This proposal is a joint experimental (Blanch) and computational (Head-Gordon) study that will examine the molecular mechanisms and the structural characteristics of protofibril and early events of amyloid fibril formation. We will employ two small proteins, the immunoglobulin-binding proteins L and G, which have low sequence identity, high structural similarity, but different folding mechanisms, to delineate the key sequence, structural, and stability determinants of protein aggregation and fibril formation. Complementary experiments and simulations using these proteins are proposed to examine the effect of mutating protein sequence on nucleation events, aggregation propensity, the kinetics of aggregation and folding, and the role of folding intermediates on aggregation. The Blanch group, using an extensive mutant library for protein L, will perform experiments to determine the kinetics of fibril formation over a range of time scales, using surface plasmon resonance and dynamic light scattering to determine protein interactions at short and intermediate time scales, and fluorescence anisotropy and thioflavin T binding to monitor the kinetics of fibril formation at longer times. The partitioning of partially-folded intermediates to native folds versus aggregation can also be examined and correlated with sequence. Our experimental efforts will be guided by simulations aimed at elucidating the sequence and structural factors that govern aggregation events. We will use coarse-grained protein models for proteins L and G developed in the Head-Gordon laboratory. These models are highly tractable, and provide complete thermodynamic and kinetic characterization of, not only folding thermodynamic and kinetics, but also complete landscape characterization of aggregation from simulations involving multiple chains. Once validated by experiment, simulations will provide a rapid screening for sequences that minimize aggregation. Using our computational results and those from other protein engineering studies, we will construct a set of guidelines for the rational design of mutations for reducing the aggregation propensity of a given protein, and test the transferability of these guidelines using a wide-range of mutants for proteins G. We have completed early studies in which experiment and simulation characterize the same mutants for stability and aggregation kinetics compared against wild-type for protein L, which provides evidence for the feasibility of the joint experimental/theoretical project proposed here. The two investigators propose a joint experimental and computational study that will examine the mechanism of protofibril and amyloid fibril formation. The model systems used are proteins G and L. The methods will include experimental characterization of the folding and aggregation pathways by surface plasmon resonance, dynamic light scattering, fluorescence anisotropy, and thioflavin T binding. A coarse-grained off-lattice simulation and atomistic molecular dynamics will also be applied, characterizing folding trajectories, kinetics, as well as thermodynamics.