Methods for the determination of the structures and dynamics of proteins by solid-state NMR spectroscopy

Protein molecules perform a vast array of functions in living organisms and the characterisation of their structures and dynamics is a key step towards a full understanding of many biological processes. Magic angle spinning (MAS) solid-state NMR (SSNMR) spectroscopy has emerged as a uniquely powerful technique for the extraction of such information at atomic resolution, with mounting successes founded on continual developments in methodology and technology. In this thesis, a number of new approaches for probing the structures and dynamics of proteins are presented, towards the aim of overcoming current challenges regarding sensitivity, spectral resolution and a shortage of quantitative experimental observables. A streamlined method for simultaneously obtaining long-distance homonuclear (13C-13C) and heteronuclear (15N-13C) contacts is introduced that relies on the third spinassisted recoupling (TSAR) mechanism. The experiment, dubbed "time-shared TSAR" (TSTSAR), effectively doubles the information content of spectra and reduces the required experimental time to that needed for just one of the equivalent PAR or PAINCP experiments. An approach for the quantitative study of large proteins and complexes is presented, relying on a combination of proton detection at "ultrafast" (≥55 kHz) MAS frequencies, sample deuteration and optional paramagnetic doping. This is successfully employed for the characterisation of a >300 kDa precipitated complex of the protein GB1 with full length human immunoglobulin (IgG), with only a few nanomoles of sample. Recent advances in MAS technology have enabled spinning frequencies of 100 kHz and above to be obtained. Using the dipeptide β-Asp-Ala, it is found that under such conditions, protons lines are narrowed to an extent similar to that achievable using contemporary homonuclear decoupling methods, leading to a time-efficient method for obtaining resolved spectra of small, natural-abundance molecules. Similar experiments with a GB1-IgG complex sample confirm the technology’s applicability to non-model biological systems, despite the tiny rotor volume of 0.7 μL (≤3 nanomoles of complex). 15N R1ρ relaxation rates are measured for the same complex and compared with identical measurements in crystalline GB1, allowing for a direct comparison between the slow (ns-ms) dynamics of the protein in different molecular environments. Motions on this time scale are found to be more prevalent in the complex, possibly evidence of an overall collective molecular motion. An approach for the measurement of aliphatic 13C relaxation rates in fully protonated samples is presented, based on a combination of ultrafast MAS rates and alternately labelled samples. Sample spinning at ≥80 kHz enables resolved 13Cα-1H correlations, forming a base for 13Cα relaxation experiments that are subsequently performed on crystalline [1,3-13C,15N]GB1 and analysed using a simple model-free (SMF) treatment. It is noted that without further data, this analysis is likely inadequate for an accurate description of the dynamics of the protein. The measurement of 13C’ R1ρ relaxation rates at ultrafast MAS rates is introduced as a probe of backbone protein dynamics in fully protonated samples. 13C and 15N R1 and R1ρ relaxation rates are measured in crystalline [U-13C,15N]GB1 and analysed using the SMF formalism. An examination of simulated spectral densities rationalises the apparent inconsistencies that arise from this and reveals that motions in GB1 occur on at least two time scales. A combined 15N/13C extended model-free (EMF) analysis is conducted for peptide plane motions in GB1, whereupon it is found that the addition of 13C data helps to remove fitting artefacts present in a 15N-only analysis

Unraveling the complexity of protein backbone dynamics with combined 13C and 15N 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 13C 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 13C′ R1ρ rates in fully protonated crystalline protein GB1 at 600 and 850 MHz 1H Larmor frequencies and compare them to 13C′ R1, 15N 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 15N data is used alone. We also discuss how internal motions characterized by different time scales contribute to 15N and 13C 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

Huntingtin exon 1 fibrils feature an interdigitated β-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 intra- and inter-molecular contacts, backbone and side chain torsion angles, relaxation measurements, and calculations of chemical shifts. These 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 co-assembled 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. Weshow 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

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 (13C–13C) and heteronuclear (15N–13C) 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

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 b-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

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 1H-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

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

Data for 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