Misfolded α-synuclein amyloid fibrils are the principal components of Lewy bodies and neurites, hallmarks of Parkinson’s disease (PD). Here we present a high-resolution structure of an α-synuclein fibril, in a form that induces robust pathology in primary neuronal culture, determined by solid-state NMR spectroscopy and validated by electron microscopy and X-ray fiber diffraction. Over 200 unique long-range distance restraints define a consensus structure with common amyloid features including parallel in-register β-sheets and hydrophobic core residues, but also substantial complexity, arising from diverse structural features: an intermolecular salt bridge, a glutamine ladder, close backbone interactions involving small residues, and several steric zippers stabilizing a novel, orthogonal Greek-key topology. These characteristics contribute to the robust propagation of this fibril form, as evidenced by structural similarity of early-onset PD mutants. The structure provides a framework for understanding the interactions of α-synuclein with other proteins and small molecules to diagnose and treat PD.

Using a set of six 1H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5–30 kDa proteins. The approach relies on perdeuteration, amide 2H/1H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary 13C/15N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.

Amphotericin has remained the powerful but highly toxic last line of defense in treating life-threatening fungal infections in humans for over 50 years with minimal development of microbial resistance. Understanding how this small molecule kills yeast is thus critical for guiding development of derivatives with an improved therapeutic index and other resistance-refractory antimicrobial agents. In the widely accepted ion channel model for its mechanism of cytocidal action, amphotericin forms aggregates inside lipid bilayers that permeabilize and kill cells. In contrast, we report that amphotericin exists primarily in the form of large, extramembranous aggregates that kill yeast by extracting ergosterol from lipid bilayers. These findings reveal that extraction of a polyfunctional lipid underlies the resistance-refractory antimicrobial action of amphotericin and suggests a roadmap for separating its cytocidal and membrane-permeabilizing activities. This new mechanistic understanding is also guiding development of the first derivatives of amphotericin that kill yeast but not human cells.

α-Synuclein (AS) fibrils are the major component of Lewy bodies, the pathological hallmark of Parkinson’s disease (PD). Here, we use results from an extensive investigation employing solid-state NMR to present a detailed structural characterization and conformational dynamics quantification of full-length AS fibrils. Our results show that the core extends with a repeated structural motif. This result disagrees with the previously proposed fold of AS fibrils obtained with limited solid-state NMR data. Additionally, our results demonstrate that the three single point mutations associated with early-onset PD—A30P, E46K and A53T—are located in structured regions. We find that E46K and A53T mutations, located in rigid β-strands of the wild-type fibrils, are associated with major and minor structural perturbations, respectively.

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NMR chemical shift tensors (CSTs) in proteins, as well as their orientations, represent an important new restraint class for protein structure refinement and determination. Here, we present the first determination of both CST magnitudes and orientations for 13 Cα and 15 N (peptide backbone) groups in a protein, the β1 IgG binding domain of protein G from Streptococcus spp., GB1. Site-specific 13 Cα and 15 N CSTs were measured using synchronously evolved recoupling experiments in which 13 C and 15 N tensors were projected onto the 1 H-13 C and 1 H-15 N vectors, respectively, and onto the 15 N-13 C vector in the case of 13 Cα. The orientations of the 13 Cα CSTs to the 1 H-13 C and 13 C-15 N vectors agreed well with the results of ab initio calculations, with an rmsd of approximately 8°. In addition, the measured 15 N tensors exhibited larger reduced anisotropies in α-helical versus β-sheet regions, with very limited variation (18 AE 4°) in the orientation of the z-axis of the 15 N CST with respect to the 1 H-15 N vector. Incorporation of the 13 Cα CST restraints into structure calculations, in combination with isotropic chemical shifts, transferred echo double resonance 13 C-15 N distances and vector angle restraints, improved the backbone rmsd to 0.16 Å (PDB ID code 2LGI) and is consistent with existing X-ray structures (0.51 Å agreement with PDB ID code 2QMT). These results demonstrate that chemical shift tensors have considerable utility in protein structure refinement, with the best structures comparable to 1.0-Å crystal structures, based upon empirical metrics such as Ramachandran geometries and χ 1 ∕χ 2 distributions, providing solid-state NMR with a powerful tool for de novo structure determination.magic-angle spinning | dihedral angles | cross validation | nanocrystal | quantum chemistry T he chemical shift is an exquisite and powerful probe of molecular structure, deriving from the interaction of molecular orbitals with an external magnetic field, B 0 . Understanding the relationships between chemical shifts and protein structure has substantial implications for modern nuclear magnetic resonance (NMR) spectroscopy, chemistry, and structural biology (1-12). The chemical shift tensor (CST) is rich with information, even when two-thirds of it is averaged to zero by molecular tumbling in solution or magic-angle spinning (MAS) of solid samples. The remaining isotopic chemical shifts remain an excellent resource for structure determination and validation, and higher-order interactions of the CST have substantial contributions to NMR relaxation (13-19). Therefore, detailed knowledge of CSTs permits a precise analysis of motion (20)(21)(22). Solid-state NMR (SSNMR) of fully aligned samples exploits amide 15 N tensor information to determine the orientations of helices relative to the bilayer (23, 24). We have previously shown that use of a force field in which experimental 13 Cα CSTs are compared with ab initio CSTs [generated as a function of backbone conformation (ϕ, ψ)] significantly improves the precision and...

