Absolute structural constraints on amyloid fibrils from solid-state NMR spectroscopy of partially oriented samples

We demonstrate that absolute, molecular-level structural information can be obtained from solid-state NMR measurements on partially oriented amyloid fibrils. Specifically, we show that the direction of the fibril axis relative to a carbonyl 13C chemical shift anisotropy (CSA) tensor can be determined from magic-angle spinning (MAS) sideband patterns in 13C NMR spectra of fibrils deposited on planar substrates. Deposition of fibrils on a planar substrate creates a highly anisotropic distribution of fibril orientations (hence, CSA tensor orientations) with most fibrils lying in the substrate plane. The anisotropic orientational distribution gives rise to distorted spinning sideband patterns in MAS spectra from which the fibril axis direction can be inferred. The experimentally determined fibril axis direction relative to the carbonyl CSA tensor of Val12 in fibrils formed by the 40-residue beta-amyloid peptide associated with Alzheimer's disease (Abeta1-40) agrees well with the predictions of a recent structural model (Petkova et al. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 16742-16747) in which Val12 is contained in a parallel beta-sheet in the cross-beta motif characteristic of amyloid fibrils.     

Intermolecular alignment in Y145Stop human prion protein amyloid fibrils probed by solid-state NMR spectroscopy

The Y145Stop mutant of human prion protein, huPrP23-144, has been linked to PrP cerebral amyloid angiopathy, an inherited amyloid disease, and also serves as a valuable in vitro model for investigating the molecular basis of amyloid strains. Prior studies of huPrP23-144 amyloid by magic-angle-spinning (MAS) solid-state NMR spectroscopy revealed a compact β-rich amyloid core region near the C-terminus and an unstructured N-terminal domain. Here, with the focus on understanding the higher-order architecture of huPrP23-144 fibrils, we probed the intermolecular alignment of β-strands within the amyloid core using MAS NMR techniques and fibrils formed from equimolar mixtures of (15)N-labeled protein and (13)C-huPrP23-144 prepared with [1,3-(13)C(2)] or [2-(13)C]glycerol. Numerous intermolecular correlations involving backbone atoms observed in 2D (15)N-(13)C spectra unequivocally suggest an overall parallel in-register alignment of the β-sheet core. Additional experiments that report on intermolecular (15)N-(13)CO and (15)N-(13)Cα dipolar couplings yielded an estimated strand spacing that is within ～10% of the distances of 4.7-4.8 ? typical for parallel β-sheets.     

Protein structure determination from 13C spin-diffusion solid-state NMR spectroscopy

Proton-driven 13C spin diffusion (PDSD) is a simple and robust two-dimensional NMR experiment. It leads to spectra with a high signal-to-noise ratio in which cross-peaks contain information about internuclear distances. We show that the total information content is sufficient to determine the atomic-resolution structure of a small protein from a single, uniformly 13C-, 15N-labeled microcrystalline sample. For the example of ubiquitin, the structure was determined by a manual procedure followed by an automatic optimization of the manual structure as well as by a fully automated structure determination approach. The relationship between internuclear distances and cross-peak intensities in the spectra is investigated.     

Homo- and heteronuclear two-dimensional covariance solid-state NMR spectroscopy with a dual-receiver system

Two-dimensional (2D) covariance NMR spectroscopy, which has originally been established to extract homonuclear correlations (HOMCOR), is extended to include heteronuclear correlations (HETCOR). In a (13)C/(15)N 2D chemical shift correlation experiment, (13)C and (15)N signals of a polycrystalline sample of (13)C, (15)N-labeled amino acid are acquired simultaneously using a dual-receiver NMR system. The data sets are rearranged for the covariance data processing, and the (13)C-(15)N heteronuclear correlations are obtained together with the (13)C-(13)C and (15)N-(15)N homonuclear correlations. The present approach retains the favorable feature of the original covariance HOMCOR that the spectral resolution along the indirect dimension is given by that of the detection dimension. As a result, much fewer amounts of data are required to obtain a well-resolved 2D spectrum compared to the case of the conventional 2D Fourier-Transformation (FT) scheme. Hence, one can significantly save the experimental time, or enhance the sensitivity by increasing the number of signal averaging within a given measurement time.     

