Contrasting disease and non-disease protein aggregation by molecular simulations

This work describes the development and application of computational models for the investigation of disease and non-disease protein aggregation. We demonstrate how the aggregate equilibrium, formation kinetics, and structural ensembles are influenced by the structural and folding properties of the monomer units. Using a coarse-grained model, we examine the influence of folding rates and mechanisms of non-disease proteins L and G on their aggregation structure and kinetics. We demonstrate that the number and spatial distribution of contacts in the denatured state correlate with aggregation rates and the identity of inter-protein contacts, and that fast forming intermediates may inhibit aggregation through the burial of "sticky" regions. To examine the driving forces underlying the transition from amorphous aggregates to cross-beta ordered amyloid fibrils, we extend this physical model and investigate the aggregation of Alzheimer's Abeta1-40 peptide. We find that the critical nucleus, the highest free-energy species on the aggregation pathway, is composed of ten ordered peptides, the minimum number necessary to stabilize the interfilament contacts defining a fibril axis that enable fast growth of a fibril. Once past the critical nucleus, the model fibril elongates by efficiently incorporating monomers at only one of two asymmetric ends, connecting local structure differences to biased elongation. Familial Alzheimer's Disease (FAD) mutations represented in the model alter the number of peptides necessary to form the critical nucleus as well as the fibril stability, suggesting a molecular mechanism for the spectrum of in vitro aggregation kinetics and morphologies associated with dramatically different FAD clinical outcomes. The region of these mutations is examined in detail through atomistic simulations and NMR analysis of the monomeric Abeta21-30 peptide. Our simulations reproduce relaxation times and ROESY cross-peaks to interpret NMR experimental population averages, but we find no evidence of majority folded structures of this fragment as previously reported. By combining coarse-grained and atomistic simulations with experimental observables, we describe the fast-forming, poorly-populated and disordered states that drive aggregation at a level of detail unattainable by experimental techniques alone, elucidating the link between atomic level properties and aggregation outcomes for the protein L and G and the Alzheimer's Abeta systems

A coarse-grained alpha-carbon protein model with anisotropic hydrogen-bonding.

We develop a sequence based alpha-carbon model to incorporate a mean field estimate of the orientation dependence of the polypeptide chain that gives rise to specific hydrogen bond pairing to stabilize alpha-helices and beta-sheets. We illustrate the success of the new protein model in capturing thermodynamic measures and folding mechanism of proteins L and G. Compared to our previous coarse-grained model, the new model shows greater folding cooperativity and improvements in designability of protein sequences, as well as predicting correct trends for kinetic rates and mechanism for proteins L and G. We believe the model is broadly applicable to other protein folding and protein-protein co-assembly processes, and does not require experimental input beyond the topology description of the native state. Even without tertiary topology information, it can also serve as a mid-resolution protein model for more exhaustive conformational search strategies that can bridge back down to atomic descriptions of the polypeptide chain

A coarse-grained alpha-carbon protein model with anisotropic hydrogen-bonding.

We develop a sequence based alpha-carbon model to incorporate a mean field estimate of the orientation dependence of the polypeptide chain that gives rise to specific hydrogen bond pairing to stabilize alpha-helices and beta-sheets. We illustrate the success of the new protein model in capturing thermodynamic measures and folding mechanism of proteins L and G. Compared to our previous coarse-grained model, the new model shows greater folding cooperativity and improvements in designability of protein sequences, as well as predicting correct trends for kinetic rates and mechanism for proteins L and G. We believe the model is broadly applicable to other protein folding and protein-protein co-assembly processes, and does not require experimental input beyond the topology description of the native state. Even without tertiary topology information, it can also serve as a mid-resolution protein model for more exhaustive conformational search strategies that can bridge back down to atomic descriptions of the polypeptide chain

Protofibril Assemblies of the Arctic, Dutch, and Flemish Mutants of the Alzheimer's Aβ1–40 Peptide

Using a coarse-grained model of the Aβ peptide, we analyze the Arctic (E22G), Dutch (E22Q), and Flemish (A21G) familial Alzheimer's disease (FAD) mutants for any changes in the stability of amyloid assemblies with respect to the wild-type (WT) sequence. Based on a structural reference state of two protofilaments aligned to create the “agitated” protofibril as determined by solid-state NMR, we determine free energy trends for Aβ assemblies for the WT and FAD familial sequences. We find that the structural characteristics and oligomer size of the critical nucleus vary dramatically among the hereditary mutants. The Arctic mutant's disorder in the turn region introduces new stabilizing interactions that better align the two protofilaments, yielding a well-defined protofibril axis at relatively small oligomer sizes with respect to WT. By contrast, the critical nucleus for the Flemish mutant is beyond the 20 chains characterized in this study, thereby showing a strong shift in the equilibrium toward monomers with respect to larger protofibril assemblies. The Dutch mutant forms more ordered protofilaments than WT, but exhibits greater disorder in protofibril structure that includes an alternative polymorph of the WT fibril. An important conclusion of this work is that the Dutch mutant does not support the agitated protofibril assembly. We discuss the implications of the structural ensembles and free energy profiles for the FAD mutants in regards to interpretation of the kinetics of fibril assembly using chromatography and dye-binding experiments

Contrasting disease and nondisease protein aggregation by molecular simulation.

