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Several studies recently showed that ex vivo fibrils from patient or animal tissue were structurally different from in vitro formed fibrils from the same polypeptide chain. Analysis of serum amyloid A (SAA) and Aβ-derived amyloid fibrils additionally revealed that ex vivo fibrils were more protease stable than in vitro fibrils. These observations gave rise to the proteolytic selection hypothesis that suggested that disease-associated amyloid fibrils were selected inside the body by their ability to resist endogenous clearance mechanisms. We here show, for more than twenty different fibril samples, that ex vivo fibrils are more protease stable than in vitro fibrils. These data support the idea of a proteolytic selection of pathogenic amyloid fibril morphologies and help to explain why only few amino acid sequences lead to amyloid diseases, although many, if not all, polypeptide chains can form amyloid fibrils in vitro.

Positron emission tomography (PET) tracer molecules like thioflavin T specifically recognize amyloid deposition in brain tissue by selective binding to hydrophobic or aromatic surface grooves on the β‐sheet surface along the fibril axis. The molecular basis of this interaction is, however, not well understood. We have employed magic angle spinning (MAS) solid‐state NMR spectroscopy to characterize Aβ‐PET tracer complexes at atomic resolution. We established a titration protocol by using bovine serum albumin as a carrier to transfer hydrophobic small molecules to Aβ(1‐40) fibrillar aggregates. The same Aβ(1‐40) amyloid fibril sample was employed in subsequent titrations to minimize systematic errors that potentially arise from sample preparation. In the experiments, the small molecules 13C‐methylated Pittsburgh compound B (PiB) as well as a novel Aβ tracer based on a diarylbithiazole (DABTA) scaffold were employed. Classical 13C‐detected as well as proton‐detected spectra of protonated and perdeuterated samples with back‐substituted protons, respectively, were acquired and analyzed. After titration of the tracers, chemical‐shift perturbations were observed in the loop region involving residues Gly25‐Lys28 and Ile32‐Gly33, thus suggesting that the PET tracer molecules interact with the loop region connecting β‐sheets β1 and β2 in Aβ fibrils. We found that titration of the PiB derivatives suppressed fibril polymorphism and stabilized the amyloid fibril structure., Binding PET tracer molecules to amyloid fibrils: PET tracers specifically recognize amyloid deposition in neurodegeneration, although the molecular basis of this interaction is not well understood. MAS solid‐state NMR spectroscopy is used to characterize Aβ‐PET tracer complexes. A bovine serum albumin protocol enables the titration of the hydrophobic molecules.

The treatment of invasive drug-resistant and potentially life-threatening fungal infections is limited to few therapeutic options that are usually associated with severe side effects. The development of new effective antimycotics with a more tolerable side effect profile is therefore of utmost clinical importance. Here, we used a combination of complementary in vitro assays and structural analytical methods to analyze the interaction of the de novo antimicrobial peptide VG16KRKP with the sterol moieties of biological cell membranes. We demonstrate that VG16KRKP disturbs the structural integrity of fungal membranes both invitro and in model membrane system containing ergosterol along with phosphatidylethanolamine lipid and exhibits broad-spectrum antifungal activity. As revealed by systematic structure-function analysis of mutated VG16KRKP analogs, a specific pattern of basic and hydrophobic amino acid side chains in the primary peptide sequence determines the selectivity of VG16KRKP for fungal specific membranes.

Human amylin forms structurally heterogeneous amyloids that have been linked to type-2 diabetes. Thus, understanding the molecular interactions governing amylin aggregation can provide mechanistic insights in its pathogenic formation. Here, we demonstrate that fibril formation of amylin is altered by synthetic amphipathic copolymer derivatives of the styrene- maleic-acid (SMAQA and SMAEA). High-speed AFM is used to follow the real-time aggregation of amylin by observing the rapid formation of de novo globular oligomers and arrestment of fibrillation by the positively-charged SMAQA. We also observed an accelerated fibril formation in the presence of the negatively-charged SMAEA. These findings were further validated by fluorescence, SOFAST-HMQC, DOSY and STD NMR experiments. Conformational analysis by CD and FT-IR revealed that the SMA copolymers modulate the conformation of amylin aggregates. While the species formed with SMAQA are α-helical, the ones formed with SMAEA are rich in β-sheet structure. The interacting interfaces between SMAEA or SMAQA and amylin are mapped by NMR and microseconds all-atom MD simulation. SMAEA displayed π-π interaction with Phe23, electrostatic π-cation interaction with His18 and hydrophobic packing with Ala13 and Val17; whereas SMAQA showed a selective interaction with amylin’s C-terminus (residues 31-37) that belongs to one of the two β-sheet regions (residues 14-19 and 31-36) involved in amylin fibrillation. Toxicity analysis showed both SMA copolymers to be non-toxic in vitro and the amylin species formed with the copolymers showed minimal deformity to zebrafish embryos. Together, this study demonstrates that chemical tools, such as copolymers, can be used to modulate amylin aggregation, alter the conformation of species.

We recently introduced RAP (reduced adjoining protonation) labelling as an easy to implement and cost-effective strategy to yield selectively methyl protonated protein samples. We show here that even though the amount of H2O employed in the bacterial growth medium is rather low, the intensities obtained in MAS solid-state NMR 1H,13C correlation spectra are comparable to spectra obtained for samples in which α-ketoisovalerate was employed as precursor. In addition to correlations for Leu and Val residues, RAP labelled samples yield also resonances for all methyl containing side chains. The labelling scheme has been employed to quantify order parameters, together with the respective asymmetry parameters. We obtain a very good correlation between the order parameters measured using a GlcRAP (glucose carbon source) and a α-ketoisovalerate labelled sample. The labelling scheme holds the potential to be very useful for the collection of long-range distance restraints among side chain atoms. Experiments are demonstrated using RAP and α-ketoisovalerate labelled samples of the α-spectrin SH3 domain, and are applied to fibrils formed from the Alzheimer’s disease Aβ1-40 peptide.

