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Simultaneous analysis of oligomer and fibril assembly kinetics reveals inhibitory effects of metastable oligomers on amyloid fibril formation., Assembly of rigid amyloid fibrils with their characteristic cross-β sheet structure is a molecular signature of numerous neurodegenerative and non-neuropathic disorders. Frequently large populations of small globular amyloid oligomers (gOs) and curvilinear fibrils (CFs) precede the formation of late-stage rigid fibrils (RFs), and have been implicated in amyloid toxicity. Yet our understanding of the origin of these metastable oligomers, their role as on-pathway precursors or off-pathway competitors, and their effects on the self-assembly of amyloid fibrils remains incomplete. Using two unrelated amyloid proteins, amyloid-β and lysozyme, we find that gO/CF formation, analogous to micelle formation by surfactants, is delineated by a “critical oligomer concentration” (COC). Below this COC, fibril assembly replicates the sigmoidal kinetics of nucleated polymerization. Upon crossing the COC, assembly kinetics becomes biphasic with gO/CF formation responsible for the lag-free initial phase, followed by a second upswing dominated by RF nucleation and growth. RF lag periods below the COC, as expected, decrease as a power law in monomer concentration. Surprisingly, the build-up of gO/CFs above the COC causes a progressive increase in RF lag periods. Our results suggest that metastable gO/CFs are off-pathway from RF formation, confined by a condition-dependent COC that is distinct from RF solubility, underlie a transition from sigmoidal to biphasic assembly kinetics and, most importantly, not only compete with RFs for the shared monomeric growth substrate but actively inhibit their nucleation and growth.

2-Deoxyribose-5-phosphate aldolase (DERA) catalyzes the reversible conversion of acetaldehyde and glyceraldehyde-3-phosphate into deoxyribose-5-phosphate. DERA is used as a biocatalyst for the synthesis of drugs such as statins and is a promising pharmaceutical target due to its involvement in nucleotide catabolism. Despite previous biochemical studies suggesting the catalytic importance of the C-terminal tyrosine residue found in several bacterial DERAs, the structural and functional basis of its participation in catalysis remains elusive because the electron density for the last eight to nine residues (i.e., the C-terminal tail) is absent in all available crystal structures. Using a combination of NMR spectroscopy and molecular dynamics simulations, we conclusively show that the rarely studied C-terminal tail of E. coli DERA (ecDERA) is intrinsically disordered and exists in equilibrium between open and catalytically relevant closed states, where the C-terminal tyrosine (Y259) enters the active site. Nuclear Overhauser effect distance restraints, obtained due to the presence of a substantial closed state population, were used to derive the solution-state structure of the ecDERA closed state. Real-time NMR hydrogen/deuterium exchange experiments reveal that Y259 is required for efficiency of the proton abstraction step of the catalytic reaction. Phosphate titration experiments show that, in addition to the phosphate-binding residues located near the active site, as observed in the available crystal structures, ecDERA contains previously unknown auxiliary phosphate-binding residues on the C-terminal tail which could facilitate in orienting Y259 in an optimal position for catalysis. Thus, we present significant insights into the structural and mechanistic importance of the ecDERA C-terminal tail and illustrate the role of conformational sampling in enzyme catalysis.

A hallmark of Alzheimer's disease (AD) is the accumulation of extracellular amyloid-v (Av) plaques in the brains of patients. N-terminally truncated pyroglutamate-modified Av (pEAv) has been described as a major compound of Av species in senile plaques. pEAv is more resistant to degradation, shows higher toxicity and has increased aggregation propensity and v-sheet stabilization compared to non-modified Av. Here we characterized recombinant pEAv(3-40) in aqueous trifluoroethanol (TFE) solution regarding its aggregation propensity and structural changes in comparison to its non-pyroglutamate-modified variant Av(1-40). Secondary structure analysis by circular dichroism spectroscopy suggests that pEAv(3-40) shows an increased tendency to form v-sheet-rich structures in 20% TFE containing solutions where Av(1-40) forms a-helices. Aggregation kinetics of pEAv(3-40) in the presence of 20% TFE monitored by thioflavin-T (ThT) assay showed a typical sigmoidal aggregation in contrast to Av(1-40), which lacks ThT positive structures under the same conditions. Transmission electron microscopy confirms that pEAv(3-40) aggregated to large fibrils and high molecular weight aggregates in spite of the presence of the helix stabilizing co-solvent TFE. High resolution NMR spectroscopy of recombinantly produced and uniformly isotope labeled [U-15N]-pEAv(3-40) in TFE containing solutions indicates that the pyroglutamate formation affects significantly the N-terminal region, which in turn leads to decreased monomer stability and increased aggregation propensity

