Nucleation, Growth, and Activation Energies for Seeded and Unseeded Aggregation of α-Chymotrypsinogen A

Abstract

The intrinsic time scales for nonnative aggregate nucleation (tau0(n)) and chain growth (tau0(g)) were determined for alpha-chymotrypsinogen A as a function of temperature under acidic conditions where the resulting aggregates do not appreciably condense. Previous results (Andrews and Roberts (2007) Biochemistry 46, 7558) indicated that the product tau0(n)tau0(g) increases with increasing temperature but could not distinguish tau0(n) and tau0(g). Separate experimental values of tau0(n) and tau0(g) are reported here from two approaches based on either (i) combining unseeded monomer loss kinetics with static light scattering of the resulting aggregates or (ii) seeded monomer loss kinetics as a function of number concentration of seed. Values of tau0(n) and tau0(g) from (i) and (ii) agree quantitatively, and indicate that nucleation has a large, negative effective activation energy (ca. -76 kcal/mol) while growth has at most a weak dependence on temperature. The results are consistent with a model in which nucleation requires significant conformational changes within a nonnative oligomer, beyond those for monomer unfolding. The results more generally illustrate the potential utility of approaches (i) and (ii) for quantitatively determining in vitro tau0(n) and tau0(g) values, as well as how the effects of seeding can be predicted purely from unseeded kinetics and static light scattering measurements prior to significant aggregate condensation.

Understanding protein adsorption phenomena at solid surfaces

“…Many different protein species were found to form such soluble clusters as proven by a broad spectrum of different analytical techniques including circular dichroism [195], static and dynamic light scattering [192,196], electron microscopy [193,197], size exclusion chromatography [173], and fluorescence methods [198]. The generally accepted formation mechanism of this kind of protein cluster includes a nucleation step resulting in a seed of one or a few aggregated monomers followed by the cluster growth through monomer addition [195,199]. Universal detailed mechanistic models for protein cluster formation, growth, and adsorption are currently under debate [200,201].…”

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.…”

“…Nucleus stoichiometry is important for predicting the sensitivity of aggregation rates to total protein concentration and monomer unfolding free energies. 8,27,35,37 Growth pathways are also important; in that, they influence the size and morphology of the resulting aggregates. 12,13 Additionally, knowledge of the rates of different steps in the pathway(s) may provide insights regarding the physics that control rate-limiting steps such as nucleation and chain polymerization.…”

“…12,13 Additionally, knowledge of the rates of different steps in the pathway(s) may provide insights regarding the physics that control rate-limiting steps such as nucleation and chain polymerization. 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.…”

“…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.…”