Sensitivity enhancement in two-dimensional solid-state NMR spectroscopy by transverse mixing.

The sensitivity of two-dimensional (2D) 13C-13C solid-state NMR spectroscopy under magic-angle spinning (MAS) is shown to be enhanced by the use of transverse polarization transfer in place of the conventional longitudinal polarization transfer. Experimental results are reported for 2D spectroscopy of a 20-residue, filament-forming peptide derived from the E. Coli RecA protein, containing five uniformly 13C-labeled residues, performed at 14.1 T with high-speed MAS and with finite-pulse radio-frequency-driven recoupling of dipolar interactions in the mixing period. Significant sensitivity enhancements observed at short mixing periods results from a more rapid build-up of cross-peaks under transverse mixing than under longitudinal mixing and from the 2 gain inherent in 2D measurements in which both orthogonal transverse polarization components in the t1 period contribute to each free-induction decay signal detected in the t2 period.     

Intermolecular structure determination of amyloid fibrils with magic-angle spinning and dynamic nuclear polarization NMR

We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this purpose, two being based on the analysis of long-range (13)C-(13)C correlation spectra and the third based on the identification of intermolecular interactions in (13)C-(15)N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3 or PI3K-SH3), that efficient (13)C-(13)C correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the proteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The (13)C-(13)C experiments are corroborated by (15)N-(13)C correlation spectra obtained from a mixed [(15)N,(12)C]/[(14)N,(13)C] sample which directly quantify interstrand distances. Furthermore, when the spectra are recorded with signal enhancement provided by dynamic nuclear polarization (DNP) at 100 K, we demonstrate a dramatic increase (from 23 to 52) in the number of intermolecular (15)N-(13)C constraints detectable in the spectra. The increase in the information content is due to the enhanced signal intensities and to the fact that dynamic processes, leading to spectral intensity losses, are quenched at low temperatures. Thus, acquisition of low temperature spectra addresses a problem that is frequently encountered in MAS spectra of proteins. In total, the experiments provide 111 intermolecular (13)C-(13)C and (15)N-(13)C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.     

Observation of the glycines in elastin using (13)C and (15)N solid-state NMR spectroscopy and isotopic labeling

We report the solid-state 13C and 15N NMR of insoluble elastin which has been synthesized in vitro with isotopically enriched glycine. Most of the glycines reside in a domain with good cross-polarization (CP) efficiencies, although surprisingly, a portion resides in an environment that is not detectable using CP. Our data indicate that much of the 13C population resides in regions of significant conformational flexibility. To support these conclusions, we present 13C and 15N cross-polarization with magic-angle-spinning (CPMAS) data in conjunction with "direct-polarization", nonspinning CP, and T1 measurements.     

Structural and dynamical characterization of fibrils from a disease-associated alanine expansion domain using proteolysis and solid-state NMR spectroscopy

The nuclear poly(A) binding protein PABPN1 possesses a natural 10 alanine stretch that can be extended to 17 Ala by codon expansion. The expansions are associated with the disease oculopharyngeal muscular dystrophy (OPMD), which is characterized histopathologically by intranuclear fibrillar deposits. Here, we have studied the Ala extended fibrillar N-terminal fragment of PABPN1, (N-(+7)Ala), comprising 152 amino acids. At natural abundance, cross-polarized 13C MAS NMR spectra are dominated by the three Ala signals with characteristic beta-sheet chemical shifts. In contrast, directly polarized 13C MAS spectra show a multitude of narrow lines, suggesting a large portion of highly mobile sites. Proteolytic cleavage of the protein combined with MALDI-TOF mass spectrometry revealed a protease-resistant peptide encompassing residues 13/14 to 50-52 with the poly-Ala stretch in the center. Measurements of the 1H-13Calpha dipolar couplings of 13C/15N-labeled N-(+7)Ala revealed high order parameters of 0.77 for the poly-Ala stretch of the fibril, while the majority of the residues of N-(+7)Ala exhibited very low order parameters between 0.06 and 0.15. Only some Gly residues that are flanking the Ala-rich region had significant order parameters of 0.47. Thus, site-specific dynamic mapping represents a useful tool to identify the topology of fibrillar proteins.     

