Jens Danielsson

High‐resolution NMR studies of the zinc‐binding site of the Alzheimer's amyloid β‐peptide

Metal binding to the amyloid β‐peptide is suggested to be involved in the pathogenesis of Alzheimers disease. We used high‐resolution NMR to study zinc binding to amyloid β‐peptide 1–40 at physiologic pH. Metal binding induces a structural change in the peptide, which is in chemical exchange on an intermediate rate, between the apo‐form and the holo‐form, with respect to the NMR timescale. This causes loss of NMR signals in the resonances affected by the binding. Heteronuclear correlation experiments, 15N‐relaxation and amide proton exchange experiments on amyloid β‐peptide 1–40 revealed that zinc binding involves the three histidines (residues 6, 13 and 14) and the N‐terminus, similar to a previously proposed copper‐binding site [Syme CD, Nadal RC, Rigby SE, Viles JH (2004) J Biol Chem279, 18169–18177]. Fluorescence experiments show that zinc shares a common binding site with copper and that the metals have similar affinities for amyloid β‐peptide. The dissociation constant Kd of zinc for the fragment amyloid β‐peptide 1–28 was measured by fluorescence, using competitive binding studies, and that for amyloid β‐peptide 1–40 was measured by NMR. Both methods gave Kd values in the micromolar range at pH 7.2 and 286 K. Zinc also has a second, weaker binding site involving residues between 23 and 28. At high metal ion concentrations, the metal‐induced aggregation should mainly have an electrostatic origin from decreased repulsion between peptides. At low metal ion concentrations, on the other hand, the metal‐induced structure of the peptide counteracts aggregation.

The Alzheimer β-peptide shows temperature-dependent transitions between left-handed 31-helix, β-strand and random coil secondary structures

The temperature‐induced structural transitions of the full length Alzheimer amyloid β‐peptide [Aβ(1–40) peptide] and fragments of it were studied using CD and 1H NMR spectroscopy. The full length peptide undergoes an overall transition from a state with a prominent population of left‐handed 31 (polyproline II; PII)‐helix at 0 °C to a random coil state at 60 °C, with an average ΔH of 6.8 ± 1.4 kJ·mol−1 per residue, obtained by fitting a Zimm–Bragg model to the CD data. The transition is noncooperative for the shortest N‐terminal fragment Aβ(1–9) and weakly cooperative for Aβ(1–40) and the longer fragments. By analysing the temperature‐dependent 3JHNHα couplings and hydrodynamic radii obtained by NMR for Aβ(1–9) and Aβ(12–28), we found that the structure transition includes more than two states. The N‐terminal hydrophilic Aβ(1–9) populates PII‐like conformations at 0 °C, then when the temperature increases, conformations with dihedral angles moving towards β‐strand at 20 °C, and approaches random coil at 60 °C. The residues in the central hydrophobic (18–28) segment show varying behaviour, but there is a significant contribution of β‐strand‐like conformations at all temperatures below 20 °C. The C‐terminal (29–40) segment was not studied by NMR, but from CD difference spectra we concluded that it is mainly in a random coil conformation at all studied temperatures. These results on structural preferences and transitions of the segments in the monomeric form of Aβ may be related to the processes leading to the aggregation and formation of fibrils in the Alzheimer plaques.

Thermodynamics of protein destabilization in live cells

Significance A key question in structural biology is how protein properties mapped out under simplified conditions in vitro transfer to the complex environment in live cells. The answer, it appears, varies. Defying predictions from steric crowding effects, experimental data have shown that cells in some cases stabilize and in other cases destabilize the native protein structures. In this study, we reconcile these seemingly conflicting results by showing that the in-cell effect on protein thermodynamics is sequence specific: The outcome depends both on the individual target protein and on its detailed host-cell environment. Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein’s interplay with the functionally optimized “interaction landscape” of the cellular interior.

A left-handed 31 helical conformation in the Alzheimer Aβ(12-28) peptide

We show for the first time that the secondary structure of the Alzheimer β‐peptide is in a temperature‐dependent equilibrium between an extended left‐handed 31 helix and a flexible random coil conformation. Circular dichroism spectra, recorded at 0.03 mM peptide concentration, show that the equilibrium is shifted towards increasing left‐handed 31 helix structure towards lower temperatures. High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study the Alzheimer peptide fragment Aβ(12–28) in aqueous solution at 0°C and higher temperatures. NMR translation diffusion measurements show that the observed peptide is in monomeric form. The chemical shift dispersion of the amide protons increases towards lower temperatures, in agreement with the increased population of a well‐ordered secondary structure. The solvent exchange rates of the amide protons at 0°C and pH 4.5 vary within at least two orders of magnitude. The lowest exchange rates (0.03–0.04 min−1) imply that the corresponding amide protons may be involved in hydrogen bonding with neighboring side chains.

