresponding disaturated series. For the latter, the order of the end segments is practically constant with acyl length, thus revealing a universal chain packing profile. We find that the DHA-containing series, while more complex, is still characterized by a universal chain packing profile, which is shifted relative to the homologous saturated series. Moreover, we show how introduction of DHA chains translates the order profile along the saturated chains, making more disordered states accessible within the bilayer central region. As a result, the area per lipid headgroup is increased as compared to disaturated bilayers. The systematic analysis of the 2H NMR data provides a basis for studies of lipid interactions with integral membrane proteins, for instance in relation to characteristic biological functions of highly unsaturated lipid membranes.     

Protein 3D structure determination by high-resolution solid-state NMR

Developments in NMR technology, sample preparation, pulse sequence methodology and structure calculation protocols have recently allowed one to progress towards structure determination at high-resolution of proteins by solid-state NMR spectroscopy. We here report solid-state NMR protocols based on magic-angle-spinning experiments, combined with modified structure calculation protocols, for structure determination of uniformly ¹³C, ¹⁵N isotopically labeled proteins. We demonstrate the use of these protocols to obtain high-resolution structures for the example of the microcrystalline Crh protein. The CHHC, DARR and PAR solid-state NMR experiments, as well as the calculation protocols using the program ARIA, are presented.     

fac-[Re(CO)3(H2O)3]+ nucleoside monophosphate adducts investigated in aqueous solution by multinuclear NMR spectroscopy

The fac-[Re(CO)3(H2O)3]+ cation, the putative DNA-binding species accounting for the biological activity of related Re(I) complexes, binds reversibly to N7 of 6-oxopurine nucleotide monophosphates (NMPs), in contrast to Pt(II) anticancer drugs. A relatively high amount of NMP is needed to convert all of the fac-[Re(CO)3(H2O)3]+ to adducts. The Re/nucleotide 1:1 adduct forms more rapidly and builds up to a higher concentration for guanosine 5'-monophosphate (5'-GMP) and inosine 5'-monophosphate (5'-IMP) than for the respective 3'-monophosphates (3'-GMP and 3'-IMP). These results are attributable to the 5'-positioning of the 5'-NMP phosphate group that allows it to approach the metal inner sphere for more favorable cation electrostatic and aqua ligand H-bonding interactions, both in the initial productive ion pair encounter complexes and in the N7-bound 1:1 adducts. A higher reactivity of 5'-GMP over 3'-GMP is known for cisplatin. In contrast, more Re/nucleotide 1:2 adduct was formed by 3'-GMP (and 3'-IMP) than by 5'-GMP (and 5'-IMP). Because the 3'-phosphate group cannot closely approach the metal inner coordination sphere, the greater stability for the 3'-GMP 1:2 adduct reflects the more favorable G N1H-phosphate interligand GMP-GMP interactions for 3'-GMP vs 5'-GMP (G=guanine base derivative). This type of interaction is known for platinum adducts. In 1:2 adducts the bound nucleotides are inequivalent, prompting us to perform mixed 5'-GMP/3'-GMP experiments, leading to the observation of major (M) and minor (m) mixed Re/5'-GMP/3'-GMP 1:1:1 adducts. The order of abundance at equilibrium in a typical experiment was M>bis 3'-GMP>m>or=bis 5'-GMP. This stability order was rationalized by invoking the phosphate interactions described above. When methionine and 5'-GMP were allowed to compete for fac-[Re(CO)3(H2O)3]+, the Re/5'-GMP 1:1 adduct was the kinetic product and the S-bound Re/methionine adduct was the thermodynamic product, a result opposite to that typically found for cisplatin.     

Molecular motions in poly(diethyl siloxane) studied by solid-state29Si NMR

Molecular motions in poly(diethyl siloxane) were studied by solid-state²⁹Si-NMR in the temperature range 180–350 K. In this temperature range two solid phases ₁ and ₂, a mesophase _m, and an amorphous isotropic phase exist. The nature of the chain mobility in the different phases was deduced from the resulting changes in the NMR line-shape governed by anisotropic chemical shift. In the intermediate solid phase ₂ its anisotropy is reduced by 25% compared with the low temperature phase ₁ due to the onset of oscillations around the chain axis and conformational transitions. In the mesophase _m the polymer chain rotates about its long axis yielding an axially symmetric chemical shift tensor opposite in sign to that in the ₁, ₂ phases. The broad transition of the mesophase into the isotropic phase is accompanied by an increase in a narrow Lorentzian line arising from the amorphous phase. The results are compared with previous¹H NMR, Raman-spectroscopy and x-ray measurements.After completion of this work we learnt that PDES has recently also been studied through¹³C-MAS and²⁹Si-NMR by Möller et al. [13].     

