In the last two decades, solid-state nuclear magnetic resonance (ssNMR) spectroscopy has transformed from a spectroscopic technique investigating small molecules and industrial polymers to a potent tool decrypting structure and underlying dynamics of complex biological systems, such as membrane proteins, fibrils, and assemblies, in near-physiological environments and temperatures. This transformation can be ascribed to improvements in hardware design, sample preparation, pulsed methods, isotope labeling strategies, resolution, and sensitivity. The fundamental engagement between nuclear spins and radio-frequency pulses in the presence of a strong static magnetic field is identical between solution and ssNMR, but the experimental procedures vastly differ because of the absence of molecular tumbling in solids. This review discusses routinely employed state-of-the-art static and MAS pulsed NMR methods relevant for biological samples with rotational correlation times exceeding 100’s of nanoseconds. Recent developments in signal filtering approaches, proton methodologies, and multiple acquisition techniques to boost sensitivity and speed up data acquisition at fast MAS are also discussed. Several examples of protein structures (globular, membrane, fibrils, and assemblies) solved with ssNMR spectroscopy have been considered. We also discuss integrated approaches to structurally characterize challenging biological systems and some newly emanating subdisciplines in ssNMR spectroscopy.

Selective recoupling of protons (SERP) is a method to selectively and quantitatively measure magnetic dipole–dipole interaction between protons and, in turn, the proton–proton distance in solid-state samples at fast magic-angle spinning. We present a bimodal operator-based Floquet approach to describe the numerically optimized SERP recoupling sequence. The description calculates the allowed terms in the first-order effective Hamiltonian, explains the origin of selectivity during recoupling, and shows how different terms are modulated as a function of the radio frequency amplitude and the phase of the sequence. Analytical and numerical simulations have been used to evaluate the effect of higher-order terms and offsets on the polarization transfer efficiency and quantitative distance measurement. The experimentally measured 1H–1H distances on a fully protonated thymol sample are ∼10%–15% shorter than those reported from diffraction studies. A semi-quantitative model combined with extensive numerical simulations is used to rationalize the effect of the third-spin and the role of different parameters in the experimentally observed shorter distances. Measurements at high magnetic fields improve the match between experimental and diffraction distances. The measurement of 1H–1H couplings at offsets different from the SERP-offset has also been explored. Experiments were also performed on a perdeuterated ubiquitin sample to demonstrate the feasibility of simultaneously measuring multiple quantitative distances and to evaluate the accuracy of the measured distance in the absence of multispin effects. The estimation of proton–proton distances provides a boost to structural characterization of small pharmaceuticals and biomolecules, given that the positions of protons are generally not well defined in x-ray structures.

α-Synuclein (α-Syn) amyloid fibrils in synucleinopathies (such as Parkinson's disease (PD), multiple system atrophy (MSA)) are structurally and functionally different, reminiscent of prion-like strains. However, how a single protein can form different fibril polymorphs in various synucleinopathies is not known. Here, we demonstrate the structure-function relationship of two distinct α-Syn fibril polymorphs, the pre-matured fibrils (PMF) and helixmatured fibrils (HMF) based on α-Syn aggregation intermediates. These polymorphs not only display the structural differences, including their fibril core structure as demonstrated by solidstate nuclear magnetic resonance (NMR) spectroscopy and H/D-exchange coupled with mass spectrometry but also possess different cellular activities such as seeding, cellular internalization, and cell-to-cell transmission. The HMF with a compact core structure exhibits low seeding potency in cells but readily internalizes and transmits from one cell to another.Whereas the less structured PMF lacks the cell-to-cell transmission ability but induces abundant α-Syn pathology and triggers the formation of aggresomes in cells. Overall, the study highlights how the conformational heterogeneity in the aggregation pathway may lead to fibril polymorphs with distinct prion-like behavior in PD. IntroductionSynucleinopathies are the group of neurological disorders, which are characterized by the presence of intracellular inclusion bodies composed of α-synuclein (α-Syn) amyloid fibrils 1 . Although α-Syn amyloids in neuronal inclusions termed as Lewy bodies (LBs) and Lewy neurites (LNs) are the characteristic feature of Parkinson's disease (PD), the α-Syn inclusions in oligodendrocytes (glial cell inclusions; GCIs) are predominant in multiple system atrophy (MSA). Previous studies have suggested that amyloid fibrils associated with various neurodegenerative disorders are infectious and exhibit 'prion-like' behavior 2,3 . For example, exogenously added α-Syn fibrils readily internalize in cells and induce the aggregation of endogenous soluble α-Syn into pathogenic insoluble LB-like inclusions 4,5 . Moreover, the LB/LN-like inclusions are also observed in animals upon receiving intracerebral injections of synthetic α-Syn fibrils or brain homogenates from old transgenic (Tg) mice with α-Syn pathology 6,7 . This suggests prion-like cellular transmission and propagation of α-Syn amyloids in PD and related disorders 8 . In this context, it has been hypothesized that α-Syn assembles into polymorphs, which may account for distinct disease phenotypes observed amongst the synucleinopathies. α-Syn fibrils from GCIs in oligodendrocytes and LBs in neurons of diseased brain samples differ significantly in their structure and exhibit distinct seeding propensity 9 .Although α-Syn fibril polymorphs mostly differ in their fibril diameter, presence of twists, and the number of protofilaments 10,11 , they share a common cross-β-sheet structure with different packing and inter-protofilament interface 12,13 as shown by cryo-...

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