[Figurre: see text]. Protein aggregation can be defined as the sacrifice of stabilizing intrachain contacts of the functional state that are replaced with interchain contacts to form non-functional states. The resulting aggregate morphologies range from amorphous structures without long-range order typical of nondisease proteins involved in inclusion bodies to highly structured fibril assemblies typical of amyloid disease proteins. In this Account, we describe the development and application of computational models for the investigation of nondisease and disease protein aggregation as illustrated for the proteins L and G and the Alzheimer's Abeta systems. In each case, we validate the models against relevant experimental observables and then expand on the experimental window to better elucidate the link between molecular properties and aggregation outcomes. Our studies show that each class of protein exhibits distinct aggregation mechanisms that are dependent on protein sequence, protein concentration, and solution conditions. Nondisease proteins can have native structural elements in the denatured state ensemble or rapidly form early folding intermediates, which offers avenues of protection against aggregation even at relatively high concentrations. The possibility that early folding intermediates may be evolutionarily selected for their protective role against unwanted aggregation could be a useful strategy for reengineering sequences to slow aggregation and increase folding yield in industrial protein production. The observed oligomeric aggregates that we see for nondisease proteins L and G may represent the nuclei for larger aggregates, not just for large amorphous inclusion bodies, but potentially as the seeds of ordered fibrillar aggregates, since most nondisease proteins can form amyloid fibrils under conditions that destabilize the native state. By contrast, amyloidogenic protein sequences such as Abeta 1-40,42 and the familial Alzheimer's disease (FAD) mutants favor aggregation into ordered fibrils once the free-energy barrier for forming a critical nucleus is crossed. However, the structural characteristics and oligomer size of the soluble nucleation species have yet to be determined experimentally for any disease peptide sequence, and the molecular mechanism of polymerization that eventually delineates a mature fibril is unknown. This is in part due to the limited experimental access to very low peptide concentrations that are required to characterize these early aggregation events, providing an opportunity for theoretical studies to bridge the gap between the monomer and fibril end points and to develop testable hypotheses. Our model shows that Abeta 1-40 requires as few as 6-10 monomer chains (depending on sequence) to begin manifesting the cross-beta order that is a signature of formation of amyloid filaments or fibrils assessed in dye-binding kinetic assays. The richness of the oligomeric structures and viable filament and fibril polymorphs that we observe may offer structural clues to disease virulence variations that are seen for the WT and hereditary mutants

Contrasting disease and nondisease protein aggregation by molecular simulation.

[Figurre: see text]. Protein aggregation can be defined as the sacrifice of stabilizing intrachain contacts of the functional state that are replaced with interchain contacts to form non-functional states. The resulting aggregate morphologies range from amorphous structures without long-range order typical of nondisease proteins involved in inclusion bodies to highly structured fibril assemblies typical of amyloid disease proteins. In this Account, we describe the development and application of computational models for the investigation of nondisease and disease protein aggregation as illustrated for the proteins L and G and the Alzheimer's Abeta systems. In each case, we validate the models against relevant experimental observables and then expand on the experimental window to better elucidate the link between molecular properties and aggregation outcomes. Our studies show that each class of protein exhibits distinct aggregation mechanisms that are dependent on protein sequence, protein concentration, and solution conditions. Nondisease proteins can have native structural elements in the denatured state ensemble or rapidly form early folding intermediates, which offers avenues of protection against aggregation even at relatively high concentrations. The possibility that early folding intermediates may be evolutionarily selected for their protective role against unwanted aggregation could be a useful strategy for reengineering sequences to slow aggregation and increase folding yield in industrial protein production. The observed oligomeric aggregates that we see for nondisease proteins L and G may represent the nuclei for larger aggregates, not just for large amorphous inclusion bodies, but potentially as the seeds of ordered fibrillar aggregates, since most nondisease proteins can form amyloid fibrils under conditions that destabilize the native state. By contrast, amyloidogenic protein sequences such as Abeta 1-40,42 and the familial Alzheimer's disease (FAD) mutants favor aggregation into ordered fibrils once the free-energy barrier for forming a critical nucleus is crossed. However, the structural characteristics and oligomer size of the soluble nucleation species have yet to be determined experimentally for any disease peptide sequence, and the molecular mechanism of polymerization that eventually delineates a mature fibril is unknown. This is in part due to the limited experimental access to very low peptide concentrations that are required to characterize these early aggregation events, providing an opportunity for theoretical studies to bridge the gap between the monomer and fibril end points and to develop testable hypotheses. Our model shows that Abeta 1-40 requires as few as 6-10 monomer chains (depending on sequence) to begin manifesting the cross-beta order that is a signature of formation of amyloid filaments or fibrils assessed in dye-binding kinetic assays. The richness of the oligomeric structures and viable filament and fibril polymorphs that we observe may offer structural clues to disease virulence variations that are seen for the WT and hereditary mutants