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Aggregation of a multidomain protein: A coagulation mechanism governs aggregation of a model IgG1 antibody under weak thermal stress
Author(s) -
Andersen Christian Beyschau,
Manno Mauro,
Rischel Christian,
Thórólfsson Matthías,
Martorana Vincenzo
Publication year - 2010
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.309
Subject(s) - protein aggregation , chemistry , dynamic light scattering , size exclusion chromatography , chemical physics , biophysics , light scattering , denaturation (fissile materials) , hydrodynamic radius , coagulation , chromatography , scattering , crystallography , analytical chemistry (journal) , materials science , biochemistry , nanotechnology , nanoparticle , physics , psychology , psychiatry , nuclear chemistry , optics , biology , enzyme
Using an IgG1 antibody as a model system, we have studied the mechanisms by which multidomain proteins aggregate at physiological pH when incubated at temperatures just below their lowest thermal transition. In this temperature interval, only minor changes to the protein conformation are observed. Light scattering consistently showed two coupled phases: an initial fast phase followed by several hours of exponential growth of the scattered intensity. This is the exact opposite of the lag‐time behavior typically observed in protein fibrillation. Dynamic light scattering showed the rapid formation of an aggregate species with a hydrodynamic radius of about 25 nm, which then increased in size throughout the experiment. Theoretical analysis of our light scattering data showed that the aggregate number density goes through a maximum in time providing compelling evidence for a coagulation mechanism in which aggregates fuse together. Both the analysis as well as size‐exclusion chromatography of incubated samples showed the actual increase in aggregate mass to be linear and reach saturation long before all molecules had been converted to aggregates. The CH2 domain is the only domain partly unfolded in the temperature interval studied, suggesting a pivotal role of this least stable domain in the aggregation process. Our results show that for multidomain proteins at temperatures below their thermal denaturation, transient unfolding of a single domain can prime the molecule for aggregation, and that the formation of large aggregates is driven by coagulation.