The aggregation of antibody light chains is linked to systemic light chain (AL) amyloidosis, a disease where amyloid deposits frequently affect the heart and the kidney. We here investigate fibrils from the λ-III FOR005 light chain (LC), which is derived from an AL-patient with severe cardiac involvement. In FOR005, five residues are mutated with respect to its closest germline gene segment IGLV3-19 and IGLJ3. All mutations are located close to the complementarity determining regions (CDRs). The sequence segments responsible for the fibril formation are not yet known. We use fibrils extracted from the heart of this particular amyloidosis patient as seeds to prepare fibrils for solid-state NMR. We show that the seeds induce the formation of a specific fibril structure from the biochemically produced protein. We have assigned the fibril core region of the FOR005-derived fibrils and characterized the secondary structure propensity of the observed amino acids. As the primary structure of the aggregated patient protein is different for every AL patient, it is important to study, analyze and report a greater number of light chain sequences associated with AL amyloidosis.

In this study, the effect of CurDAc, a water-soluble curcumin derivative, on the formation and stability of amyloid fibers is revealed. CurDAc interaction with amyloid is structurally selective, which is reflected in a strong interference with hIAPP aggregation while showing weaker interactions with human-calcitonin and amyloid-β(1–40) in comparison. Remarkably, CurDAc also exhibited potent fiber disaggregation for hIAPP generating a toxic oligomeric species.

Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we study the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that reflect the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt β-sheet conformation in patient protein fibrils. We identify glycine-49 that is mutated with respect to the germline sequence into arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity for the C-terminal residues 80-102 which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra show 13Cα chemical shifts that are highly similar to patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C-terminus in the fibril state, while the overall fibril topology is retained.

Systemic antibody light chains (AL) amyloidosis is characterized by deposition of amyloid fibrils derived from a particular antibody light chain. Cardiac involvement is a major risk factor for mortality. Using MAS solid-state NMR, we studied the fibril structure of a recombinant light chain fragment corresponding to the fibril protein from patient FOR005, together with fibrils formed by protein sequence variants that are derived from the closest germline (GL) sequence. Both analyzed fibril structures were seeded with ex-vivo amyloid fibrils purified from the explanted heart of this patient. We find that residues 11-42 and 69-102 adopt β-sheet conformation in patient protein fibrils. We identify arginine-49 as a key residue that forms a salt bridge to aspartate-25 in the patient protein fibril structure. In the germline sequence, this residue is replaced by a glycine. Fibrils from the GL protein and from the patient protein harboring the single point mutation R49G can be both heterologously seeded using patient ex-vivo fibrils. Seeded R49G fibrils show an increased heterogeneity in the C-terminal residues 80-102, which is reflected by the disappearance of all resonances of these residues. By contrast, residues 11-42 and 69-77, which are visible in the MAS solid-state NMR spectra, show 13Cα chemical shifts that are highly like patient fibrils. The mutation R49G thus induces a conformational heterogeneity at the C terminus in the fibril state, whereas the overall fibril topology is retained. These findings imply that patient mutations in FOR005 can stabilize the fibril structure.

The tumor suppressor p16INK4A induces cell cycle arrest and senescence in response to oncogenic transformation and is therefore frequently lost in cancer. p16INK4A is also known to accumulate under conditions of oxidative stress. Thus, we hypothesized it could potentially be regulated by reversible oxidation of cysteines (redox signaling). Here we report that oxidation of the single cysteine in p16INK4A in human cells occurs under relatively mild oxidizing conditions and leads to disulfide-dependent dimerization. p16INK4A is an all α-helical protein, but we find that upon cysteine-dependent dimerization, p16INK4A undergoes a dramatic structural rearrangement and forms aggregates that have the typical features of amyloid fibrils, including binding of diagnostic dyes, presence of cross-β sheet structure, and typical dimensions found in electron microscopy. p16INK4A amyloid formation abolishes its function as a Cyclin Dependent Kinase 4/6 inhibitor. Collectively, these observations mechanistically link the cellular redox state to the inactivation of p16INK4A through the formation of amyloid fibrils., Graphical abstract Image 1, Highlights • The only cysteine in human p16INK4A is prone to oxidation in vitro and in vivo. • p16INK4A forms disulfide-dependent homodimers upon oxidation. • The all α-helical p16INK4A protein aggregates as β-amyloids upon oxidation. • Oxidation and subsequent β-amyloid formation impair p16INK4A function.

Despite their importance for antibody architecture and design, the principles governing antibody domain stability are still not understood in sufficient detail. Here, to address this question, we chose a domain from the invariant part of IgG, the C(H)2 domain. We found that compared with other Ig domains, the isolated C(H)2 domain is a surprisingly unstable monomer, exhibiting a melting temperature of ∼44 °C. We further show that the presence of an additional C-terminal lysine in a C(H)2 variant substantially increases the melting temperature by ∼14 °C relative to C(H)2 WT. To explore the molecular mechanism of this effect, we employed biophysical approaches to probe structural features of C(H)2. The results revealed that Lys(101) is key for the formation of three secondary structure elements: the very C-terminal β-strand and two adjacent α-helices. We also noted that a dipole interaction between Lys(101) and the nearby α-helix, is important for stabilizing the C(H)2 architecture by protecting the hydrophobic core. Interestingly, this interaction between the α-helix and C-terminal charged residues is highly conserved in antibody domains, suggesting that it represents a general mechanism for maintaining their integrity. We conclude that the observed interactions involving terminal residues have practical applications for defining domain boundaries in the development of antibody therapeutics and diagnostics.