Biophysical Studies of the Amyloid β-Peptide: Interactions with Metal Ions and Small Molecules

Alzheimers disease is the most common of the protein misfolding (“amyloid”) diseases. The deposits in the brains of afflicted patients contain as a major fraction an aggregated insoluble form of the so‐called amyloid β‐peptides (Aβ peptides): fragments of the amyloid precursor protein of 39–43 residues in length. This review focuses on biophysical studies of the Aβ peptides: that is, of the aggregation pathways and intermediates observed during aggregation, of the molecular structures observed along these pathways, and of the interactions of Aβ with Cu and Zn ions and with small molecules that modify the aggregation pathways. Particular emphasis is placed on studies based on high‐resolution and solid‐state NMR methods. Theoretical studies relating to the interactions are also included. An emerging picture is that of Aβ peptides in aqueous solution undergoing hydrophobic collapse together with identical partners. There then follows a relatively slow process leading to more ordered secondary and tertiary (quaternary) structures in the growing aggregates. These aggregates eventually assemble into elongated fibrils visible by electron microscopy. Small molecules or metal ions that interfere with the aggregation processes give rise to a variety of aggregation products that may be studied in vitro and considered in relation to observations in cell cultures or in vivo. Although the heterogeneous nature of the processes makes detailed structural studies difficult, knowledge and understanding of the underlying physical chemistry might provide a basis for future therapeutic strategies against the disease. A final part of the review deals with the interactions that may occur between the Aβ peptides and the prion protein, where the latter is involved in other protein misfolding diseases.

Fibrillation precursor of superoxide dismutase 1 revealed by gradual tuning of the protein-folding equilibrium

Although superoxide dismutase 1 (SOD1) stands out as a relatively soluble protein in vitro, it can be made to fibrillate by mechanical agitation. The mechanism of this fibrillation process is yet poorly understood, but attains considerable interest due to SOD1’s involvement in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this study, we map out the apoSOD1 fibrillation process from how it competes with the global folding events at increasing concentrations of urea: We determine how the fibrillation lag time (τlag) and maximum growth rate (νmax) depend on gradual titration of the folding equilibrium, from the native to the unfolded state. The results show that the agitation-induced fibrillation of apoSOD1 uses globally unfolded precursors and relies on fragmentation-assisted growth. Mutational screening and fibrillation m-values (∂ log τlag/∂[urea] and ∂ log νmax/∂[urea]) indicate moreover that the fibrillation pathway proceeds via a diffusely bound transient complex that responds to the global physiochemical properties of the SOD1 sequence. Fibrillation of apoSOD1, as it bifurcates from the denatured ensemble, seems thus mechanistically analogous to that of disordered peptides, save the competing folding transition to the native state. Finally, we examine by comparison with in vivo data to what extent this mode of fibrillation, originating from selective amplification of mechanically brittle aggregates by sample agitation, captures the mechanism of pathological SOD1 aggregation in ALS.

Zinc as chaperone-mimicking agent for retardation of amyloid β peptide fibril formation

