Magic-angle spinning solid-state NMR (SSNMR) studies of the beta1 immunoglobulin binding domain of protein G (GB1) are presented. Chemical shift correlation spectra at 11.7 T (500 MHz 1H frequency) were employed to identify signals specific to each amino acid residue type and to establish backbone connectivities. High sensitivity and resolution facilitated the detection and assignment of every 15N and 13C site, including the N-terminal (M1) 15NH3, the C-terminal (E56) 13C', and side-chain resonances from residues exhibiting fast-limit conformational exchange near room temperature. The assigned spectra lend novel insight into the structure and dynamics of microcrystalline GB1. Secondary isotropic chemical shifts report on conformation, enabling a detailed comparison of the microcrystalline state with the conformation of single crystals and the protein in solution; the consistency of backbone conformation in these three preparations is the best among proteins studied so far. Signal intensities and line widths vary as a function of amino acid position and temperature. High-resolution spectra are observed near room temperature (280 K) and at <180 K, whereas resolution and sensitivity greatly degrade substantially near 210 K; the magnitude of this effect is greatest among the side chains of residues at the intermolecular interface of the microcrystal lattice, which we attribute to intermediate-rate translational diffusion of solvent molecules near the glass transition. These features of GB1 will enable its use as an excellent model protein not only for SSNMR methods development but also for fundamental studies of protein thermodynamics in the solid state.

Remarkable progress in solid-state NMR has enabled complete structure determination of uniformly labeled proteins in the size range of 5-10 kDa. Expanding these applications to larger or mass-limited systems requires further improvements in spectral sensitivity, for which inverse detection of 13C and 15N signals with 1H is one promising approach. Proton detection has previously been demonstrated to offer sensitivity benefits in the limit of sparse protonation or with approximately 30 kHz magic-angle spinning (MAS). Here we focus on experimental schemes for proteins with approximately 100% protonation. Full protonation simplifies sample preparation and permits more complete chemical shift information to be obtained from a single sample. We demonstrate experimental schemes using the fully protonated, uniformly 13C,15N-labeled protein GB1 at 40 kHz MAS rate with 1.6-mm rotors. At 500 MHz proton frequency, 1-ppm proton line widths were observed (500 +/- 150 Hz), and the sensitivity was enhanced by 3 and 4 times, respectively, versus direct 13C and 15N detection. The enhanced sensitivity enabled a family of 3D experiments for spectral assignment to be performed in a time-efficient manner with less than a micromole of protein. CANH, CONH, and NCAH 3D spectra provided sufficient resolution and sensitivity to make full backbone and partial side-chain proton assignments. At 750 MHz proton frequency and 40 kHz MAS rate, proton line widths improve further in an absolute sense (360 +/- 115 Hz). Sensitivity and resolution increase in a better than linear manner with increasing magnetic field, resulting in 14 times greater sensitivity for 1H detection relative to that of 15N detection.

High-sensitivity proton-detected experiments in solid-state NMR have been recently demonstrated in proton-diluted proteins as well as fully protonated samples under fast magic-angle spinning. One key element for performing successful proton detection is effective solvent suppression achieved by pulsed field gradients (PFG) and/or saturation pulses. Here we report a high-performance solvent suppression method that attenuates multiple solvent signals simultaneously by more than a factor of 10,000, achieved by an optimized combination of homospoil gradients and supercycled saturation pulses. This method, which we call Multiple Intense Solvent Suppression Intended for Sensitive Spectroscopic Investigation of Protonated Proteins, Instantly (MISSISSIPPI), can be applied without a PFG probe. It opens up new opportunities for two-dimensional heteronuclear correlation spectroscopy of hydrated proteins at natural abundance as well as high-sensitivity and multidimensional experimental investigation of protein-solvent interactions.

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α-Synuclein (AS) fibrils are the main protein component of Lewy Bodies, the pathological hallmark of Parkinson’s disease and other related disorders. AS forms helices that bind phospholipid membranes with high affinity, but no atomic level data for AS aggregation in the presence of lipids is yet available. Here, we present direct evidence of a conversion from α-helical conformation to β-sheet fibrils in the presence of anionic phospholipid vesicles and direct conversion to β-sheet fibrils in their absence. We have trapped intermediate states throughout the fibril formation pathways to examine the structural changes using solid-state NMR spectroscopy and electron microscopy. The comparison between mature AS fibrils formed in aqueous buffer and those derived in the presence of anionic phospholipids demonstrates no major changes in the overall fibril fold. However, a site-specific comparison of these fibrillar states demonstrates major perturbations in the N-terminal domain with a partial disruption of the long β-strand located in the 40’s and small perturbations in residues located in the “non-β amyloid component” (NAC) domain. Combining all these results, we propose a model for AS fibrillogenesis in the presence of phospholipid vesicles.

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Magische Methoden: Die Kombination von schneller Drehung im magischen Winkel, Isotopenverdünnung und hoher Magnetfeldstärke ergibt gut aufgelöste Festkörper‐1H‐NMR‐Spektren, die effizient eingesetzt wurden, um Proteinstrukturen zu lösen. Mit den neuen Techniken genügen nun drei Tage für die Datensammlung, die Zuordnung der Signale zu den Protonen und die Erstellung einer hochaufgelösten Struktur von mikrokristallinem GB1.

Solid-state NMR has emerged as an important tool for structural biology and chemistry, capable of solving atomic-resolution structures for proteins in membrane-bound and aggregated states. Proton detection methods have been recently realized under fast magic-angle spinning conditions, providing large sensitivity enhancements for efficient examination of uniformly labeled proteins. The first and often most challenging step of protein structure determination by NMR is the site-specific resonance assignment. Here we demonstrate resonance assignments based on high-sensitivity proton-detected three-dimensional experiments for samples of different physical states, including a fully-protonated small protein (GB1, 6 kDa), a deuterated microcrystalline protein (DsbA, 21 kDa), a membrane protein (DsbB, 20 kDa) prepared in a lipid environment, and the extended core of a fibrillar protein (α-synuclein, 14 kDa). In our implementation of these experiments, including CONH, CO(CA)NH, CANH, CA(CO)NH, CBCANH, and CBCA(CO)NH, dipolar-based polarization transfer methods have been chosen for optimal efficiency for relatively high protonation levels (full protonation or 100 % amide proton), fast magic-angle spinning conditions (40 kHz) and moderate proton decoupling power levels. Each H–N pair correlates exclusively to either intra- or inter-residue carbons, but not both, to maximize spectral resolution. Experiment time can be reduced by at least a factor of 10 by using proton detection in comparison to carbon detection. These high-sensitivity experiments are especially important for membrane proteins, which often have rather low expression yield. Proton-detection based experiments are expected to play an important role in accelerating protein structure elucidation by solid-state NMR with the improved sensitivity and resolution.