Structure of alpha-synuclein fibrils derived from human Lewy body dementia tissue.

The defining feature of Parkinson disease (PD) and Lewy body dementia (LBD) is the accumulation of alpha-synuclein (Asyn) fibrils in Lewy bodies and Lewy neurites. Here we develop and validate a method to amplify Asyn fibrils extracted from LBD postmortem tissue samples and use solid state nuclear magnetic resonance (SSNMR) studies to determine atomic resolution structure. Amplified LBD Asyn fibrils comprise a mixture of single protofilament and two protofilament fibrils with very low twist. The protofilament fold is highly similar to the fold determined by a recent cryo-electron microscopy study for a minority population of twisted single protofilament fibrils extracted from LBD tissue. These results expand the structural characterization of LBD Asyn fibrils and approaches for studying disease mechanisms, imaging agents and therapeutics targeting Asyn.

Solid-state NMR analysis of membrane proteins and protein aggregates by proton detected spectroscopy.

Solid-state NMR has emerged as an important tool for structural biology and chemistry, capable of solving atomic-resolution structures for proteins in membrane-bound and aggregated states. Proton detection methods have been recently realized under fast magic-angle spinning conditions, providing large sensitivity enhancements for efficient examination of uniformly labeled proteins. The first and often most challenging step of protein structure determination by NMR is the site-specific resonance assignment. Here we demonstrate resonance assignments based on high-sensitivity proton-detected three-dimensional experiments for samples of different physical states, including a fully-protonated small protein (GB1, 6 kDa), a deuterated microcrystalline protein (DsbA, 21 kDa), a membrane protein (DsbB, 20 kDa) prepared in a lipid environment, and the extended core of a fibrillar protein (α-synuclein, 14 kDa). In our implementation of these experiments, including CONH, CO(CA)NH, CANH, CA(CO)NH, CBCANH, and CBCA(CO)NH, dipolar-based polarization transfer methods have been chosen for optimal efficiency for relatively high protonation levels (full protonation or 100 % amide proton), fast magic-angle spinning conditions (40 kHz) and moderate proton decoupling power levels. Each H-N pair correlates exclusively to either intra- or inter-residue carbons, but not both, to maximize spectral resolution. Experiment time can be reduced by at least a factor of 10 by using proton detection in comparison to carbon detection. These high-sensitivity experiments are especially important for membrane proteins, which often have rather low expression yield. Proton-detection based experiments are expected to play an important role in accelerating protein structure elucidation by solid-state NMR with the improved sensitivity and resolution.

Solid-state NMR study of the charge-transfer complex between ubiquinone-8 and disulfide bond generating membrane protein DsbB.

Ubiquinone (Coenzyme Q) plays an important role in the mitochondrial respiratory chain and also acts as an antioxidant in its reduced form, protecting cellular membranes from peroxidation. De novo disulfide bond generation in the E. coli periplasm involves a transient complex consisting of DsbA, DsbB, and ubiquinone (UQ). It is hypothesized that a charge-transfer complex intermediate is formed between the UQ ring and the DsbB-C44 thiolate during the reoxidation of DsbA, which gives a distinctive ~500 nm absorbance band. No enzymological precedent exists for an UQ-protein thiolate charge-transfer complex, and definitive evidence of this unique reaction pathway for DsbB has not been fully demonstrated. In order to study the UQ-8-DsbB complex in the presence of native lipids, we have prepared isotopically labeled samples of precipitated DsbB (WT and C41S) with endogenous UQ-8 and lipids, and we have applied advanced multidimensional solid-state NMR methods. Double-quantum filter and dipolar dephasing experiments facilitated assignments of UQ isoprenoid chain resonances not previously observed and headgroup sites important for the characterization of the UQ redox states: methyls (~20 ppm), methoxys (~60 ppm), olefin carbons (120-140 ppm), and carbonyls (150-160 ppm). Upon increasing the DsbB(C41S) pH from 5.5 to 8.0, we observed a 10.8 ppm upfield shift for the UQ C1 and C4 carbonyls indicating an increase of electron density on the carbonyls. This observation is consistent with the deprotonation of the DsbB-C44 thiolate at pH 8.0 and provides direct evidence of the charge-transfer complex formation. A similar trend was noted for the UQ chemical shifts of the DsbA(C33S)-DsbB(WT) heterodimer, confirming that the charge-transfer complex is unperturbed by the DsbB(C41S) mutant used to mimic the intermediate state of the disulfide bond generating reaction pathway.

