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

Abstract

Thermally induced aggregates of alpha-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 (approximately 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 beta-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 alpha-chymotrypsinogen A, the scaling behavior is combined with the kinetics of aggregation to deduce separate values for the characteristic timescales for nucleation (tau(n)) and growth (tau(g)), as well as the stoichiometry of the nucleus (x). The analysis illustrates a general procedure to noninvasively and quantitatively determine tau(n), tau(g), and x for soluble (chain polymer) aggregates, as well as the relationship between tau(n)/tau(g) and aggregate M(w).

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment

“…The long term loss of monomer is generally predicted through quantitative monitoring of aggregation under accelerated and stress conditions over weeks to months. Recent progress has been made in: (i) the development of detailed kinetic models [2,4,5,34,42,49,60,96]; (ii) correlating aggregation kinetics with protein structure and folding [11,15,16,32,35,52,53,54,67,88]; (iii) and with native-state protein-protein interaction measurements [36,46,57,74,75,79,82].…”

“…Aggregation can be described through models incorporating nucleation and subsequent growth steps such as Lumry-Eyring nucleation polymerization models [1,2,9,50,96]. Nucleation generally refers to the steps prior to formation of the smallest net irreversible aggregate, which can include partial unfolding, reversible association of partially unfolded intermediates, and conformational rearrangements.…”

Nonnative Aggregation of an IgG1 Antibody in Acidic Conditions, Part 2: Nucleation and Growth Kinetics with Competing Growth Mechanisms

“…12 A number of theoretical and experimental models capture some of these steps in the multistep process of aggregation. [20][21][22][23][24][25][26][27][28][29] The Lumry-Eyring nucleated polymerization (LENP) model 30,31 and its predecessors 28,[32][33][34] incorporate these steps in a framework that has been used qualitatively and quantitatively to infer details about aggregation mechanisms for alpha-chymotrypsinogen (aCgn) A, 13,35,36 bovine granulocyte-colony stimulating factor, 36,37 and beta-lactoglobulin. 34,38 Mechanistic details of interest may include the stoichiometry of the aggregate nucleus, the dominant pathway(s) for aggregate growth, and quantitative rates for different stages in the aggregation process.…”

“…12,35 The LENP model 30,31 and its simpler counterparts 5,20,23,24,26 have been shown to explain the aggregation of relatively small, single-domain, and/or globular proteins. 8,13,35,36,39 Similarly, nucleated polymerization mechanisms have been used to explain aggregation mechanisms and rates for disease-related polypeptides. [40][41][42] However, for large, multidomain proteins such as monoclonal antibodies, aggregation has been much less systematically examined within this context.…”

Aerosol dynamics in porous media

“…Nucleation plays an important role in atmospheric processes [45,24] and it is a crucial concept in the production of various polymer, alloy and ceramic materials [1,25].…”