Bolalipid membrane structure revealed by solid-state 2H NMR spectroscopy

Membranes made from three specifically deuterium-labeled ether-linked bolalipids, [1',1',20',20'-2H4]C20BAS-PC, [2',2',19',19'-2H4]C20BAS-PC, or [10',11'-2H2]C20BAS-PC, were analyzed by 2H NMR spectroscopy. Unlike more common monopolar, ester-linked phospholipids, C20BAS-PC exhibits a high degree of orientational order throughout the membrane and the sn-1 chain of the lipid initially penetrates the bilayer at an orientation different from that of the bilayer normal, resulting in inequivalent deuterium atoms at the C1 position. The approximate hydrophobic layer thickness and area per lipid are 18.4 A and 60.4 A2, respectively, at 25 degrees C, and their respective thermal expansion coefficients are within 20% of the monopolar phospholipid, DLPC.     

Self-assembled ionophores from isoguanosine: diffusion NMR spectroscopy clarifies cation's and anion's influence on supramolecular structure

Cation-templated self-assembly of the lipophilic isoguanosine (isoG 1) with different monovalent cations (M(+)=Li(+), Na(+), K(+), NH(4) (+), and Cs(+)) was studied in solvents of different polarity by using diffusion NMR spectroscopy. Previous studies that did not use diffusion NMR techniques concluded that isoG 1 forms both pentamers (isoG 1)(5)M(+) and decamers (isoG 1)(10)M(+) in the presence of alkali-metal cations. The present diffusion NMR studies demonstrate, however, that isoG 1 does not form (isoG 1)(5)M(+) pentamers. In fact, the diffusion NMR data indicates that both doubly charged decamers of formula (isoG 1)(10)2 M(+) and singly charged decamers, (isoG 1)(10)M(+), are formed with lithium, sodium, potassium, and ammonium tetraphenylborate salts (LiB(Ph)(4), KB(Ph)(4), NaB(Ph)(4) and NH(4)B(Ph)(4)), depending on the isoG 1:salt stoichiometry of the solution. In the presence of CsB(Ph)(4), isoG 1 affords only the singly charged decamers (isoG 1)(10)Cs(+). By monitoring the diffusion coefficient of the B(Ph)(4) (-) ion in the different mixtures of solvents, we also concluded that the anion is more strongly associated to the doubly charged decamers (isoG 1)(10)2 M(+) than to the singly charged decamers (isoG 1)(10)M(+). The (isoG 1)(10)2 M(+) species can, however, exist in solution without the mediation of the anion. This last conclusion was supported by the finding that the doubly charged decamers (isoG 1)(10)2 M(+) also prevail in 1:1 CD(3)CN:CDCl(3), a solvent mixture in which the B(Ph)(4) (-) ion does not interact significantly with the self-assembled complex. These diffusion measurements, which have provided new and improved structural information about these decameric isoG 1 assemblies, demonstrate the utility of combining diffusion NMR techniques with conventional NMR methods in seeking to characterize labile, multicomponent, supramolecular systems in solution, especially those with high symmetry.     

Determination of global structure from distance and orientation constraints in biological solids using solid-state NMR spectroscopy

We report the results from a new solid-state NMR experiment, DANTE-REDOR, which can determine global secondary structure in uniformly (13C,15N)-enriched systems by simultaneously measuring distance and orientation constraints. Following a heteronuclear spin-pair selection using a DANTE pulse train, the magnitude and orientation of the internuclear dipole vector, within the chemical shift anisotropy (CSA) frame of the observed nucleus, are determined by tracking the dephasing of individual spinning sidebands under magic angle spinning. The efficacy of the experiment is demonstrated by measuring the imidazole side-chain orientation in U-[13C6,15N3]-L-histidine x HCl x H2O.     