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Membranes play key regulatory roles in biological processes, with bilayer composition exerting marked effects on binding affinities and catalytic activities of a number of membrane-associated proteins. In particular, proteins involved in diverse processes such as vesicle fusion, intracellular signaling cascades, and blood coagulation interact specifically with anionic lipids such as phosphatidylserine (PS) in the presence of Ca 2+ ions. While Ca 2+ is suspected to induce PS clustering in mixed phospholipid bilayers, the detailed structural effects of this ion on anionic lipids are not established. In this study, combining magic angle spinning (MAS) solid-state NMR (SSNMR) measurements of isotopically labeled serine headgroups in mixed lipid bilayers with molecular dynamics (MD) simulations of PS lipid bilayers in the presence of different counterions, we provide site-resolved insights into the effects of Ca 2+ on the structure and dynamics of lipid bilayers. Ca 2+ -induced conformational changes of PS in mixed bilayers are observed in both liposomes and Nanodiscs, a nanoscale membrane-mimetic of bilayer patches. Site-resolved multidimensional correlation SSNMR spectra of bilayers containing 13 C, 15 Nlabeled PS demonstrate that Ca 2+ ions promote two major PS headgroup conformations, which are well resolved in two-dimensional 13 C-13 C, 15 N-13 C and 31 P-13 C spectra. The results of MD simulations performed on PS lipid bilayers in the presence or absence of Ca 2+ provide an atomic view of the conformational effects underlying the observed spectra.In healthy cells, phosphatidylserine (PS) resides on the inner leaflet of the plasma membrane (1) and represents 10-20% of all plasma membrane lipids (2,3). PS both imparts a negative surface potential for nonspecific binding of cationic proteins (4,5) and recruits several proteins through specific interactions, frequently involving Ca 2+ (6). Externalization of PS in activated platelets and apoptotic cells constitutes a signal eliciting coagulation and † This work was supported by the National Institute of General Medical Sciences, NIH (R01-GM075937 and R01-GM079530 to C.M.R., and R01-GM086749 and R01-GM067887 to E.T.), the National Center for Research Resources, NIH (P41-RR05969 to E.T.), the National Heart Lung and Blood Institute, NIH (R01 HL47014 to J.H.M. and R01 HL103999 to J.H.M. and C.M.R.), and by the American Heart Association (0920045G to R.D.H.). * To whom correspondence should be addressed: Chad Rienstra, Dept. of Chemistry, University of Illinois at Urbana-Champaign, 600 S Mathews Ave, Box 50-6, Urbana, IL 61801, Phone: 217-244-4655. Fax: 217-244-3186. rienstra@scs.uiuc.edu. # These two authors contributed equally to this work. NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2012 March 29. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript phagocytosis, respectively (7,8). It is well documented that relatively high concentrations of Ca 2+ can exert dramatic effects on membranes contain...