Mapping an Aggregation Nucleus One Protein at a Time

Small transient protein aggregates are the bottleneck through which partly unfolded protein monomers must funnel before they can form large stable aggregates. The exact size of the “thermodynamically stable nucleus” for aggregation is difficult to determine, as are the free-energy changes for addition or subtraction of individual monomers from the nucleus. Here, we measured the thermodynamic nucleus size and free energy for a well-defined protein construct. We used tethered trimers and tetramers of the aggregation-prone protein U1A. The protein’s folding kinetics served as a “clock” for aggregation dynamics. As shown previously, at n = 2, the transient aggregate is least stable compared to the native state. At n = 4, the aggregate state finally becomes thermodynamically more stable than the native state. Quantitative aggregation nucleus data provide key input for the next generation of coarse-grained and all-atom simulations of early stages of protein aggregation.