High-resolution membrane protein structure by joint calculations with solid-state NMR and X-ray experimental data.

X-ray diffraction and nuclear magnetic resonance spectroscopy (NMR) are the staple methods for revealing atomic structures of proteins. Since crystals of biomolecular assemblies and membrane proteins often diffract weakly and such large systems encroach upon the molecular tumbling limit of solution NMR, new methods are essential to extend structures of such systems to high resolution. Here we present a method that incorporates solid-state NMR restraints alongside of X-ray reflections to the conventional model building and refinement steps of structure calculations. Using the 3.7 Å crystal structure of the integral membrane protein complex DsbB-DsbA as a test case yielded a significantly improved backbone precision of 0.92 Å in the transmembrane region, a 58% enhancement from using X-ray reflections alone. Furthermore, addition of solid-state NMR restraints greatly improved the overall quality of the structure by promoting 22% of DsbB transmembrane residues into the most favored regions of Ramachandran space in comparison to the crystal structure. This method is widely applicable to any protein system where X-ray data are available, and is particularly useful for the study of weakly diffracting crystals.

High resolution NMR spectroscopy of nanocrystalline proteins at ultra-high magnetic field.

Magic-angle spinning (MAS) solid-state NMR (SSNMR) spectroscopy of uniformly-(13)C,(15)N labeled protein samples provides insight into atomic-resolution chemistry and structure. Data collection efficiency has advanced remarkably in the last decade; however, the study of larger proteins is still challenged by relatively low resolution in comparison to solution NMR. In this study, we present a systematic analysis of SSNMR protein spectra acquired at 11.7, 17.6 and 21.1 Tesla ((1)H frequencies of 500, 750, and 900 MHz). For two protein systems--GB1, a 6 kDa nanocrystalline protein and DsbA, a 21 kDa nanocrystalline protein--line narrowing is demonstrated in all spectral regions with increasing field. Resolution enhancement is greatest in the aliphatic region, including methine, methylene and methyl sites. The resolution for GB1 increases markedly as a function of field, and for DsbA, resolution in the C-C region increases by 42%, according to the number of peaks that can be uniquely picked and integrated in the 900 MHz spectra when compared to the 500 MHz spectra. Additionally, chemical exchange is uniquely observed in the highest field spectra for at least two isoleucine C delta 1 sites in DsbA. These results further illustrate the benefits of high-field MAS SSNMR spectroscopy for protein structural studies.

Membrane-bound diiron carboxylate proteins.

Four proteins have been identified recently as diiron carboxylate proteins on the basis of conservation of six amino acids (four carboxylate residues and two histidines) constituting an iron-binding motif. Unlike previously identified proteins with this motif, biochemical studies indicate that each of these proteins is membrane bound, although homology modeling rules out a transmembrane mode of binding. Therefore, the predicted structure of each protein [the alternative oxidase (AOX), the plastid terminal oxidase (PTOX), the diiron 5-demethoxyquinone hydroxylase (DMQ hydroxylase), and the aerobic Mg-protoporphyrin IX monomethylester hydroxylase (MME hydroxylase)] is that of a protein bound monotopically to one leaflet of the membrane bilayer. Three of these enzymes utilize a quinol substrate, with two oxidizing the quinol (AOX and PTOX) and one hydroxylating it (DMQ hydroxylase). MME hydroxylase is involved in synthesis of the isocyclic ring of chlorophyll. Two enzymes are involved in respiration (AOX and, indirectly, the diiron DMQ hydroxylase through ubiquinone biosynthesis) and two in photosynthesis, through their roles in carotenoid and chlorophyll biosynthesis (PTOX and MME hydroxylase, respectively). We discuss what is known about each enzyme as well as our expectations based on their identification as interfacially bound proteins with a diiron carboxylate active site.

Chemical shift assignment of the transmembrane helices of DsbB, a 20-kDa integral membrane enzyme, by 3D magic-angle spinning NMR spectroscopy.