The small heat shock protein αA-crystallin is a molecular chaperone important for the optical properties of the vertebrate eye lens. It forms heterogeneous oligomeric ensembles. We determined the structures of human αA-crystallin oligomers by combining cryo-electron microscopy, cross-linking/mass spectrometry, NMR spectroscopy and molecular modeling. The different oligomers can be interconverted by the addition or subtraction of tetramers, leading to mainly 12-, 16- and 20-meric assemblies in which interactions between N-terminal regions are important. Cross-dimer domain-swapping of the C-terminal region is a determinant of αA-crystallin heterogeneity. Human αA-crystallin contains two cysteines, which can form an intramolecular disulfide in vivo. Oxidation in vitro requires conformational changes and oligomer dissociation. The oxidized oligomers, which are larger than reduced αA-crystallin and destabilized against unfolding, are active chaperones and can transfer the disulfide to destabilized substrate proteins. The insight into the structure and function of αA-crystallin provides a basis for understanding its role in the eye lens. Oligomers of human αA-crystallin are characterized structurally via a hybrid approach, combining cryo-EM, cross-linking/mass spectrometry, NMR and modeling, providing insight into their dynamic behavior and heterogeneity and revealing that oxidized oligomers can also act as chaperones.

In this study, the effect of CurDAc, a water-soluble curcumin derivative, on the formation and stability of amyloid fibers is revealed. CurDAc interaction with amyloid is structurally selective which is reflected in a strong interference with hIAPP aggregation, while showing weaker interactions with human-calcitonin and Amyloid-β1-40 in comparison. Remarkably, CurDAc also exhibited potent fiber disaggregation for hIAPP generating a toxic oligomeric species.

Designed peptides derived from the islet amyloid polypeptide (IAPP) cross‐amyloid interaction surface with Aβ (termed interaction surface mimics or ISMs) have been shown to be highly potent inhibitors of Aβ amyloid self‐assembly. However, the molecular mechanism of their function is not well understood. Using solution‐state and solid‐state NMR spectroscopy in combination with ensemble‐averaged dynamics simulations and other biophysical methods including TEM, fluorescence spectroscopy and microscopy, and DLS, we characterize ISM structural preferences and interactions. We find that the ISM peptide R3‐GI is highly dynamic, can adopt a β‐like structure, and oligomerizes into colloid‐like assemblies in a process that is reminiscent of liquid–liquid phase separation (LLPS). Our results suggest that such assemblies yield multivalent surfaces for interactions with Aβ40. Sequestration of substrates into these colloid‐like structures provides a mechanistic basis for ISM function and the design of novel potent anti‐amyloid molecules., ISM inhibitors are highly dynamic assemblies and exchange between monomeric and high‐molecular‐weight states. In both states, the ISM peptide adopts a β‐loop‐like structure that provides a suitable surface for sequestration of Aβ40 in the colloidal state. ISM inhibitors thus exploit multivalency by self‐association to yield high substrate avidity.

Using NMR to probe transient binding of Aβ(1–40) monomers to fibers, we find partially bound conformations with the highest degree of interaction near F19-K28 and a lesser degree of interaction near the C-terminus (L34-G37). This represents a shift away from the KLVFFA recognition sequence (residues 16–21) currently used for inhibitor design.

Polymorphism is a key feature of amyloid fibril structures but it remains challenging to explain these variations for a particular sample. Here, we report electron cryomicroscopy-based reconstructions from different fibril morphologies formed by a peptide fragment from an amyloidogenic immunoglobulin light chain. The observed fibril morphologies vary in the number and cross-sectional arrangement of a structurally conserved building block. A comparison with the theoretically possible constellations reveals the experimentally observed spectrum of fibril morphologies to be governed by opposing sets of forces that primarily arise from the β-sheet twist, as well as peptide–peptide interactions within the fibril cross-section. Our results provide a framework for rationalizing and predicting the structure and polymorphism of cross-β fibrils, and suggest that a small number of physical parameters control the observed fibril architectures., Amyloid fibril structures can display polymorphism. Here the authors reveal the cryo-EM structures of several different fibril morphologies of a peptide derived from an amyloidogenic immunoglobulin light chain and present a mathematical analysis of physical factors that influence fibril polymorphism.

A novel role for S100B, a recognized brain distress marker, as a chaperone-like suppressor of Aβ42 aggregation and toxicity., Amyloid-β (Aβ) aggregation and neuroinflammation are consistent features in Alzheimer’s disease (AD) and strong candidates for the initiation of neurodegeneration. S100B is one of the most abundant proinflammatory proteins that is chronically up-regulated in AD and is found associated with senile plaques. This recognized biomarker for brain distress may, thus, play roles in amyloid aggregation which remain to be determined. We report a novel role for the neuronal S100B protein as suppressor of Aβ42 aggregation and toxicity. We determined the structural details of the interaction between monomeric Aβ42 and S100B, which is favored by calcium binding to S100B, possibly involving conformational switching of disordered Aβ42 into an α-helical conformer, which locks aggregation. From nuclear magnetic resonance experiments, we show that this dynamic interaction occurs at a promiscuous peptide-binding region within the interfacial cleft of the S100B homodimer. This physical interaction is coupled to a functional role in the inhibition of Aβ42 aggregation and toxicity and is tuned by calcium binding to S100B. S100B delays the onset of Aβ42 aggregation by interacting with Aβ42 monomers inhibiting primary nucleation, and the calcium-bound state substantially affects secondary nucleation by inhibiting fibril surface–catalyzed reactions through S100B binding to growing Aβ42 oligomers and fibrils. S100B protects cells from Aβ42-mediated toxicity, rescuing cell viability and decreasing apoptosis induced by Aβ42 in cell cultures. Together, our findings suggest that molecular targeting of S100B could be translated into development of novel approaches to ameliorate AD neurodegeneration.