Intermolecular packing and alignment in an ordered beta-hairpin antimicrobial peptide aggregate from 2D solid-state NMR

The aggregation and packing of a membrane-disruptive beta-hairpin antimicrobial peptide, protegrin-1 (PG-1), in the solid state are investigated to understand its oligomerization and hydrogen-bonding propensity. Incubation of PG-1 in phosphate buffer saline produced well-ordered nanometer-scale aggregates, as indicated by 13C and 15N NMR line widths, chemical shifts, and electron microscopy. Two-dimensional 13C and 1H spin diffusion experiments using C-terminus strand and N-terminus strand labeled peptides indicate that the beta-hairpin molecules in these ordered aggregates are oriented parallel to each other with like strands lining the intermolecular interface. In comparison, disordered and lyophilized peptide samples are randomly packed with both parallel and antiparallel alignments. The PG-1 aggregates show significant immobilization of the Phe ring near the beta-turn, further supporting the structural ordering. The intermolecular packing of PG-1 found in the solid state is consistent with its oligomerization in lipid bilayers. This solid-state aggregation approach may be useful for determining the quaternary structure of peptides in general and for gaining insights into the oligomerization of antimicrobial peptides in lipid bilayers in particular.     

Structure and thermochromic solid-state phase transition of poly (3-alkylthiophene)

In order to clarify the structural changes that occur in the thermochromic phase transition of poly (3-dodecylthiophene) [P3DT] and poly (3-hexylthiophene) [P3HT], the temperature dependence of x-ray diffraction and Fourier transform infrared spectra was measured. (1) Orthogonal unit-cell parameters were determined at room temperature: a=25.83 Å, b=7.75 Å, c (fiber axis)=7.77 Å for P3DT and a=16.63 Å, b=7.75 Å, and c=7.77 Å for P3HT. A large variation of the a-axis length between P3DT and P3HT indicates the extended trans conformation for the alkyl side chains which are oriented along the lateral a-axis direction. (2) The interplanar spacing, intensity, and integral width of the x-ray (h00) and (00l) reflections were found to change drastically in the transition region. (3) Polarized infrared measurements at high temperature revealed a marked increase of the gauche band intensity for the alkyl side group modes followed by a decrease in the band intensity of the thiophene ring modes. (4) The layer reflections of the x-ray fiber diagram become diffuse at high temperatures, indicating that the transition occurs in a liquidcrystalline manner with the orientation of the main chain axes preserved but with almost no axial correlation between the neighboring main chains. These results provide experimental support for the structural model proposed earlier: as the temperature increases, the trans-type side chains begin to disorder by introduction of gauche bonds. This disordering disrupts the regularity of the main chain conformation and decreases the effective length of the polythiophene conjugated system.     

Altering the polymorphic product distribution in a solid-state dehydration process by rapid sample rotation in a solid-state NMR probe

There is currently considerable interest in the phenomenon of polymorphism in organic molecular solids, and a key issue in this field is to understand the experimental techniques and procedures that may be employed to obtain new polymorphic forms of a given molecule. This paper demonstrates that the polymorphic form of a material (sodium acetate) obtained by a solid-state dehydration process (starting from sodium acetate trihydrate) can be altered by carrying out the dehydration process under conditions of rapid (several kilohertz) sample rotation in a solid-state magic-angle-spinning NMR probe. This observation suggests a new opportunity to influence the outcome of solid-state dehydration/desolvation processes and, in particular, to alter the polymorphic form of the product obtained.     

Nanopore formation by self-assembly of the model genetically engineered elastin-like polymer [(VPGVG)2(VPGEG)(VPGVG)2]15

The self-assembly characteristics of the model genetically engineered elastin-like polymer [(VPGVG)2(VPGEG)(VPGVG)2]15 have been studied in this work. An AFM study of the topology of polymer films deposited from acid and basic solutions on a hydrophobic silicon substrate has been carried out. Under acidic conditions, polymer deposition results in a flat surface with no particular topological features. However, from basic solutions, polymer deposition clearly shows an aperiodic pattern of nanopores ( approximately 70 nm width and separated about 150 nm). This dramatic dependence of film topology on pH is explained in terms of the different polarity of the free gamma-carboxyl group of the glutamic acid. In the carboxylate form, this moiety shows a markedly higher polarity than the rest of the polymer domains and the substrate itself. Under these conditions, the charged carboxylates impede hydrophobic contact with their surroundings, which is the predominant assembly pathway for this type of polymer. The charged domains, along with their hydration sphere, are then segregated from the hydrophobic surroundings giving rise to nanopores.     

Local environment and distribution of alkali ions in polyelectrolyte complexes studied by solid-state NMR

