Nonnative Protein Polymers: Structure, Morphology, and Relation to Nucleation and Growth

Abstract

Thermally induced aggregates of α-chymotrypsinogen A and bovine granulocyte-colony stimulating factor in acidic solutions were characterized by a combination of static and dynamic light scattering, spectroscopy, transmission electron microscopy, and monomer loss kinetics. The resulting soluble, high-molecular weight aggregates (∼10 3–10 5 kDa) are linear, semiflexible polymer chains that do not appreciably associate with one another under the conditions at which they were formed, with classic power-law scaling of the radius of gyration and hydrodynamic radius with weight-average molecular weight ( M w). Aggregates in both systems are composed of nonnative monomers with elevated levels of β-sheet secondary structure, and bind thioflavine T. In general, the aggregate size distributions showed low polydispersity by light scattering. Together with the inverse scaling of M w with protein concentration, the results clearly indicate that aggregation proceeds via nucleated (chain) polymerization. For α-chymotrypsinogen A, the scaling behavior is combined with the kinetics of aggregation to deduce separate values for the characteristic timescales for nucleation ( τ n) and growth ( τ g), as well as the stoichiometry of the nucleus ( x). The analysis illustrates a general procedure to noninvasively and quantitatively determine τ n, τ g, and x for soluble (chain polymer) aggregates, as well as the relationship between τ n/ τ g and aggregate M w.