The antibody light chain (LC) consists of two domains and is essential for antigen binding in mature immunoglobulins. The two domains are connected by a highly conserved linker that comprises the structurally important Arg108 residue. In antibody light chain (AL) amyloidosis, a severe protein amyloid disease, the LC and its N-terminal variable domain (V-L) convert to fibrils deposited in the tissues causing organ failure. Understanding the factors shaping the architecture of the LC is important for basic science, biotechnology and for deciphering the principles that lead to fibril formation. In this study, we examined the structure and properties of LC variants with a mutated or extended linker. We show that under destabilizing conditions, the linker modulates the amyloidogenicity of the LC. The fibril formation propensity of LC linker variants and their susceptibility to proteolysis directly correlate implying an interplay between the two LC domains. Using NMR and residual dipolar coupling-based simulations, we found that the linker residue Arg108 is a key factor regulating the relative orientation of the VL and CL domains, keeping them in a bent and dense, but still flexible conformation. Thus, inter-domain contacts and the relative orientation of VL and CL to each other are of major importance for maintaining the structural integrity of the full-length LC. (C) 2018 Elsevier Ltd. All rights reserved.

Solid-state nuclear magnetic resonance (NMR) has recently emerged as a method of choice to study structural and dynamic properties of large biomolecular complexes at atomic resolution. Indeed, recent technological and methodological developments have enabled the study of ever more complex systems in the solid-state. However, to explore multicomponent protein complexes by NMR, specific labeling schemes need to be developed that are dependent on the biological question to be answered. We show here how to reconstitute an isotopically labeled protein within the unlabeled 50S or 70S ribosomal subunit. In particular, we focus on the 63-residue ribosomal protein L29 (~7 kDa), which is located at the exit of the tunnel of the large 50S ribosomal subunit (~1.5 MDa). The aim of this work is the preparation of a suitable sample to investigate allosteric conformational changes in a ribosomal protein that are induced by the nascent polypeptide chain and that trigger the interaction with different chaperones (e.g., trigger factor or SRP).

The design of inhibitors of protein-protein interactions mediating amyloid self-assembly is a major challenge mainly due to the dynamic nature of the involved structures and interfaces. Interactions of amyloidogenic polypeptides with other proteins are important modulators of self-assembly. Here we present a hot-segment-linking approach to design a series of mimics of the IAPP cross-amyloid interaction surface with Aβ (ISMs) as nanomolar inhibitors of amyloidogenesis and cytotoxicity of Aβ, IAPP, or both polypeptides. The nature of the linker determines ISM structure and inhibitory function including both potency and target selectivity. Importantly, ISMs effectively suppress both self- and cross-seeded IAPP self-assembly. Our results provide a novel class of highly potent peptide leads for targeting protein aggregation in Alzheimer's disease, type 2 diabetes, or both diseases and a chemical approach to inhibit amyloid self-assembly and pathogenic interactions of other proteins as well.

Type II diabetes (T2D) is characterized by diminished insulin production and resistance of cells to insulin. Among others, endoplasmic reticulum (ER) stress is a principal factor contributing to T2D and induces a shift towards a more reducing cellular environment. At the same time, peripheral insulin resistance triggers the over-production of regulatory hormones such as insulin and human islet amyloid polypeptide (hIAPP). We show that the differential aggregation of reduced and oxidized hIAPP assists to maintain the redox equilibrium by restoring redox equivalents. Aggregation thus induces redox balancing which can assist initially to counteract ER stress. Failure of the protein degradation machinery might finally result in β-cell disruption and cell death. We further present a structural characterization of hIAPP in solution, demonstrating that the N-terminus of the oxidized peptide has a high propensity to form an α-helical structure which is lacking in the reduced state of hIAPP. In healthy cells, this residual structure prevents the conversion into amyloidogenic aggregates.

Antibody light chain amyloidosis is a rare disease caused by fibril formation of secreted immunoglobulin light chains (LCs). The huge variety of antibody sequences puts a serious challenge to drug discovery. The green tea polyphenol epigallocatechin-3-gallate (EGCG) is known to interfere with fibril formation in general. Here we present solution- and solid-state NMR studies as well as MD simulations to characterise the interaction of EGCG with LC variable domains. We identified two distinct EGCG binding sites, both of which include a proline as an important recognition element. The binding sites were confirmed by site-directed mutagenesis and solid-state NMR analysis. The EGCG-induced protein complexes are unstructured. We propose a general mechanistic model for EGCG binding to a conserved site in LCs. We find that EGCG reacts selectively with amyloidogenic mutants. This makes this compound a promising lead structure, that can handle the immense sequence variability of antibody LCs.

Membrane-assisted amyloid formation is implicated in human diseases, and many of the aggregating species accelerate amyloid formation and induce cell death. While structures of membrane-associated intermediates would provide tremendous insights into the pathology and aid in the design of compounds to potentially treat the diseases, it has not been feasible to overcome the challenges posed by the cell membrane. Here, we use NMR experimental constraints to solve the structure of a type-2 diabetes related human islet amyloid polypeptide intermediate stabilized in nanodiscs. ROSETTA and MD simulations resulted in a unique β-strand structure distinct from the conventional amyloid β-hairpin and revealed that the nucleating NFGAIL region remains flexible and accessible within this isolated intermediate, suggesting a mechanism by which membrane-associated aggregation may be propagated. The ability of nanodiscs to trap amyloid intermediates as demonstrated could become one of the most powerful approaches to dissect the complicated misfolding pathways of protein aggregation.

