Correction to: Characterization of fibril dynamics on three timescales by solid-state NMR.

In our recent publication (Smith et al., J Biomol NMR 65:171-191, 2016) on the dynamics of HET-s(218-289), we reported on page 176, that calculation of solid-state NMR R1ρ rate constants using analytical equations based on Redfield theory (Kurbanov et al., J Chem Phys 135:184104:184101-184109, 2011) failed when the correlation time of motion becomes too long.

Partially-deuterated samples of HET-s(218-289) fibrils: assignment and deuterium isotope effect.

Fast magic-angle spinning and partial sample deuteration allows direct detection of 1H in solid-state NMR, yielding significant gains in mass sensitivity. In order to further analyze the spectra, 1H detection requires assignment of the 1H resonances. In this work, resonance assignments of backbone HN and Hα are presented for HET-s(218-289) fibrils, based on the existing assignment of Cα, Cß, C', and N resonances. The samples used are partially deuterated for higher spectral resolution, and the shifts in resonance frequencies of Cα and Cß due to the deuterium isotope effect are investigated. It is shown that the deuterium isotope effect can be estimated and used for assigning resonances of deuterated samples in solid-state NMR, based on known resonances of the protonated protein.

Characterization of fibril dynamics on three timescales by solid-state NMR.

A multi-timescale analysis of the backbone dynamics of HET-s (218-289) fibrils is described based on multiple site-specific R 1 and R 1ρ data sets and S (2) measurements via REDOR for most backbone (15)N and (13)Cα nuclei. (15)N and (13)Cα data are fitted with motions at three timescales. Slow motion is found, indicating a global fibril motion. We further investigate the effect of (13)C-(13)C transfer in measurement of (13)Cα R 1. Finally, we show that it is necessary to go beyond the Redfield approximation for slow motions in order to obtain accurate numerical values for R 1ρ.

De†novo 3D structure determination from sub-milligram protein samples by solid-state 100†kHz MAS NMR spectroscopy.

Solid-state NMR spectroscopy is an emerging tool for structural studies of crystalline, membrane-associated, sedimented, and fibrillar proteins. A major limitation for many studies is still the large amount of sample needed for the experiments, typically several isotopically labeled samples of 10-20 mg each. Here we show that a new NMR probe, pushing magic-angle sample rotation to frequencies around 100 kHz, makes it possible to narrow the proton resonance lines sufficiently to provide the necessary sensitivity and spectral resolution for efficient and sensitive proton detection. Using restraints from such spectra, a well-defined de novo structure of the model protein ubiquitin was obtained from two samples of roughly 500 µg protein each. This proof of principle opens new avenues for structural studies of proteins available in microgram, or tens of nanomoles, quantities that are, for example, typically achieved for eukaryotic membrane proteins by in-cell or cell-free expression.

Influence of dsDNA fragment length on particle binding in an evanescent field biosensing system.

Particle labels are widely used in affinity-based biosensing due to the high detection signal per label, the high stability, and the convenient biofunctionalization of particles. In this paper we address the question how the time-course of particle binding and the resulting signals depend on the length of captured target molecules. As a model system we used fragments of dsDNA with lengths of 105 bp (36 nm), 290 bp (99 nm) and 590 bp (201 nm), detected in an evanescent-field optomagnetic biosensing system. On both ends the fragments were provided with small-molecule tags to allow binding of the fragments to protein-coated particles and to the capture molecules at the sensor surface. For isolated single particles bound to the surface, we observe that the optical scattering signal per particle depends only weakly on the fragment length, which we attribute to the pivoting motion that allows the particles to get closer to the surface. Our data show a strong influence of the fragment length on the particle binding: the binding rate of particles to the sensor surface is an order of magnitude higher for the longest dsDNA fragments compared to the smallest fragment studied in this paper. We attribute the enhanced binding rate to the length and motional freedom of the fragments. These results generate a new dimension for the design of assays and systems in particle-based biosensing.