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International audience; A pathological hallmark of Huntington's disease (HD) is the formation of neuronal protein deposits containing mutant huntingtin fragments with expanded polyglutamine (polyQ) domains. Prior studies have shown the strengths of solid-state NMR (ssNMR) to probe the atomic structure of such aggregates, but have required in vitro isotopic labeling. Herein, we present an approach for the structural fingerprinting of fibrils through ssNMR at natural isotopic abundance (NA). These methods will enable the spectroscopic fingerprinting of unlabeled (e.g., ex vivo) protein aggregates and the extraction of valuable new long-range 13 C− 13 C distance constraints

Chiral nanoparticle assemblies are an interesting class of materials whose chiroptical properties make them attractive for a variety of applications. Here, C18-(PEPAu(M-ox))2 (PEPAu(M-ox) = AYSSGAPPM(ox)PPF) is shown to direct the assembly of single-helical gold nanoparticle superstructures that exhibit exceptionally strong chiroptical activity at the plasmon frequency with absolute g-factor values up to 0.04. Transmission electron microscopy (TEM) and cryogenic electron tomography (cryo-ET) results indicate that the single helices have a periodic pitch of approximately 100 nm and consist of oblong gold nanoparticles. The morphology and assembled structure of C18-(PEPAu(M-ox))2 are studied using TEM, atomic force microscopy (AFM), Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD) spectroscopy, X-ray diffraction (XRD), and solid-state nuclear magnetic resonance (ssNMR) spectroscopy. TEM and AFM reveal that C18-(PEPAu(M-ox))2 assembles into linear amyloid-like 1D helical ribbons having structural parameters that correlate to those of the single-helical gold nanoparticle superstructures. FTIR, CD, XRD, and ssNMR indicate the presence of cross-β and polyproline II secondary structures. A molecular assembly model is presented that takes into account all experimental observations and that supports the single-helical nanoparticle assembly architecture. This model provides the basis for the design of future nanoparticle assemblies having programmable structures and properties.

Dynamic nuclear polarization (DNP) permits a approximately 10(2)-10(3) enhancement of the nuclear spin polarization and therefore increases sensitivity in nuclear magnetic resonance (NMR) experiments. Here, we demonstrate the efficient transfer of DNP-enhanced (1)H polarization from an aqueous, radical-containing solvent matrix into peptide crystals via (1)H-(1)H spin diffusion across the matrix-crystal interface. The samples consist of nanocrystals of the amyloid-forming peptide GNNQQNY(7-13), derived from the yeast prion protein Sup35p, dispersed in a glycerol-water matrix containing a biradical polarizing agent, TOTAPOL. These crystals have an average width of 100-200 nm, and their known crystal structure suggests that the size of the biradical precludes its penetration into the crystal lattice; therefore, intimate contact of the molecules in the nanocrystal core with the polarizing agent is unlikely. This is supported by the observed differences between the time-dependent growth of the enhanced polarization in the solvent versus the nanocrystals. Nevertheless, DNP-enhanced magic-angle spinning (MAS) spectra recorded at 5 T and 90 K exhibit an average signal enhancement epsilon approximately 120. This is slightly lower than the DNP enhancement of the solvent mixture surrounding the crystals (epsilon approximately 160), and we show that it is consistent with spin diffusion across the solvent-matrix interface. In particular, we correlate the expected DNP enhancement to several properties of the sample, such as crystal size, the nuclear T(1), and the average (1)H-(1)H spin diffusion constant. The enhanced (1)H polarization was subsequently transferred to (13)C and (15)N via cross-polarization, and allowed rapid acquisition of two-dimensional (13)C-(13)C correlation data.

Sup35p is a prion protein found in yeast that contains a prion-forming domain characterized by a repetitive sequence rich in Gln, Asn, Tyr, and Gly amino acid residues. The peptide GNNQQNY7-13 is one of the shortest segments of this domain found to form amyloid fibrils, in a fashion similar to the protein itself. Upon dissolution in water, GNNQQNY displays a concentration-dependent polymorphism, forming monoclinic and orthorhombic crystals at low concentrations and amyloid fibrils at higher concentrations. We prepared nanocrystals of both space groups as well as fibril samples that reproducibly contain three (coexisting) structural forms and examined the specimens with magic angle spinning (MAS) solid-state nuclear magnetic resonance. 13C and 15N MAS spectra of both nanocrystals and fibrils reveal narrow resonances indicative of a high level of microscopic sample homogeneity that permitted resonance assignments of all five species. We observed variations in chemical shift among the three dominant forms of the fibrils which were indicated by the presence of three distinct, self-consistent sets of correlated NMR signals. Similarly, the monoclinic and orthorhombic crystals exhibit chemical shifts that differ from one another and from the fibrils. Collectively, the chemical shift data suggest that the peptide assumes five conformations in the crystals and fibrils that differ from one another in subtle but distinct ways. This includes variations in the mobility of the aromatic Tyr ring. The data also suggest that various structures assumed by the peptide may be correlated to the "steric zipper" observed in the monoclinic crystals.

We have used ab initio calculations to compute all of the tensor elements of the electric field gradient for each carbon-deuterium bond in the ring of deuterated 3-methyl-indole. Previous analyses have ignored the smaller tensor elements perpendicular to principal component Vzz which is aligned with the C-2H bond (local bond z-axis). At each ring position, the smallest element Vxx is in the molecular plane and Vyy is normal to the plane of the ring. The asymmetry parameter η = (|Vyy|-|Vxx|)/|Vzz| ranges from 0.07 at C4 to 0.11 at C2. We used the perpendicular (off-bond) tensor elements, in concert with an improved understanding of the indole ring geometry1, to analyze prototype 2H-NMR spectra from well-oriented, hydrated peptide/lipid samples. For each of the 4 tryptophans of membrane-spanning gramicidin A (gA)2 channels, the inclusion of the perpendicular elements changes the deduced ring tilt by nearly 10° and increases the ring principal order parameter Szz for overall ‘wobble’ with respect to the membrane normal (molecular z-axis). With the improved analysis, the magnitude of Szz for the outermost indole rings of Trp13 and Trp15 is indistinguishable from that observed previously for backbone atoms (0.93 ± 0.03). For the Trp9 and Trp11 rings, which are slightly more buried within the membrane, Szz is slightly lower (0.86 ± 0.03). The results show that the perpendicular elements are important for the detailed analysis of 2H-NMR spectra from aromatic ring systems.

We have used experimental deuterium NMR spectra from labeled tryptophans in membrane-spanning gramicidin A (gA)(1) channels to refine the geometry of the indole ring and, specifically, the C2-(2)H bond direction. By using partial exchange in a cold organic acid, we were able to selectively deuterate ring positions C2 and C5 and, thereby, define unambiguous spectral assignments. In a backbone-independent analysis, the assigned spectra from four distinct labeled tryptophans were used to assess the geometry of the planar indole ring. We found that the C2-(2)H bond makes an angle of about 6 degrees with respect to the normal to the indole ring bridge, and the experimental geometry was confirmed by density functional calculations using a 6-311G** basis set. The precisely determined ring geometry and the experimental spectra in turn are the foundation for calculations of the orientation of each tryptophan indole ring, with respect to the bilayer membrane normal, and of a principal order parameter S(zz) for each ring. The results have general significance for revising the tryptophan ring geometry that is used in protein molecular modeling, as well as for the analysis of tryptophan ring orientations in membrane-spanning proteins. The experimental precision in the definition of the indole ring geometry demonstrates yet another practical application emanating from fundamental research on the robust gramicidin channel.