Co-translational protein folding is not yet well understood despite the availability of high-resolution ribosome crystal structures. We present first solid-state NMR data on non-mobile regions of a prokaryotic ribosomal complex. Localized chemical shift perturbations and line broadening are observed for the backbone amide resonances corresponding to the regions in the trigger factor ribosome-binding domain that are involved in direct contact with the ribosome or undergo conformational changes upon ribosome binding. This large asymmetric protein complex (1.4 MDa) becomes accessible for NMR investigations by the combined use of proton detection and high MAS frequencies (60 kHz). The presented results open new perspectives for the understanding of the mechanism of large molecular machineries.

The green tea compound epigallocatechin-3-gallate (EGCG) inhibits Alzheimer's disease β-amyloid peptide (Aβ) neurotoxicity. Solution-state NMR allows probing initial EGCG-Aβ interactions. We show that EGCG-induced Aβ oligomers adopt a well-defined structure and are amenable for magic angle spinning solid-state NMR investigations. We find that EGCG interferes with the aromatic hydrophobic core of Aβ. The C-terminal part of the Aβ peptide (residues 22-39) adopts a β-sheet conformation, whereas the N-terminus (residues 1-20) is unstructured. The characteristic salt bridge involving residues D23 and K28 is present in the structure of these oligomeric Aβ aggregates as well. The structural analysis of small-molecule-induced amyloid aggregates will open new perspectives for Alzheimer's disease drug development.

High resolution proton spectra are obtained in MAS solid-state NMR in case samples are prepared using perdeuterated protein and D 2 O in the recrystallization buffer. Deuteration reduces drastically 1 H, 1 H dipolar interactions and allows to obtain amide proton line widths on the order of 20 Hz. Similarly, high-resolution proton spectra of aliphatic groups can be obtained if specifically labeled precursors for biosynthesis of methyl containing side chains are used, or if limited amounts of H 2 O in the bacterial growth medium is employed. This review summarizes recent spectroscopic developments to access structure and dynamics of biomacromolecules in the solid-state, and shows a number of applications to amyloid fibrils and membrane proteins.

NMR studies involving perdeuterated proteins focus in general on exchangeable amide protons. However, non-exchangeable sites contain as well a small amount of protons as the employed precursors for protein biosynthesis are not completely proton depleted. The degree of methyl group protonation is in the order of 9% for CD 2 H using >97% deuterium enriched glucose. We show in this manuscript that this small amount of residual protonation is sufficient to perform 2D and 3D MAS solid-state NMR experiments. In particular, we suggest a HCCH-TOBSY type experiment which we successfully employ to assign the methyl resonances in aliphatic side chains in a perdeuterated sample of the SH3 domain of chicken α-spectrin.

Assignment of proteins in MAS (magic angle spinning) solid-state NMR relies so far on correlations among heteronuclei. This strategy is based on well dispersed resonances in the (15)N dimension. In many complex cases like membrane proteins or amyloid fibrils, an additional frequency dimension is desirable in order to spread the amide resonances. We show here that proton detected HNCO, HNCA, and HNCACB type experiments can successfully be implemented in the solid-state. Coherences are sufficiently long lived to allow pulse schemes of a duration greater than 70 ms before incrementation of the first indirect dimension. The achieved resolution is comparable to the resolution obtained in solution-state NMR experiments. We demonstrate the experiments using a triply labeled sample of the SH3 domain of chicken alpha-spectrin, which was re-crystallized in H(2)O/D(2)O using a ratio of 1/9. We employ paramagnetic relaxation enhancement (PRE) using EDTA chelated Cu(II) to enable rapid data acquisition.

We found previously by fluorescence resonance energy transfer experiments that amyloid precursor protein (APP) homodimerizes in living cells. APP homodimerization is likely to be mediated by two sites of the ectodomain and a third site within the transmembrane sequence of APP. We have now investigated the role of the N-terminal growth factor-like domain in APP dimerization by NMR, biochemical, and cell biological approaches. Under nonreducing conditions, the N-terminal domain of APP formed SDS-labile and SDS-stable complexes. The presence of SDS was sufficient to convert native APP dimers entirely into monomers. Addition of an excess of a synthetic peptide (APP residues 91-116) containing the disulfide bridge-stabilized loop inhibited cross-linking of pre-existing SDS-labile APP ectodomain dimers. Surface plasmon resonance analysis revealed that this peptide specifically bound to the N-terminal domain of APP and that binding was entirely dependent on the oxidation of the thiol groups. By solution-state NMR we detected small chemical shift changes indicating that the loop peptide interacted with a large protein surface rather than binding to a defined pocket. Finally, we studied the effect of the loop peptide added to the medium of living cells. Whereas the levels of alpha-secretory APP increased, soluble beta-cleaved APP levels decreased. Because Abeta40 and Abeta42 decreased to similar levels as soluble beta-cleaved APP, we conclude either that beta-secretase binding to APP was impaired or that the peptide allosterically affected APP processing. We suggest that APP acquires a loop-mediated homodimeric state that is further stabilized by interactions of hydrophobic residues of neighboring domains.

Previously, Ishii et al., could show that chelated paramagnetic ions can be employed to significantly decrease the recycle delay of a MAS solid-state NMR experiment [N.P. Wickramasinghe, M. Kotecha, A. Samoson, J. Past, Y. Ishii, Sensitivity enhancement in C-13 solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing H-1 T-1 relaxation, J. Magn. Reson. 184 (2007) 350–356]. Application of the method is limited to very robust samples, for which sample stability is not compromised by RF induced heating. In addition, probe integrity might be perturbed in standard MAS PRE experiments due to the use of very short duty cycles. We show that these deleterious effects can be avoided if perdeuterated proteins are employed that have been re-crystallized from D2O:H2O = 9:1 containing buffer solutions. The experiments are demonstrated using the SH3 domain of chicken a-spectrin as a model system. The labeling scheme allows to record proton detected 1 H, 15 N correlation spectra with very high resolution in the absence of heteronuclear dipolar decoupling. Cu–edta as a doping reagent yields a reduction of the recycle delay by up to a factor of 15. In particular, we find that the 1 H T1 for the bulk H N magnetization is reduced from 4.4 s to 0.3 s if the Cu–edta concentration is increased from 0 mM to 250 mM. Possible perturbations like chemical shift changes or line broadening due to the paramagnetic chelate complex are minimal. No degradation of our samples was observed in the course of the experiments. 2007 Elsevier Inc. All rights reserved.

