Conformational conversion may precede or follow aggregate elongation on alternative pathways of amyloid protofibril formation.

A major goal in the study of protein aggregation is to understand how the conformational heterogeneity characteristic of the process leads to structurally distinct amyloid fibrils. The small protein barstar is known to form amyloid protofibrils in multiple steps at low pH: a small oligomer, the A-form, first transforms into a larger spherical higher oligomeric intermediate (HOI), which then self-associates to form the elongated protofibril. To determine how the conformational conversion reaction during aggregation is coupled to the process of protofibril formation, cysteine-scanning mutagenesis was first used to identify specific residue positions in the protein sequence, which are important in defining the nature of the aggregation process. Two classes of mutant proteins, which are distinguished by their kinetics of aggregation at high protein concentration, have been identified: Class I mutant proteins undergo conformational conversion, as measured by an increase in thioflavin T binding ability and an increase in circular dichroism at 216 nm, significantly faster than Class II mutant proteins. At low protein concentration, the rates of conformational conversion are, however, identical for both classes of mutant proteins. At high protein concentration, the two classes of mutant proteins can be further distinguished on the basis of their rates of protofibril growth, as determined from dynamic light-scattering measurements. For Class I mutant proteins, protofibril elongation occurs at the same, or slightly faster, rate than conformational conversion. For Class II mutant proteins, protofibril elongation is significantly slower than conformational conversion. Dynamic light scattering measurements and atomic force microscopy imaging indicate that for the Class I mutant proteins, conformational conversion occurs concurrently with the self-association of prefibrillar HOIs into protofibrils. On the other hand, for the Class II mutant proteins, the prefibrillar HOI first undergoes conformational conversion, and the conformationally converted HOIs then self-associate to form protofibrils. The two classes of mutant proteins appear, therefore, to use structurally distinct pathways to form amyloid protofibrils. On one pathway, conformational conversion occurs along with, or after, elongation of the oligomers; on the other pathway, conformational conversion precedes elongation of the oligomers. Single mutations in the protein can cause aggregation to switch from one pathway to the other. Importantly, the protofibrils formed by the two classes of mutant proteins have significantly different diameters and different internal structures.