1H line width dependence on MAS speed in solid state NMR - Comparison of experiment and simulation.

Recent developments in magic angle spinning (MAS) technology permit spinning frequencies of ≥100 kHz. We examine the effect of such fast MAS rates upon nuclear magnetic resonance proton line widths in the multi-spin system of ß-Asp-Ala crystal. We perform powder pattern simulations employing Fokker-Plank approach with periodic boundary conditions and 1H-chemical shift tensors calculated using the bond polarization theory. The theoretical predictions mirror well the experimental results. Both approaches demonstrate that homogeneous broadening has a linear-quadratic dependency on the inverse of the MAS spinning frequency and that, at the faster end of the spinning frequencies, the residual spectral line broadening becomes dominated by chemical shift distributions and susceptibility effects even for crystalline systems.

Characterization of Protein-Protein Interfaces in Large Complexes by Solid-State NMR Solvent Paramagnetic Relaxation Enhancements.

Solid-state NMR is becoming a viable alternative for obtaining information about structures and dynamics of large biomolecular complexes, including ones that are not accessible to other high-resolution biophysical techniques. In this context, methods for probing protein-protein interfaces at atomic resolution are highly desirable. Solvent paramagnetic relaxation enhancements (sPREs) proved to be a powerful method for probing protein-protein interfaces in large complexes in solution but have not been employed toward this goal in the solid state. We demonstrate that 1H and 15N relaxation-based sPREs provide a powerful tool for characterizing intermolecular interactions in large assemblies in the solid state. We present approaches for measuring sPREs in practically the entire range of magic angle spinning frequencies used for biomolecular studies and discuss their benefits and limitations. We validate the approach on crystalline GB1, with our experimental results in good agreement with theoretical predictions. Finally, we use sPREs to characterize protein-protein interfaces in the GB1 complex with immunoglobulin G (IgG). Our results suggest the potential existence of an additional binding site and provide new insights into GB1:IgG complex structure that amend and revise the current model available from studies with IgG fragments. We demonstrate sPREs as a practical, widely applicable, robust, and very sensitive technique for determining intermolecular interaction interfaces in large biomolecular complexes in the solid state.

Huntingtin exon 1 fibrils feature an interdigitated ß-hairpin-based polyglutamine core.

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 ß-hairpin-containing ß-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 ß-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.

Intermolecular Interactions and Protein Dynamics by Solid-State NMR Spectroscopy.

Understanding the dynamics of interacting proteins is a crucial step toward describing many biophysical processes. Here we investigate the backbone dynamics for protein GB1 in two different assemblies: crystalline GB1 and the precipitated GB1-antibody complex with a molecular weight of more than 300 kDa. We perform these measurements on samples containing as little as eight nanomoles of GB1. From measurements of site-specific (15) N relaxation rates including relaxation dispersion we obtain snapshots of dynamics spanning nine orders of magnitude in terms of the time scale. A comparison of measurements for GB1 in either environment reveals that while many of the dynamic features of the protein are conserved between them (in particular for the fast picosecond-nanosecond motions), much greater differences occur for slow motions with motions in the >500 ns range being more prevalent in the complex. The data suggest that GB1 can potentially undergo a small-amplitude overall anisotropic motion sampling the interaction interface in the complex.

Unraveling the complexity of protein backbone dynamics with combined (13)C and (15)N solid-state NMR relaxation measurements.

Typically, protein dynamics involve a complex hierarchy of motions occurring on different time scales between conformations separated by a range of different energy barriers. NMR relaxation can in principle provide a site-specific picture of both the time scales and amplitudes of these motions, but independent relaxation rates sensitive to fluctuations in different time scale ranges are required to obtain a faithful representation of the underlying dynamic complexity. This is especially pertinent for relaxation measurements in the solid state, which report on dynamics in a broader window of time scales by more than 3 orders of magnitudes compared to solution NMR relaxation. To aid in unraveling the intricacies of biomolecular dynamics we introduce (13)C spin-lattice relaxation in the rotating frame (R1ρ) as a probe of backbone nanosecond-microsecond motions in proteins in the solid state. We present measurements of (13)C'R1ρ rates in fully protonated crystalline protein GB1 at 600 and 850 MHz (1)H Larmor frequencies and compare them to (13)C'R1, (15)N R1 and R1ρ measured under the same conditions. The addition of carbon relaxation data to the model free analysis of nitrogen relaxation data leads to greatly improved characterization of time scales of protein backbone motions, minimizing the occurrence of fitting artifacts that may be present when (15)N data is used alone. We also discuss how internal motions characterized by different time scales contribute to (15)N and (13)C relaxation rates in the solid state and solution state, leading to fundamental differences between them, as well as phenomena such as underestimation of picosecond-range motions in the solid state and nanosecond-range motions in solution.

Intermolecular Interactions and Protein Dynamics by Solid-State NMR Spectroscopy.

Understanding the dynamics of interacting proteins is a crucial step toward describing many biophysical processes. Here we investigate the backbone dynamics for protein GB1 in two different assemblies: crystalline GB1 and the precipitated GB1-antibody complex with a molecular weight of more than 300 kDa. We perform these measurements on samples containing as little as eight nanomoles of GB1. From measurements of site-specific 15N relaxation rates including relaxation dispersion we obtain snapshots of dynamics spanning nine orders of magnitude in terms of the time scale. A comparison of measurements for GB1 in either environment reveals that while many of the dynamic features of the protein are conserved between them (in particular for the fast picosecond-nanosecond motions), much greater differences occur for slow motions with motions in the >500 ns range being more prevalent in the complex. The data suggest that GB1 can potentially undergo a small-amplitude overall anisotropic motion sampling the interaction interface in the complex.

Solid-state NMR of a protein in a precipitated complex with a full-length antibody.

NMR spectroscopy is a prime technique for characterizing atomic-resolution structures and dynamics of biomolecular complexes but for such systems faces challenges of sensitivity and spectral resolution. We demonstrate that the application of (1)H-detected experiments at magic-angle spinning frequencies of >50 kHz enables the recording, in a matter of minutes to hours, of solid-state NMR spectra suitable for quantitative analysis of protein complexes present in quantities as small as a few nanomoles (tens of micrograms for the observed component). This approach enables direct structure determination and quantitative dynamics measurements in domains of protein complexes with masses of hundreds of kilodaltons. Protein-protein interaction interfaces can be mapped out by comparison of the chemical shifts of proteins within solid-state complexes with those of the same constituent proteins free in solution. We employed this methodology to characterize a >300 kDa complex of GB1 with full-length human immunoglobulin, where we found that sample preparation by simple precipitation yields spectra of exceptional quality, a feature that is likely to be shared with some other precipitating complexes. Finally, we investigated extensions of our methodology to spinning frequencies of up to 100 kHz.

Simultaneous acquisition of homonuclear and heteronuclear long-distance contacts with time-shared third spin assisted recoupling.

We present a time-shared Third Spin Assisted Recoupling (TSTSAR) experiment that allows for simultaneous acquisition of homonuclear ((13)C-(13)C) and heteronuclear ((15)N-(13)C) long-distance contacts in biomolecular solids under magic angle spinning. TSTSAR leads to substantial time savings and increases the information content of 2D correlation spectra.