International audience; Small heat-shock proteins, including αB-crystallin (αB), play an important part in protein homeostasis, because their ATP-independent chaperone activity inhibits uncontrolled protein aggregation. Mechanistic details of human αB, particularly in its client-bound state, have been elusive so far, owing to the high molecular weight and the heterogeneity of these complexes. Here we provide structural insights into this highly dynamic assembly and show, by using state-of-the-art NMR spectroscopy, that the αB complex is assembled from asymmetric building blocks. Interaction studies demonstrated that the fibril-forming Alzheimer's disease Aβ1-40 peptide preferentially binds to a hydrophobic edge of the central β-sandwich of αB. In contrast, the amorphously aggregating client lysozyme is captured by the partially disordered N-terminal domain of αB. We suggest that αB uses its inherent structural plasticity to expose distinct binding interfaces and thus interact with a wide range of structurally variable clients.

The aggregation of mostly antibody light chain variable (VL) domains into amyloid fibrils in various tissues is the main cause of death in systemic amyloid light chain amyloidosis. Point mutations within the domain are important to shift the VL into the fibrillar pathway, but why and how only some site-specific mutations achieve this still remains elusive. We show here that both destabilizing and surprisingly stable mutants readily predispose an amyloid-resistant VL domain to amyloid formation. The decreased thermodynamic stability of the destabilizing mutant results in the accumulation of non-native intermediates that readily populate the amyloid state. Interestingly, the stable mutants establish site-specific non-native interactions with especially nearby serine/threonine residues that unexpectedly do not affect the folding behavior of the VL domain but rather readily induce and stabilize the fibril structure, a previously unrecognized mechanism. These findings provide a new concept for the molecular mechanism of amyloid fibril formation.

Alzheimer disease is the most severe neurodegenerative disease worldwide. In the past years, a plethora of small molecules interfering with amyloid-β (Aβ) aggregation has been reported. However, their mode of interaction with amyloid fibers is not understood. Non-steroidal anti-inflammatory drugs (NSAIDs) are known γ-secretase modulators; they influence Aβ populations. It has been suggested that NSAIDs are pleiotrophic and can interact with more than one pathomechanism. Here we present a magic angle spinning solid-state NMR study demonstrating that the NSAID sulindac sulfide interacts specifically with Alzheimer disease Aβ fibrils. We find that sulindac sulfide does not induce drastic architectural changes in the fibrillar structure but intercalates between the two β-strands of the amyloid fibril and binds to hydrophobic cavities, which are found consistently in all analyzed structures. The characteristic Asp(23)-Lys(28) salt bridge is not affected upon interacting with sulindac sulfide. The primary binding site is located in the vicinity of residue Gly(33), a residue involved in Met(35) oxidation. The results presented here will assist the search for pharmacologically active molecules that can potentially be employed as lead structures to guide the design of small molecules for the treatment of Alzheimer disease.

The association of light chains (LCs) and heavy chains is the basis for functional antibodies that are essential for adaptive immune responses. However, in some cases, LCs and especially fragments consisting of the LC variable (VL) domain are pathologically deposited in fatal aggregation diseases. The two domains of the LC are connected by a highly conserved linker. We show here that, unexpectedly, the linker residue Arg108 affects the conformational stability and folding of both VLκ and LC constant (CLκ) domains. Interestingly, the extension of VL by Arg108 results in its resistance to amyloid formation, which suggests that the nature of the truncation of the LC plays a crucial role in disease progression. Increased solvation due to the exposed charged C-terminal Arg108 residue explains its stabilizing effects on the VL domain. For the CL domain, the interaction of N-terminal loop residues with Arg108 is important for the integrity of the domain, as the disruption of this interaction results in fluctuation, partial opening of the protein's interior and the exposure of hydrophobic residues that destabilize the domain. This establishes new principles for antibody domain architecture and amyloidogenicity.

We present in this paper 2H,13C MAS correlation experiments that are performed on a uniformly 2H,13C,15N labeled sample of Nac-Val, and on the uniformly 2H,15N labeled dipeptide Nac-Val-Leu-OH. The experiments involve the measurement of 2H T1 relaxation times at two different magnetic fields, as well as the measurement of the 2H tensor parameters by evolution of the 2H chemical shift. The data are interpreted quantitatively to differentiate between different side chain motional models.

In this Communication, we demonstrate the use of deuteration together with back substitution of exchangeable protons as a means of attenuating the strong 1H-1H couplings that broaden 1H magic angle spinning (MAS) spectra of solids. The approach facilitates 15N-1H correlation experiments as well as experiments for the measurement of 1H-1H distances. The distance measurement relies on the excellent resolution in the 1H MAS spectrum and homonuclear double quantum recoupling techniques. The 1H-1H dipolar recoupling can be analyzed in an analytical fashion by fitting the data to a 2- or 3-spin system. The experiments are performed on a sample of the dipeptide N-Ac-Val-Leu-OH, which was synthesized from uniformly [2H, 15N] labeled materials and back-exchanged in H2O.

