Quantification of Rapid Cooling of Glycerol/Water Solutions Based on Photoluminescence from Thioflavin T.

Rapid cooling to a solid state allows intermediates in chemical and biomolecular processes that occur in solution near room temperature to be trapped for subsequent measurements by magnetic resonance spectroscopies, electron microscopy, or other techniques. In time-resolved solid state nuclear magnetic resonance and rapid freeze-quench electron paramagnetic resonance studies, solutions are typically frozen by spraying into a cold bath or onto a cold metal surface. Although simulations suggest freezing on millisecond or submillisecond time scales, direct experimental measurements of cooling rates have been elusive.

Conformations of a low-complexity protein in homogeneous and phase-separated frozen solutions.

Solutions of the intrinsically disordered, low-complexity domain of the FUS protein (FUS-LC) undergo liquid-liquid phase separation (LLPS) below a temperature T. To investigate whether local conformational distributions are detectably different in the homogeneous (i.e.

Optimization of N-C double-resonance NMR experiments under low temperature magic angle spinning dynamic nuclear polarization conditions.

Dynamic nuclear polarization (DNP) enhanced magic angle spinning (MAS) solid-state NMR carried out at 25 K enables rapid acquisition of multi-dimensional C-N correlation spectra for protein structure studies and resonance assignment. Under commonly used DNP conditions, solvent deuteration reduces H-N cross polarization (CP) efficiencies, necessitates more careful optimization, and requires longer high-power N radio-frequency pulses. The sensitivity of 2D heteronuclear correlation experiments is potentially impaired.

Conformations of a Low-Complexity Protein in Homogeneous and Phase-Separated Frozen Solutions.

Unlabelled: Solutions of the intrinsically disordered, low-complexity domain of the FUS protein (FUS-LC) undergo liquid-liquid phase separation (LLPS) below temperatures T in the 20-40° C range. To investigate whether local conformational distributions are detectably different in the homogeneous and phase-separated states of FUS-LC, we performed solid state nuclear magnetic resonance (ssNMR) measurements on solutions that were frozen on sub-millisecond time scales after equilibration at temperatures well above (50° C) or well below (4° C) T. Measurements were performed at 25 K with signal enhancements from dynamic nuclear polarization.

Structure of Amyloid Peptide Ribbons Characterized by Electron Microscopy, Atomic Force Microscopy, and Solid-State Nuclear Magnetic Resonance.

Polypeptides often self-assemble to form amyloid fibrils, which contain cross-β structural motifs and are typically 5-15 nm in width and micrometers in length. In many cases, short segments of longer amyloid-forming protein or peptide sequences also form cross-β assemblies but with distinctive ribbon-like morphologies that are characterized by a well-defined thickness (on the order of 5 nm) in one lateral dimension and a variable width (typically 10-100 nm) in the other. Here, we use a novel combination of data from solid-state nuclear magnetic resonance (ssNMR), dark-field transmission electron microscopy (TEM), atomic force microscopy (AFM), and cryogenic electron microscopy (cryoEM) to investigate the structures within amyloid ribbons formed by residues 14-23 and residues 11-25 of the Alzheimer's disease-associated amyloid-β peptide (Aβ and Aβ).