Polyelectrolyte complexes (PECs) formed by the addition of substoichiometric amounts of (poly(diallyldimethyl ammonium chloride)) (PDADMAC) solutions to sodium or lithium poly(styrene sulfonate) (Na- or Li-PSS) solution contain adjustable amounts of charge balancing Li(+) or Na(+) cations, which possess ionic mobility of interest for solid electrolyte applications. Very little is known regarding the local environments and the spatial distributions of these cations and their interactions with the polyelectrolyte chains in these amorphous materials. To address such issues, the present work develops a comprehensive solid state NMR strategy based on complementary high-resolution magic-angle spinning (MAS) NMR and various dipolar spectroscopic techniques. (6,7)Li and (23)Na chemical shifts measured on a series of PECs with general composition described by B((2x-1))PSS(x)PDADMA((1-x)) (B = Li or Na and 0.53 ≤x≤ 1) reveal composition-independent local cation environments. In contrast, (7)Li{(6)Li} spin echo double resonance (SEDOR) experiments measured on (6)Li enriched materials and (7)Li{(1)H} rotational echo double resonance (REDOR) experiments are consistent with an approximately random ion distribution. The same conclusion is suggested by (23)Na{(1)H} REDOR measurements on the analogous sodium containing system indicating a non-segregated PEC structure. In apparent contrast to this conclusion, (23)Na spin echo decay spectroscopy yields nearly constant dipolar second moments over a wide composition range. This can be explained by considering that the (23)Na spin echo decays are affected by both (23)Na-(23)Na homonuclear dipolar couplings and (23)Na-(1)H heteronuclear dipolar interactions in the presence of strong homonuclear (1)H-(1)H spin exchange. In protonated Na-PSS both contributions are of comparable magnitude. In the PECs the contribution from (23)Na-(23)Na interactions decreases, while that from (23)Na-(1)H dipolar couplings with the protons from the PDADMA chains increases with decreasing Na content, resulting in superimposed opposite dependences on the ion concentration. All results for Li and Na containing PECs point at a non-phase separated polymer network with uniform ionic sites of very similar environment. The cations can be viewed as randomly distributed and located close to the polyion sulfate groups.     

Lamellar structure in poly(ala-gly) determined by solid-state NMR and statistical mechanical calculations

Lamellar structure of poly(Ala-Gly) or (AG)n in the solid was examined using 13C solid-state NMR and statistical mechanical approaches. Two doubly labeled versions, [1-13C]Gly14[1-13C]Ala15- and [1-13C]Gly18[1-13C]Ala19 of (AG)15 were examined by two-dimensional (2D) 13C spin diffusion NMR in the solid state. In addition five doubly labeled [15N,13C]-versions of the same peptide, (AG) 15 and 15 versions labeled [3-13C] in each of the successive Ala residues were utilized for REDOR and 13C CP/MAS NMR measurements, respectively. The observed spin diffusion NMR spectra were consistent with a structure containing a combination of distorted beta-turns with a large distribution of the torsion angles and antiparallel beta-sheets. The relative proportion of the distorted beta-turn form was evaluated by examination of 13C CP/MAS NMR spectra of [3-13C]Ala-(AG)15. In addition, REDOR determinations showed five kinds of atomic distances between doubly labeled 13C and 15N nuclei which were also interpreted in terms of a combination of beta-sheets and beta-turns. Our statistical mechanical analysis is in excellent agreement with our Ala Cbeta 13C CP/MAS NMR data strongly suggesting that (AG)15 has a lamellar structure.     

Structural characterization of an anhydrous polymorph of paclitaxel by solid-state NMR

The three-dimensional structure of a unique polymorph of the anticancer drug paclitaxel (Taxol) is established using solid state NMR (SSNMR) tensor ((13)C & (15)N) and heteronuclear correlation ((1)H-(13)C) data. The polymorph has two molecules per asymmetric unit (Z' = 2) and is thus the first conformational characterization with Z' > 1 established solely by SSNMR. Experimental data are correlated with structure through a series of computational models that extensively sample all conformations. For each computational model, corresponding tensor values are computed to supply comparisons with experimental information which, in turn, establishes paclitaxel's structure. Heteronuclear correlation data at thirteen key positions provide shift assignments to the asymmetric unit for each comparison. The two distinct molecules of the asymmetric unit possess nearly identical baccatin III moieties with matching conformations of the C10 acetyl moiety and, specifically, the torsion angle formed by C30-O-C10-C9. Additionally, both are found to exhibit an extended conformation of the phenylisoserine sidechain at C13 with notable differences in the dihedral angles centered around the rotation axes of O-C13, C2'-C1' and C3'-C2'.     

Ion-binding study by 17O solid-state NMR spectroscopy in the model peptide Gly-Gly-Gly at 19.6 T

Li(+) and Ca(2+) binding to the carbonyl oxygen sites of a model peptide system has been studied by (17)O solid-state NMR spectroscopy. (17)O chemical shift (CS) and quadrupole coupling (QC) tensors are determined in four Gly-(Gly-(17)O)-Gly polymorphs by a combination of stationary and fast magic-angle spinning (MAS) methods at high magnetic field, 19.6 T. In the crystal lattice, the carbonyl oxygen of the central glycyl residue in two gly-gly-gly polymorphs form intermolecular hydrogen bonds with amides, whereas the corresponding carbonyl oxygens of the other two polymorphs form interactions with Li(+) and Ca(2+) ions. This permits a comparison of perturbations on (17)O NMR properties by ion binding and intermolecular hydrogen bonding. High quality spectra are augmented by density functional theory (DFT) calculations on large molecular clusters to gain additional theoretical insights and to aid in the spectral simulations. Ion binding significantly decreases the two (17)O chemical shift tensor components in the peptide plane, delta(11) and delta(22), and, thus, a substantial change in the isotropic chemical shift. In addition, quadrupole coupling constants are decreased by up to 1 MHz. The effects of ion binding are found to be almost an order of magnitude greater than those induced by hydrogen bonding.