The Stability of Monomeric Intermediates Controls Amyloid Formation: A[beta]25-35 and its N27Q Mutant

INTRODUCTION

Polypeptide chains can exist in many structural forms, such as unfolded, natively folded, and misfolded. The native folded state is the biological functional state. Some proteins exist in the natively disordered state; however, they transform into the folded state by favorable binding interactions. Misfolded proteins may form disordered aggregates or ordered amyloid fibrils, which are irreversible and toxic (1). Various intermediate states relate to the transformation between the native and the amyloid states. The structure and stabilities of the intermediates can be dependent on or independent of the folding/unfolding processes (2,3). Some of the intermediates may assemble to form soluble oligomers, which then lead to amyloid fibrils (4). The prion disease is a well known example where the normal form of the prion proteins (PrP^sup C^) converts to misfolded PrP^sup Sc^ intermediates, which then form amyloids (5). Amino acid mutations in the protein sequence can affect both folding and amyloid formation processes (6).

Nature optimizes the protein sequence to escape amyloid formation (1,4,7). It appears to do that in two ways-by stabilizing the folded state and destabilizing the amyloid state. The structure and stabilities of the intermediates, especially those linked to the amyloid pathway, may also perturb nature's selection. The intermediates are usually more flexible and broadly distributed; it is thus probably harder to control intermediates than to control the folded and amyloid states. In addition, it is unclear whether one should stabilize or destabilize intermediates to modulate amyloid formation.

The possible effects of the intermediate stability may account for amyloid-related diseases. Alzheimer's disease is a neurodegenerative disorder characterized by the dysfunction and death of nerve cells responsible for the storage and processing of information (8). The disease is mainly related to the altered proteolytic processing of the amyloid precursor protein, which leads to aggregation of neurotoxic forms of the amyloid ß (Aß)-peptide. One characteristic of Alzheimer's disease is the extracellular aggregation of the Aß peptide. Both the full-length form (Aß1-40 and Aß1-42) and the key fragment (Aß25-35) form fibrils that are neurotoxic (8-10). Recently, alternative propositions have been put forward to explain the pathogenesis of Alzheimer's disease with the possibility that a fraction of these Aß peptides stay at the membrane lipid bilayer after they are generated (11). One critical mechanism of the cytotoxicity is that the amyloid proteins/peptides form unregulated ion channels in membranes (12). Ion channels formed by the Alzheimer's peptide have been implicated in Alzheimer's disease pathophysiology (13,14). In the pore-formation/ion-channel mechanisms, it also appears that small oligomers play critical roles.

The sequence of Aß25-35 (GSNKGAIIGLM) has a positively charged N-terminus and a hydrophobic C-terminus. The solution structures of the Aß25-35 are a mixture of random coil, ß-strand, and a-helix (15,16). Hydrogen/deuterium (H/D)-exchange NMR experiments indicate that the Aß25-35 amyloid fibrils have a core formed from residues 28-35, with residues 31 and 32 being the most protected (17). Even though the H/D-exchange NMR results indicate that N27 is only marginally protected in the Aß25-35 amyloid fibril, the Aß25-35 Asn^sup 27^Gln mutant does not form amyloids (18). It seems that the difference in amyloid formation for the Aß25-35 and Asn^sup 27^Gln mutant does not come from the perturbation of the amyloid fibril core. To understand why the N27Q mutation blocks in vitro amyloid formation we carried out exhaustive simulation studies of both Aß25-35 and Asn^sup 27^Gln mutant sequences to investigate 1), the stability of candidate amyloid oligomers and 2), distributions of free energies for candidate intermediate monomer states with partial secondary structure formation. Indeed, our molecular dynamics simulations did not show destabilization effects of the Asn^sup 27^Gln of the oligomer clusters of Aß25-35. In contrast, the relative conformational stabilities of the Aß25-35 monomers are altered in the Asn^sup 27^Gln mutant, which may slow the amyloid formation process.

In particular, we find a single-mode distribution of the model free energies for the Aß25-35 peptide in the region of high extension favorable for amyloid formation, whereas the mutant peptide has a two-mode ensemble with energies bracketing those of the Aß25-35 peptide. The mode of the energy landscape may be used to explain the different behavior of amyloid formation. Using Kramer's theory of barrier crossing and a Morse-function-like energy landscape, we show that the change of the stability of the intermediates dramatically increases or decreases the rate of amyloid formation. By considering the barrier-crossing times for a three-state free energy functional with fixed globally stable disordered and metastable "amyloid" minima but a tunable intermediate state, we argue that this observation can explain the blocking of amyloid formation by the mutant sequence. The key to the argument is that weakly stable intermediates too easily flip back to the disordered minimum, whereas highly stable intermediates kinetically trap, so only intermediates with medium stability can make the transition to the amyloid form.

METHODS

Energy landscape functions and rate of barrier crossing