In vitro protein-folding studies using chemical denaturants such as urea are indispensible in elucidating the forces and mechanisms determining the stability, structure, and dynamics of water-soluble proteins. By contrast, α-helical membrane-associated proteins largely evade such approaches because they are resilient to extensive unfolding. We have used optical and NMR spectroscopy to provide an atomistic-level dissection of the effects of urea on the structure and dynamics of the α-helical membrane-associated protein Mistic as well as its interactions with detergent and solvent molecules. In the presence of the zwitterionic detergent lauryl dimethylamine oxide, increasing concentrations of urea result in a complex sequence of conformational changes that go beyond simple two-state unfolding. Exploiting this finding, we report the first high-resolution structural models of the urea denaturation process of an α-helical membrane-associated protein and its completely unfolded state, which contains almost no regular secondary structure but nevertheless retains a topology close to that of the folded state.

In a recent study we described the second periplasmic loop P2 of the transmembrane protein MalF (MalF-P2) of the maltose ATP-binding cassette transporter (MalFGK(2)-E) as an important element in the recognition of substrate by the maltose-binding protein MalE. In this study, we focus on MalE and find that MalE undergoes a structural rearrangement after addition of MalF-P2. Analysis of residual dipolar couplings (RDCs) shows that binding of MalF-P2 induces a semiopen state of MalE in the presence and absence of maltose, whereas maltose is retained in the binding pocket. These data are in agreement with paramagnetic relaxation enhancement experiments. After addition of MalF-P2, an increased solvent accessibility for residues in the vicinity of the maltose-binding site of MalE is observed. MalF-P2 is thus not only responsible for substrate recognition, but also directly involved in activation of substrate transport. The observation that substrate-bound and substrate-free MalE in the presence of MalF-P2 adopts a similar semiopen state hints at the origin of the futile ATP hydrolysis of MalFGK(2)-E.

The human small heat-shock protein αB-crystallin (αB) rescues misfolded proteins from irreversible aggregation during cellular stress. Binding of Cu(II) was shown to modulate the oligomeric architecture and the chaperone activity of αB. However, the mechanistic basis of this stimulation is so far not understood. We provide here first structural insights into this Cu(II)-mediated modulation of chaperone function using NMR spectroscopy and other biophysical approaches. We show that the α-crystallin domain is the elementary Cu(II)-binding unit specifically coordinating one Cu(II) ion with picomolar binding affinity. Putative Cu(II) ligands are His(83), His(104), His(111), and Asp(109) at the dimer interface. These loop residues are conserved among different metazoans, but also for human αA-crystallin, HSP20, and HSP27. The involvement of Asp(109) has direct implications for dimer stability, because this residue forms a salt bridge with the disease-related Arg(120) of the neighboring monomer. Furthermore, we observe structural reorganization of strands β2-β3 triggered by Cu(II) binding. This N-terminal region is known to mediate both the intermolecular arrangement in αB oligomers and the binding of client proteins. In the presence of Cu(II), the size and the heterogeneity of αB multimers are increased. At the same time, Cu(II) increases the chaperone activity of αB toward the lens-specific protein β(L)-crystallin. We therefore suggest that Cu(II) binding unblocks potential client binding sites and alters quaternary dynamics of both the dimeric building block as well as the higher order assemblies of αB.

We study the uniformly 13C,15N isotopically enriched Escherichia coli multidrug resistance transporter EmrE using MAS solid-state NMR. Solid-state NMR can provide complementary structural information as the method allows studying membrane proteins in their native environment as no detergent is required for reconstitution. We compare the spectra obtained from wildtype EmrE to those obtained from the mutant EmrE-E14C. To resolve the critical amino acid E14, glutamic/aspartic acid selective experiments are carried out. These experiments allow to assign the chemical shift of the carboxylic carbon of E14. In addition, spectra are analyzed which are obtained in the presence and absence of the ligand TPP+.

Molecular chaperones are a group of proteins that bind transiently to nascent polypeptide chains and are thought to function by preventing aggregation by maintaining polypeptides in conformations competent for folding and subunit assembly. On the other hand, specific chaperones are considered to be involved in the mechanism of prion propagation and others are found to colocalize in plaques of patients suffering from neurodegenerative diseases. Solution-state NMR spectroscopy can provide insight into the interactions between the misfolding protein and the chaperone at atomic resolution. In particular, experimental results for Sup35, a yeast prion protein, and β-amyloid, which is responsible for Alzheimer's disease, and their interactions with Hsp104 and αB-crystallin, respectively, are discussed.

In analogy to human prions, a domain of the translation-termination protein in Saccharomyces cerevisiae, Sup35, can switch its conformation from a soluble functional state, [psi-], to a conformation, [PSI+], that facilitates aggregation and impairs its native function. Overexpression of the molecular chaperone Hsp104 abolishes the [PSI+] phenotype and restores the normal function of Sup35. We have recently shown that Hsp104 interacts preferably with low oligomeric species of a Sup35 derived peptide, Sup35[5-26]; however, due to possible exchange between different oligomeric states, it was not possible to obtain information on the distribution and stability of the oligomeric state. We show here, that low-molecular-weight oligomers (Sup35[5-26])n (n approximately = 4-6) are indeed important for the fibril formation and disassembly process. We find that Hsp104 is able to disaggregate Sup35[5-26] fibrils by substraction of hexameric to decameric Sup35[5-26] oligomers. This disaggregation effect does not require assistance from other chaperones and is independent of ATP at high Hsp104 concentrations. Furthermore, we demonstrate that critical oligomers have a preference for alpha-helical conformations. The conformational reorganization into beta-sheet structures seems to occur only upon incorporation of these oligomers into fibrillar structures. The results are demonstrated by using an equilibrium dialysis experiment that employed different molecular-weight cut-off membranes. A combination of thioflavin-T (ThT) fluorescence and UV measurements allowed the quantification of fibril formation and the amount of peptide diffusing out of the dialysis bag. CD and NMR spectroscopy data were combined to obtain structural information.