Significance One histologic hallmark of Alzheimer’s disease is the self-assembly of amyloid β peptide (Aβ) into insoluble amyloid aggregates. This aggregation process is strongly dependent on environmental conditions and metal ions, such as zinc, have been shown to modulate Aβ aggregation. To understand the underlying molecular mechanism of how zinc affects fibril formation we analyzed the aggregation kinetics and could conclude that zinc causes a significant reduction in elongation rate (i.e., monomer addition to the fibril ends). We used NMR methods to elucidate the details of zinc binding and we found that the N terminus of Aβ transiently folds around the zinc ion, forming a metastable dynamic complex. Metal ions have emerged to play a key role in the aggregation process of amyloid β (Aβ) peptide that is closely related to the pathogenesis of Alzheimer’s disease. A detailed understanding of the underlying mechanistic process of peptide–metal interactions, however, has been challenging to obtain. By applying a combination of NMR relaxation dispersion and fluorescence kinetics methods we have investigated quantitatively the thermodynamic Aβ–Zn2+ binding features as well as how Zn2+ modulates the nucleation mechanism of the aggregation process. Our results show that, under near-physiological conditions, substoichiometric amounts of Zn2+ effectively retard the generation of amyloid fibrils. A global kinetic profile analysis reveals that in the absence of zinc Aβ40 aggregation is driven by a monomer-dependent secondary nucleation process in addition to fibril-end elongation. In the presence of Zn2+, the elongation rate is reduced, resulting in reduction of the aggregation rate, but not a complete inhibition of amyloid formation. We show that Zn2+ transiently binds to residues in the N terminus of the monomeric peptide. A thermodynamic analysis supports a model where the N terminus is folded around the Zn2+ ion, forming a marginally stable, short-lived folded Aβ40 species. This conformation is highly dynamic and only a few percent of the peptide molecules adopt this structure at any given time point. Our findings suggest that the folded Aβ40–Zn2+ complex modulates the fibril ends, where elongation takes place, which efficiently retards fibril formation. In this conceptual framework we propose that zinc adopts the role of a minimal antiaggregation chaperone for Aβ40.

Folding without charges

Surface charges of proteins have in several cases been found to function as “structural gatekeepers,” which avoid unwanted interactions by negative design, for example, in the control of protein aggregation and binding. The question is then if side-chain charges, due to their desolvation penalties, play a corresponding role in protein folding by avoiding competing, misfolded traps? To find out, we removed all 32 side-chain charges from the 101-residue protein S6 from Thermus thermophilus. The results show that the charge-depleted S6 variant not only retains its native structure and cooperative folding transition, but folds also faster than the wild-type protein. In addition, charge removal unleashes pronounced aggregation on longer timescales. S6 provides thus an example where the bias toward native contacts of a naturally evolved protein sequence is independent of charges, and point at a fundamental difference in the codes for folding and intermolecular interaction: specificity in folding is governed primarily by hydrophobic packing and hydrogen bonding, whereas solubility and binding relies critically on the interplay of side-chain charges.

Cooperative formation of native-like tertiary contacts in the ensemble of unfolded states of a four-helix protein

In studies of the ensembles of unfolded structures of a four-helix bundle protein, we have detected the presence of potential precursors of native tertiary structures. These observations were based on the perturbation of NMR chemical shifts of the protein backbone atoms by single site mutations. Some mutations change the chemical shifts of residues remote from the site of mutation indicating the presence of an interaction between the mutated and the remote residues, suggesting that the formation of helix segments and helix-helix interactions is cooperative. We can begin to track down the folding mechanism of this protein using only experimental data by combining the information available for the rate limiting structure formation during the folding process with measurements of the site specific hydrogen bond formation in the burst phase, and with the existence prior to the folding reaction of tertiary structures in the ensemble of otherwise unfolded structures observed in the present study.

The hairpin conformation of the amyloid β peptide is an important structural motif along the aggregation pathway

The amyloid β (Aβ) peptides are 39–42 residue-long peptides found in the senile plaques in the brains of Alzheimer’s disease (AD) patients. These peptides self-aggregate in aqueous solution, going from soluble and mainly unstructured monomers to insoluble ordered fibrils. The aggregation process(es) are strongly influenced by environmental conditions. Several lines of evidence indicate that the neurotoxic species are the intermediate oligomeric states appearing along the aggregation pathways. This minireview summarizes recent findings, mainly based on solution and solid-state NMR experiments and electron microscopy, which investigate the molecular structures and characteristics of the Aβ peptides at different stages along the aggregation pathways. We conclude that a hairpin-like conformation constitutes a common motif for the Aβ peptides in most of the described structures. There are certain variations in different hairpin conformations, for example regarding H-bonding partners, which could be one reason for the molecular heterogeneity observed in the aggregated systems. Interacting hairpins are the building blocks of the insoluble fibrils, again with variations in how hairpins are organized in the cross-section of the fibril, perpendicular to the fibril axis. The secondary structure propensities can be seen already in peptide monomers in solution. Unfortunately, detailed structural information about the intermediate oligomeric states is presently not available. In the review, special attention is given to metal ion interactions, particularly the binding constants and ligand structures of Aβ complexes with Cu(II) and Zn(II), since these ions affect the aggregation process(es) and are considered to be involved in the molecular mechanisms underlying AD pathology.