Alzheimer's disease (AD) is the leading cause of dementia in the elderly and is characterized by memory loss and cognitive decline. Pathological hallmark of AD brains are intracellular neurofibrillary tangles and extracellular amyloid plaques. The major component of these plaques is the highly heterogeneous amyloid-beta (A beta) peptide, varying in length and modification. In recent years pyroglutamate-modified amyloid-beta (pEA beta) peptides have increasingly moved into the focus since they have been described to be the predominant species of all N-terminally truncated A beta. Compared to unmodified A beta, pEA beta is known to show increased hydrophobicity, higher toxicity, faster aggregation and beta-sheet stabilization and is more resistant to degradation. Nuclear magnetic resonance (NMR) spectroscopy is a particularly powerful method to investigate the conformations of pEA beta isoforms in solution and to study peptide/ligand interactions for drug development. However, biophysical characterization of pEA beta and comparison to its non-modified variant has so far been seriously hampered by the lack of highly pure recombinant and isotope-enriched protein. Here we present, to our knowledge, for the first time a reproducible protocol for the production of pEA beta from a recombinant precursor expressed in E. coli in natural isotope abundance as well as in uniformly [U-N-15]-or [U-C-13, N-15]-labeled form, with yields of up to 15 mg/l E. coli culture broth. The chemical state of the purified protein was evaluated by RP-HPLC and formation of pyroglutamate was verified by mass spectroscopy. The recombinant pyroglutamate-modified A beta peptides showed characteristic sigmoidal aggregation kinetics as monitored by thioflavin-T assays. The quality and quantity of produced pEA beta 40 and pEA beta 42 allowed us to perform heteronuclear multidimensional NMR spectroscopy in solution and to sequence-specifically assign the backbone resonances under near-physiological conditions. Our results suggest that the presented method will be useful in obtaining cost-effective high-quality recombinant pEA beta 40 and pEA beta 42 for further physiological and biochemical studies.

Protein folding intermediates have been implicated in amyloid fibril formation involved in neurodegenerative disorders. However, the structural mechanisms by which intermediates initiate fibrillar aggregation have remained largely elusive. We used CPMG relaxation dispersion NMR spectroscopy to determine the atomic-resolution structure of a 2% populated, on-pathway folding intermediate of the A39V/N53P/V55L Fyn SH3 domain. To this end, backbone chemical shifts and RDCs/RCSAs of the "invisible" intermediate reconstructed from CPMG experiments were used as experimental input for chemical shift restrained molecular dynamics simulations via the CamShift approach [1]. The C-terminus remains disordered in this intermediate [2], thereby exposing the aggregation-prone N-terminal strand. Accordingly, mutants lacking the C-terminus and thus mimicking the intermediate fail to safeguard the folding route and spontaneously form beta-sheet-rich fibrillar aggregates with a diameter of several nm and affinity for Congo red. The structure provides a detailed picture of the non-native interactions stabilizing an aggregation-prone intermediate under native conditions and insight into how such an intermediate can derail folding and initiate fibrillation.

Protein folding intermediates are implicated in amyloid fibril formation but difficult to characterize. CPMG NMR relaxation dispersion experiments allowed us to detect a previously unknown intermediate on the folding pathway of the Fyn SH3 A39V/N53P/V55L and to reconstruct chemical shifts and RDCs/RCSAs for this 2% populated intermediate. Calculation of the high-resolution structure of the “invisible” intermediate from these experimental restraints using the Camshift strategy revealed a native-like arrangement of 4 of the 5 native beta-strands stabilized by several non-native long-range interactions. By contrast, the C-terminus remains disordered, leaving an aggregation-prone strand exposed. Accordingly, mutants mimicking this intermediate spontaneously form amyloid fibrils. This structure provides a detailed picture of how an intermediate can facilitate both, folding but also misfolding/aggregation.