The Escherichia coli inner membrane enzyme DsbB catalyzes disulfide bond formation in periplasmic proteins, by transferring electrons to ubiquinone from DsbA, which in turn directly oxidizes cysteines in substrate proteins. We have previously shown that DsbB can be prepared in a state that gives highly resolved magic-angle spinning (MAS) NMR spectra. Here we report sequential 13C and 15N chemical shift assignments for the majority of the residues in the transmembrane helices, achieved by three-dimensional (3D) correlation experiments on a uniformly 13C, 15N-labeled sample at 750-MHz 1H frequency. We also present a four-dimensional (4D) correlation spectrum, which confirms assignments in some highly congested regions of the 3D spectra. Overall, our results show the potential to assign larger membrane proteins using 3D and 4D correlation experiments and form the basis of further structural and dynamical studies of DsbB by MAS NMR.

High-yield expression and purification of isotopically labeled cytochrome P450 monooxygenases for solid-state NMR spectroscopy.

Cytochrome P450 monooxygenases (P450s), which represent the major group of drug metabolizing enzymes in humans, also catalyze important synthetic and detoxicative reactions in insects, plants and many microbes. Flexibilities in their catalytic sites and membrane associations are thought to play central roles in substrate binding and catalytic specificity. To date, Escherichia coli expression strategies for structural analysis of eukaryotic membrane-bound P450s by X-ray crystallography have necessitated full or partial removal of their N-terminal signal anchor domain and, often, replacement of residues more peripherally associated with the membrane (such as the F-G loop region). Even with these modifications, investigations of P450 structural flexibility remain challenging with multiple single crystal conditions needed to identify spatial variations between substrate-free and different substrate-bound forms. To overcome these limitations, we have developed methods for the efficient expression of 13C- and 15N-labeled P450s and analysis of their structures by magic-angle spinning solid-state NMR (SSNMR) spectroscopy. In the presence of co-expressed GroEL and GroES chaperones, full-length (53 kDa) Arabidopsis 13C,15N-labeled His4CYP98A3 is expressed at yields of 2-4 mg per liter of minimal media without the necessity of generating side chain modifications or N-terminal deletions. Precipitated His4CYP98A3 generates high quality SSNMR spectra consistent with a homogeneous, folded protein. These data highlight the potential of these methodologies to contribute to the structural analysis of membrane-bound proteins.

An efficient method and device for transfer of semisolid materials into solid-state NMR spectroscopy rotors.

The study of mass-limited biological samples by magic angle spinning (MAS) solid-state NMR spectroscopy critically relies upon the high-yield transfer of material from a biological preparation into the MAS rotor. This issue is particularly important for maintaining biological activity and hydration of semi-solid samples such as membrane proteins in lipid bilayers, pharmaceutical formulations, microcrystalline proteins and protein fibrils. Here we present protocols and designs for rotor-packing devices specifically suited for packing hydrated samples into Pencil-style 1.6 mm, 3.2 mm standard, and 3.2 mm limited speed MAS rotors. The devices are modular and therefore readily adaptable to other rotor and/or ultracentrifugation tube geometries.

Solid-state NMR structure of a pathogenic fibril of full-length human α-synuclein.

Misfolded α-synuclein amyloid fibrils are the principal components of Lewy bodies and neurites, hallmarks of Parkinson's disease (PD). 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 EM and X-ray fiber diffraction. Over 200 unique long-range distance restraints define a consensus structure with common amyloid features including parallel, in-register ß-sheets and hydrophobic-core residues, and with substantial complexity arising from diverse structural features including an intermolecular salt bridge, a glutamine ladder, close backbone interactions involving small residues, and several steric zippers stabilizing a new orthogonal Greek-key topology. These characteristics contribute to the robust propagation of this fibril form, as supported by the 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 aid in PD diagnosis and treatment.

Control of protein orientation on gold nanoparticles.

Gold nanoparticles (Au NPs) have attracted much attention due to their potential applications in nano-medicine. While numerous studies have quantified biomolecular adsorption to Au NPs in terms of equilibrium binding constants, far less is known about biomolecular orientation on nanoparticle surfaces. In this study, the binding of the protein α-synuclein to citrate and (16-mercaptohexadecyl) trimethylammonium bromide (MTAB) coated 12 nm Au NPs is examined by heteronuclear single quantum coherence NMR spectroscopy to provide site-specific measurements of protein-nanoparticle binding. Molecular dynamics simulations support the orientation assignments, which show N-terminus binding to the Au NP for citrate-capped NPs, and C-terminus binding for the MTAB-capped NPs.

Screening for functional expression and overexpression of a family of diiron-containing interfacial membrane proteins using the univector recombination system.