Polymerization of the soluble beta-amyloid peptide into highly ordered fibrils is hypothesized to be a causative event in the development of Alzheimer's disease. Understanding the interactions of Abeta with inhibitors on an atomic level is fundamental for the development of diagnostics and therapeutic approaches, and can provide, in addition, important indirect information of the amyloid fibril structure. We have shown recently that trRDCs can be measured in solution state NMR for peptide ligands binding weakly to amyloid fibrils. We present here the structures for two inhibitor peptides, LPFFD and DPFFL, and their structural models bound to fibrillar Abeta(14-23) and Abeta(1-40) based on transferred nuclear Overhauser effect (trNOE) and transferred residual dipolar coupling (trRDC) data. In a first step, the inhibitor peptide structure is calculated on the basis of trNOE data; the trRDC data are then validated on the basis of the trNOE-derived structure using the program PALES. The orientation of the peptide inhibitors with respect to Abeta fibrils is obtained from trRDC data, assuming that Abeta fibrils orient such that the fibril axis is aligned in parallel with the magnetic field. The trRDC-derived alignment tensor of the peptide ligand is then used as a restraint for molecular dynamics docking studies. We find that the structure with the lowest rmsd value is in agreement with a model in which the inhibitor peptide binds to the long side of an amyloid fibril. Especially, we detect interactions involving the hydrophobic core, residues K16 and E22/D23 of the Abeta sequence. Structural differences are observed for binding of the inhibitor peptide to Abeta14-23 and Abeta1-40 fibrils, respectively, indicating different fibril structure. We expect this approach to be useful in the rational design of amyloid ligands with improved binding characteristics.

Spectral resolution imposes a major problem on the evaluation of MAS solid-state NMR experiments as larger biomolecular systems are concerned. We show in this communication that decoupling of the (13)C-(13)C homonuclear scalar couplings during stroboscopic detection can be successfully applied to increase the spectral resolution up to a factor of 2-2.5 and sensitivity up to a factor of 1.2. We expect that this approach will be useful for the study of large biomolecular systems like membrane proteins and amyloidogenic peptides and proteins where spectral overlap is critical. The experiments are demonstrated on a uniformly (13)C,(15)N-labelled sample of Nac-Val-Leu-OH and applied to a uniformly (13)C,(15)N-enriched sample of a hexameric amyloidogenic peptide.

The [ PSI + ] determinant of Saccharomyces cerevisiae , consisting of the cytosolic translation termination factor Sup35, is a prion-type genetic element that induces an inheritable conformational change and converts the Sup35 protein into amyloid fibers. The molecular chaperone Hsp104 is required to maintain self-replication of [ PSI + ]. We observe in vitro that addition of catalytic amounts of Hsp104 to the prion-determining region of the NM domain of Sup35, Sup35 5–26 , results in the dissociation of oligomeric Sup35 into monomeric species. Several intermediates of Sup35 5–26 could be detected during this process. Strong interactions are found between Hsp104 and hexameric/tetrameric Sup35 5–26 , whereas the intermediate and monomeric “release” forms show a decreased affinity with respect to Hsp104, as monitored by saturation transfer difference and diffusion-ordered NMR spectroscopic experiments. Interactions are mediated mostly by the side chains of Gln, Asn, and Tyr residues in Sup35 5–26 . No interaction can be detected between Hsp104 and higher oligomeric states (≥8) of Sup35 5–26 . Taking into account the fact that Hsp104 is required for maintenance of [ PSI + ], we suggest that low-oligomeric-weight species of Sup35 are important for prion propagation in yeast.

We describe new correlation experiments suitable for determining long-range 1H-1H distances in 2H,15N-labeled peptides and proteins. The approach uses perdeuteration together with back substitution of exchangeable protons during sample preparation as a means of attenuating the strong 1H-1H dipolar couplings that broaden 1H magic angle spinning (MAS) spectra of solids. In the approach described here, we retain 100% of the 1H sensitivity by labeling and detecting all exchangeable sites. This is in contrast to homonuclear multiple pulse decoupling sequences that are applied during detection and that compromise sensitivity because of the requirement of sampling between pulses. As a result 1H detection provides a gain in sensitivity of5 compared to the 15N detected version of the experiment (at a MAS frequency of 13.5kHz). The pulse schemes make use of the favorable dispersion of the amide 15Ns resonances in the protein backbone. The experiments are demonstrated on a sample of the uniformly 2H,15N-labeled dipeptide N-Ac-Val-Leu-OH and are analogous to the solution-state suite of HSQC-NOESY experiments. In this compound the 1H amide linewidths at 750MHz vary from approximately 0.67 ppm at omega(r)/2pi approximately 5kHz to approximately 0.20 ppm at omega(r)/2pi approximately 30kHz, indicating that useful resolution is available in the 1H spectrum via this approach. Since the experiments circumvent the problem of dipolar truncation in the 1H-1H spin system, they should make it possible to measure long-range distances in a uniformly labeled environment. Thus, we expect the experiments to be useful in constraining the global fold of a protein.

We present a novel solid-state magic angle-spinning NMR method for measuring the NH(i)-NH(i+1) projection angletheta;(i,i+1) in peptides. The experiment is applicable to uniformly (15)N-labeled peptides and is demonstrated on the chemotactic tripeptide N-formyl-l-Met-l-Leu-l-Phe. The projection angletheta;(i,i+1) is directly related to the peptide backbone torsion anglesphi;(i) and psi(i). The method utilizes the T-MREV recoupling scheme to restore (15)N-(1)H interactions, and proton-mediated spin diffusion to establish (15)N-(15)N correlations. T-MREV has recently been shown to increase the dynamic range of the (15)N-(1)H recoupling by gamma-encoding, and permits an accurate determination of the recoupled NH dipolar interaction. The results are interpreted in a quasi-analytical fashion that permits efficient extraction of the structural parameters.