Dependence on solution conditions of aggregation and amyloid formation by an SH3 domain

Computational studies of proteins have significantly improved our understanding of protein folding. These studies are normally carried out by using chains in isolation. However, in many systems of practical interest, proteins fold in the presence of other molecules. To obtain insight into folding in such situations, we compare the thermodynamics of folding for a Miyazawa-Jernigan model 64-mer in isolation to results obtained in the presence of additional chains. The melting temperature falls as the chain concentration increases. In multichain systems, free-energy landscapes for folding show an increased preference for misfolded states. Misfolding is accompanied by an increase in interprotein interactions; however, near the folding temperature, the transition from folded chains to misfolded and associated chains is entropically driven. A majority of the most probable interprotein contacts are also native contacts, suggesting that native topology plays a role in early stages of aggregation.computer simulation ͉ protein aggregation ͉ lattice model L attice-model proteins have played a key role in developing our understanding of protein folding. These model proteins contain enough detail to capture the essential physics of the folding process, yet are amenable to rigorous calculation of free-energy landscapes used to describe the folding pathway. Such calculations have provided a conceptual solution to the Levinthal Paradox, which ponders the ability of a protein to navigate a vast amount of conformational space to reach its native state on time scales of seconds or less (1-3). The funnellike nature of the free-energy landscapes, first calculated from lattice models, shows that proteins only need to sample a small fraction of conformations to reach the native state. The energetic bias toward the native state exists because native interactions are, on average, more stable than nonnative ones. Similar features are observed in folding landscapes generated from experiments, validating results from model calculations (1).Most computational studies of protein folding have examined a single chain in isolation (4-7). However, in many systems of practical interest, including in vivo folding, proteins fold in crowded environments. In such situations, interactions with other biological molecules compete with the intraprotein interactions that bias a protein's conformation toward its native state. Some biological molecules, such as molecular chaperones, promote folding (8). However, intermolecular interactions can also induce misfolding and aggregation (9, 10), resulting in loss of protein function (11). Further, protein aggregates can be toxic. Protein association has been linked to Ͼ20 human diseases, including Alzheimer's, Parkinson's, and Huntington's diseases (12, 13).We report simulations for systems containing one-, two-, or four-lattice model 64-mers. This chain length is greater than that in most model studies of multichain systems and correspondingly provides a more realistic surface area͞volume ratio than that for sm...

Section: Resultssupporting

confidence: 87%

Section: Discussionsupporting

confidence: 90%

Protein-folding landscapes in multichain systems

Cellmer

Bratko

Prausnitz

et al. 2005

“…Unfolded polypeptide chains represent, therefore, plausible candidates for the precursor structures of these amyloid fibrils. (iii) Increasing experimental and theoretical evidence (even for all ␤ proteins or domains) suggests that the amyloid fibril precursors are more expanded than well defined and compact partially folded states (33)(34)(35). (iv) ''Unfolded'' conformations (including acidic urea-denatured AMB) can have discernible structure, in particular they seem to possess a bias toward extended main chain conformations (26,(36)(37)(38).…”

Section: Discussionmentioning

confidence: 99%

Myoglobin forms amyloid fibrils by association of unfolded polypeptide segments

Fändrich¹,

Forge²,

Buder³

et al. 2003

267

238

Observations that ␤-sheet proteins form amyloid fibrils under at least partially denaturing conditions has raised questions as to whether these fibrils assemble by docking of preformed ␤-structure or by association of unfolded polypeptide segments. By using ␣-helical protein apomyoglobin, we show that the ease of fibril assembly correlates with the extent of denaturation. By contrast, monomeric ␤-sheet intermediates could not be observed under the conditions of fibril formation. These data suggest that amyloid fibril formation from apomyoglobin depends on disordered polypeptide segments and conditions that are selectively unfavorable to folding. However, it is inevitable that such conditions often stabilize protein folding intermediates.

“…PI3-SH3 has been shown to form highly ordered structures upon incubation at low pH and low ionic strength that have the key characteristics of amyloid fibrils associated with human disease (14,15). The most well characterized example of the amyloid fibrils formed by PI3-SH3 comprises a double helix of protofilament pairs, in which the great majority of the 84 amino acid-residue protein is contained within the fibril structure, as shown from measurements of hydrogen/deuterium exchange kinetics and controlled proteolysis (16)(17)(18).…”

mentioning

confidence: 99%

Direct characterization of amyloidogenic oligomers by single-molecule fluorescence

Orte

Birkett

Clarke

et al. 2008

180

208

A key issue in understanding the pathogenic conditions associated with the aberrant aggregation of misfolded proteins is the identification and characterization of species formed during the aggregation process. Probing the nature of such species has, however, proved to be extremely challenging to conventional techniques because of their transient and heterogeneous character. We describe here the application of a two-color single-molecule fluorescence technique to examine the assembly of oligomeric species formed during the aggregation of the SH3 domain of PI3 kinase. The single-molecule experiments show that the species formed at the stage of the reaction where aggregates have previously been found to be maximally cytotoxic are a heterogeneous ensemble of oligomers with a median size of 38 ؎ 10 molecules. This number is remarkably similar to estimates from bulk measurements of the critical size of species observed to seed ordered fibril formation and of the most infective form of prion particles. Moreover, although the size distribution of the SH3 oligomers remains virtually constant as the time of aggregation increases, their stability increases substantially. These findings together provide direct evidence for a general mechanism of amyloid aggregation in which the stable cross-␤ structure emerges via internal reorganization of disordered oligomers formed during the lag phase of the self-assembly reaction.amyloid aggregation ͉ amyloid oligomers ͉ two-color coincidence spectroscopy ͉ PI3-SH3 domain ͉ neurodegenerative diseases