The large number of uncharacterized genes emerging from genome sequencing projects has resulted in a need for quick and reliable screening methods for protein expression parameters. We have utilized the univector plasmid recombination system (as previously reported) to develop a series of vectors for rapid screening for expression in Escherichia coli. A high level of recombinant protein expression is a requirement for purification of protein for structural determination and other purposes. In other applications, successful complementation of a missing enzyme activity in E. coli, as well as directed evolution studies and metabolic engineering, often require a much lower level of protein expression. In this report we describe the construction of a number of new pHOST vectors that can be screened for both low- and high-level expression. We isolated a mutant vector for MBP fusions that exhibited a more optimal level of expression for complementation of aerobic respiration in hemA(-) E. coli, our functional assay for the alternative oxidase. We then demonstrated the use of our system to rapidly screen for both optimal functional expression and optimal overexpression of the alternative oxidase as well as two other members of a family of membrane-bound diiron carboxylate proteins, the plastid terminal oxidase and 5-demethoxyquinone hydroxylase.

Solid-state NMR study of a 41 kDa membrane protein complex DsbA/DsbB.

The disulfide bond generation system in E. coli is led by a periplasmic protein, DsbA, and an integral membrane protein, DsbB. Here we present a solid-state NMR (SSNMR) study of a 41 kDa membrane protein complex DsbA/DsbB precipitated in the presence of native lipids to investigate conformational changes and dynamics that occur upon transient complex formation within the electron transfer pathway. Chemical shift changes in the periplasmic enzyme DsbA in three states (wild type, C33S mutant, and in complex with DsbB) reveal structural and/or dynamic information. We report a 4.9 ppm (15)N chemical shift change observed for Pro31 in the active site between the wild type and C33S mutant of DsbA. Additionally, the Pro31 residue remains elusive in the DsbA/DsbB complex, indicating that the dynamics change drastically in the active site between the three states of DsbA. Using three-dimensional SSNMR spectra, partial (13)C and (15)N de novo chemical shift assignments throughout DsbA in the DsbA/DsbB complex were compared with the shifts from DsbA alone to map site-specific chemical shift perturbations. These results demonstrate that there are further structural and dynamic changes of DsbA in the native membrane observed by SSNMR, beyond the differences between the crystal structures of DsbA and the DsbA/DsbB complex.

Structure of the disulfide bond generating membrane protein DsbB in the lipid bilayer.

The integral membrane protein DsbB in Escherichia coli is responsible for oxidizing the periplasmic protein DsbA, which forms disulfide bonds in substrate proteins. We have developed a high-resolution structural model by combining experimental X-ray and solid-state NMR with molecular dynamics (MD) simulations. We embedded the high-resolution DsbB structure, derived from the joint calculation with X-ray reflections and solid-state NMR restraints, into the lipid bilayer and performed MD simulations to provide a mechanistic view of DsbB function in the membrane. Further, we revealed the membrane topology of DsbB by selective proton spin diffusion experiments, which directly probe the correlations of DsbB with water and lipid acyl chains. NMR data also support the model of a flexible periplasmic loop and an interhelical hydrogen bond between Glu26 and Tyr153.

Solid-State NMR of a Large Membrane Protein by Paramagnetic Relaxation Enhancement.

Membrane proteins play an important role in many biological functions. Solid-state NMR spectroscopy is uniquely suited for studying structure and dynamics of membrane proteins in a membranous environment. The major challenge to obtain high quality solid-state NMR spectra of membrane proteins is sensitivity, due to limited quantities of labeled high-molecular-weight proteins. Here we demonstrate the incorporation of paramagnetic metal (Cu(2+)) ions, through either EDTA or a chelator lipid, into membrane protein samples for rapid data collection under fast magic-angle spinning (MAS) and low power (1)H decoupling. Spectral sensitivity of DsbB (20 kDa), an integral membrane protein, more than doubles in the same experimental time due to (1)H T(1) relaxation enhancement by Cu(2+) ions, with DsbB native fold and active site intact. This technique can be implemented to acquire multidimensional solid-state NMR spectra for chemical shift assignments and structure elucidation of large membrane proteins with small sample quantities.

Assignment strategies for large proteins by magic-angle spinning NMR: the 21-kDa disulfide-bond-forming enzyme DsbA.

