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.

A hierarchy of protein motions Functioning proteins are not static but explore complex conformational energy landscapes. Lewandowski et al. used multinuclear solid-state nuclear magnetic resonance experiments to measure protein motion over a broad range of temperatures and time scales. Above 160 K there was a strong coupling between solvent and protein motion. The hierarchy of motions as the temperature increased revealed the dynamic modes that relate solvent, sidechain, and backbone motion. Science , this issue p. 578

We introduce a homonuclear version of third spin assisted recoupling, a second-order mechanism that can be used for polarization transfer between (13)C or (15)N spins in magic angle spinning (MAS) NMR experiments, particularly at high spinning frequencies employed in contemporary high field MAS experiments. The resulting sequence, which we refer to as proton assisted recoupling (PAR), relies on a cross-term between (1)H-(13)C (or (1)H-(15)N) couplings to mediate zero quantum (13)C-(13)C (or (15)N-(15)N recoupling). In particular, using average Hamiltonian theory we derive an effective Hamiltonian for PAR and show that the transfer is mediated by trilinear terms of the form C(1) (+/-)C(2) (-/+)H(Z) for (13)C-(13)C recoupling experiments (or N(1) (+/-)N(2) (-/+)H(Z) for (15)N-(15)N). We use analytical and numerical simulations to explain the structure of the PAR optimization maps and to delineate the PAR matching conditions. We also detail the PAR polarization transfer dependence with respect to the local molecular geometry and explain the observed reduction in dipolar truncation. Finally, we demonstrate the utility of PAR in structural studies of proteins with (13)C-(13)C spectra of uniformly (13)C, (15)N labeled microcrystalline Crh, a 85 amino acid model protein that forms a domain swapped dimer (MW=2 x 10.4 kDa). The spectra, which were acquired at high MAS frequencies (omega(r)2pi>20 kHz) and magnetic fields (750-900 MHz (1)H frequencies) using moderate rf fields, exhibit numerous cross peaks corresponding to long (up to 6-7 A) (13)C-(13)C distances which are particularly useful in protein structure determination. Using results from PAR spectra we calculate the structure of the Crh protein.

Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington's disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin-containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of "intrinsic" polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.solid-state NMR | Huntington's disease | amyloid disease | protein aggregation | amyloid

There is an urgent need to understand the behavior of the novel coronavirus (SARS-COV-2), which is the causative agent of COVID-19, and to develop point-of-care diagnostics. Here, a glyconanoparticle platform is used to discover that N -acetyl neuraminic acid has affinity toward the SARS-COV-2 spike glycoprotein, demonstrating its glycan-binding function. Optimization of the particle size and coating enabled detection of the spike glycoprotein in lateral flow and showed selectivity over the SARS-COV-1 spike protein. Using a virus-like particle and a pseudotyped lentivirus model, paper-based lateral flow detection was demonstrated in under 30 min, showing the potential of this system as a low-cost detection platform.

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.

This communication presents a solid-state NMR 15 N-13 C polarization transfer scheme applicable at high B 0 and high MAS frequencies, requiring moderate r.f. powers (~50 kHz 13 C/ 15 N) and mixing time (1-6 ms). The sequence, PAIN-CP, involves the abundant nearby protons in the heteronuclear recoupling dynamics, and provides a new tool for obtaining long distance 15 N-13 C contacts. It should be of major interest for biomolecular structural studies.Polarization transfer 1-15 between different nuclear species mediated by dipolar couplings is used extensively in magic angle spinning (MAS) 16 experiments to perform chemical shift assignments, and to measure distances 5-7, 11, 17-21 and torsion angles. 22-25 Heteronuclear dipolar recoupling sequences can be classified into two categories depending on their behavior with respect to dipolar truncation. The first group includes the double CP sequence (DCP26) and its variants (SPICP27, RFDRCP 4 , iDCP 9 ) which lead to non-commuting terms in the effective Hamiltonian, and thus are mainly used to perform one-bond transfers (NCO, NCA) sometimes followed by a homonuclear 13 C-13 C recoupling period for obtaining 15 N-13 C multiple-bond contacts. 28, 29 The second group of sequences (REDOR5, TEDOR 1 / REPT 19 , GATE 17 ) yields a longitudinal effective Hamiltonian and enables measurement of long distances (< 4 Å). 20, 21High magnetic fields (>600 MHz) are an important experimental component for improving sensitivity and resolution in biomolecular MAS experiments involving 15 N-13 C magnetization transfer, provided that experiments can be performed at high MAS frequencies (>15 kHz) to compensate for increases in chemical shift anisotropies (CSA). Unfortunately, the application of the sequences mentioned above becomes problematic in this regime as the applied high r.f. powers lead to increased sample heating, jeopardize the integrity of the probe, but often do not provide sufficient 1 H decoupling.Here we present an efficient 15 N-13 C heteronuclear recoupling technique that involves nearby protons in the transfer and is applicable at high MAS frequency (ω r /2π>20 kHz). This new scheme, Proton Assisted Insensitive Nuclei Cross Polarization (PAIN-CP), reduces dipolar truncation and therefore is particularly well suited for obtaining long distance contacts. PAIN-CP demonstrates that the involvement of protons in the polarization transfer between low-γ nuclei does not have to be deleterious in nature; on the contrary 1 H's can be used to enhance the rate and efficiency of the transfer. The PAIN-CP experiment utilizes a mechanism which we refer to as Third Spin Assisted Recoupling (TSAR). Its extension to the homonuclear case is straightforward and will be discussed elsewhere. Note that the use of 1 H irradiation has been reported previously for 13 C-13 C recoupling experiments, 30-32 but that the underlying mechanism is different. Even though PAIN-CP and DCP 26 have similar pulse sequences (see Figure 1 and Supporting Information Section 7-8 (S.I.-7, 8)), they rely on v...

We demonstrate that a quantitative measure of slow molecular motions in solid proteins can be accessed by measuring site-specific (15)N rotating-frame relaxation rates at high magic-angle-spinning frequencies.