Chemical shifts for the unusual DNA structure in Pf1 bacteriophage from dynamic-nuclear-polarization-enhanced solid-state NMR spectroscopy

Solid-state NMR spectra, including dynamic nuclear polarization enhanced 400 MHz spectra acquired at 100 K, as well as non-DNP spectra at a variety of field strengths and at temperatures in the range 213-243 K, have allowed the assignment of the (13)C and (15)N resonances of the unusual DNA structure in the Pf1 virion. The (13)C chemical shifts of C3' and C5', considered to be key reporters of deoxyribose conformation, fall near or beyond the edges of their respective ranges in available databases. The (13)C and (15)N chemical shifts of the DNA bases have above-average values for AC4, AC5, CC5, TC2, and TC5, and below average values for AC8, GC8, and GN2, pointing to an absence of Watson-Crick hydrogen bonding, yet the presence of some type of aromatic ring interaction. Crosspeaks between Tyr40 of the coat protein and several DNA atoms suggest that Tyr40 is involved in this ring interaction. In addition, these crosspeak resonances and several deoxyribose resonances are multiply split, presumably through the effects of ordered but differing interactions between capsid protein subunits and each type of nucleotide in each of the two DNA strands. Overall, these observations characterize and support the DNA model proposed by Liu and Day and refined by Tsuboi et al., which calls for the most highly stretched and twisted naturally occurring DNA yet encountered.     

Heterogeneous structure of silk fibers from Bombyx mori resolved by 13C solid-state NMR spectroscopy

The molecular conformation of silk fibrion is characterized by solid-state 13C NMR before spinning (silk I structure) and after spinning (silk II structure). We compare native silk fibers with the quasi-crystalline Cp-fraction and a synthetic model peptide (Ala-Gly)15, both of which can be converted either into silk I by dialysis from 9 M LiBr or into silk II by treatment with formic acid. Our results demonstrate that silk II fibers are intrinsically heterogeneous, consisting of beta-sheets, distorted beta-turns, and distorted beta-sheets. This higher-order heterogeneity is revealed by the 13C-NMR Cbeta-peak of Ala, indicating that the Ala side chains are stacked partially in parallel and partially face-to-face, at a ratio of 1:2.     

Determining secondary structure in spider dragline silk by carbon-carbon correlation solid-state NMR spectroscopy

Two-dimensional (2D) (13)C-(13)C NMR correlation spectra were collected on (13)C-enriched dragline silk fibers produced from Nephila clavipes spiders. The 2D NMR spectra were acquired under fast magic-angle spinning (MAS) and dipolar-assisted rotational resonance (DARR) recoupling to enhance magnetization transfer between (13)C spins. Spectra obtained with short (150 ms) recoupling periods were utilized to extract distinct chemical shifts for all carbon resonances of each labeled amino acid in the silk spectra, resulting in a complete resonance assignment. The NMR results presented here permit extraction of the precise chemical shift of the carbonyl environment for each (13)C-labeled amino acid in spider silk for the first time. Spectra collected with longer recoupling periods (1 s) were implemented to detect intermolecular magnetization exchange between neighboring amino acids. This information is used to ascribe NMR resonances to the specific repetitive amino acid motifs prevalent in spider silk proteins. These results indicate that glycine and alanine are both present in two distinct structural environments: a disordered 3(1)-helical conformation and an ordered beta-sheet structure. The former can be ascribed to the Gly-Gly-Ala motif while the latter is assigned to the poly(Ala) and poly(Gly-Ala) domains.     

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.     

Drug-dendrimer supramolecular complexation studied from molecular dynamics simulations and NMR spectroscopy

Fully atomistic molecular dynamics (MD) simulations and NMR spectroscopy were employed to get insights about the molecular details of drug-dendrimer supramolecular association phenomena, using piroxicam (PRX) and the third generation poly(amido amine) (PAMAM-G3) dendrimer as model systems. Theoretical results concerning the complex stoichiometry suggest that PRX forms drug-dendrimer complexes of the type 24:1 at pH 7.0. This result was validated with the experimental quantities obtained from aqueous solubility profiles, which led to an empiric stoichiometry of 23:1 for the PRX:PAMAM-G3 system. The predicted binding mode between PRX and PAMAM-G3 accounts for the preferred encapsulation of the drug inside dendrimer cavities, which is mainly driven by van der Waals and hydrogen bonding interactions, and to a lesser extent, for the external association of the guest through electrostatic contacts with the positively charged amino groups of PAMAM periphery. The binding mode obtained from MD simulations was confirmed with 2D-NOESY experiments, which evidence the preferred internal complexation of PRX with PAMAM-G3. The predominance of internal encapsulation over external contacts in the PRX:PAMAM-G3 system differs from the general behaviour expected for acidic anionic guests, for which external electrostatic interactions with the positively charged PAMAM surface have been postulated as the most relevant factor for drug association.