Nonnative protein polymers: structure, morphology, and relation to nucleation and growth.
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).
Weiss
William F
WF
Department of Chemical Engineering, University of Delaware, Newark, Delaware, USA.
Hodgdon
Travis K
TK
Kaler
Eric W
EW
Lenhoff
Abraham M
AM
Roberts
Christopher J
CJ
D002918:Chymotrypsin / Q000737:chemistry* / Q000648:ultrastructure*; D003198:Computer Simulation; D003460:Crystallization / Q000379:methods*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000648:ultrastructure*; D008956:Models, Chemical; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000648:ultrastructure*; D011487:Protein Conformation
D046912:Multiprotein Complexes; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D002918:Chymotrypsin; C117608:alpha-chymotrypsin
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