We present strategies for chemical shift assignments of large proteins by magic-angle spinning solid-state NMR, using the 21-kDa disulfide-bond-forming enzyme DsbA as prototype. Previous studies have demonstrated that complete de novo assignments are possible for proteins up to approximately 17 kDa, and partial assignments have been performed for several larger proteins. Here we show that combinations of isotopic labeling strategies, high field correlation spectroscopy, and three-dimensional (3D) and four-dimensional (4D) backbone correlation experiments yield highly confident assignments for more than 90% of backbone resonances in DsbA. Samples were prepared as nanocrystalline precipitates by a dialysis procedure, resulting in heterogeneous linewidths below 0.2 ppm. Thus, high magnetic fields, selective decoupling pulse sequences, and sparse isotopic labeling all improved spectral resolution. Assignments by amino acid type were facilitated by particular combinations of pulse sequences and isotopic labeling; for example, transferred echo double resonance experiments enhanced sensitivity for Pro and Gly residues; [2-(13)C]glycerol labeling clarified Val, Ile, and Leu assignments; in-phase anti-phase correlation spectra enabled interpretation of otherwise crowded Glx/Asx side-chain regions; and 3D NCACX experiments on [2-(13)C]glycerol samples provided unique sets of aromatic (Phe, Tyr, and Trp) correlations. Together with high-sensitivity CANCOCA 4D experiments and CANCOCX 3D experiments, unambiguous backbone walks could be performed throughout the majority of the sequence. At 189 residues, DsbA represents the largest monomeric unit for which essentially complete solid-state NMR assignments have so far been achieved. These results will facilitate studies of nanocrystalline DsbA structure and dynamics and will enable analysis of its 41-kDa covalent complex with the membrane protein DsbB, for which we demonstrate a high-resolution two-dimensional (13)C-(13)C spectrum.

Partial (13)C and (15)N chemical-shift assignments of the disulfide-bond-forming enzyme DsbB by 3D magic-angle spinning NMR spectroscopy.

DsbB is a 20 kDa Escherichia coli inner-membrane protein that catalyzes disulfide-bond formation in periplasmic proteins. We report highly resolved, multidimensional magic-angle spinning NMR spectra at 750 MHz (1)H frequency, which enable partial (13)C and (15)N chemical-shift assignments of the signals. The narrow line widths observed indicate excellent microscopic order of the protein sample, suitable for full structure determination by solid-state NMR. Experiments were performed exclusively on uniformly (13)C,(15)N-labeled DsbB. Chemical-shift-correlation experiments based on dipolar transfer yielded strong signals in the 3D spectra, many of which have been site-specifically assigned to the four transmembrane helices of DsbB. Significant numbers of additional residues have been assigned to stretches of amino acids, although not yet placed in the amino acid sequence. We also report the temperature dependence of signal intensities from -50 degrees C to 0 degrees C, a range over which samples of DsbB are highly stable. Structural and dynamic information derived from SSNMR studies can give insight into DsbB in a state that so far has not been successfully crystallized.

EPR studies of the mitochondrial alternative oxidase. Evidence for a diiron carboxylate center.

The alternative oxidase (AOX) is a ubiquinol oxidase found in the mitochondrial respiratory chain of plants as well as some fungi and protists. It has been predicted to contain a coupled diiron center on the basis of a conserved sequence motif consisting of the proposed iron ligands, four glutamate and two histidine residues. However, this prediction has not been experimentally verified. Here we report the high level expression of the Arabidopsis thaliana alternative oxidase AOX1a as a maltose-binding protein fusion in Escherichia coli. Reduction and reoxidation of a sample of isolated E. coli membranes containing the alternative oxidase generated an EPR signal characteristic of a mixed-valent Fe(II)/Fe(III) binuclear iron center. The high anisotropy of the signal, the low value of the g-average tensor, and a small exchange coupling (-J) suggest that the iron center is hydroxo-bridged. A reduced membrane preparation yielded a parallel mode EPR signal with a g-value of about 15. In AOX containing a mutation of a putative glutamate ligand of the diiron center (E222A or E273A) the EPR signals are absent. These data provide evidence for an antiferromagnetic-coupled binuclear iron center, and together with the conserved sequence motif, identify the alternative oxidase as belonging to the growing family of diiron carboxylate proteins. The alternative oxidase is the first integral membrane protein in this family, and adds a new catalytic activity (ubiquinol oxidation) to this group of enzymatically diverse proteins.