Effect of environmental conditions on aggregation and fibril formation of barstar

Abstract

The dependence on environmental conditions of the assembly of barstar into amyloid fibrils was investigated starting from the nonnative, partially folded state at low pH (A-state). The kinetics of this process was monitored by CD spectroscopy and static and dynamic light scattering. The morphology of the fibrils was visualized by electron microscopy, while the existence of the typical cross-beta structure substantiated by solution X-ray scattering. At room temperature, barstar in the A-state is unable to form amyloid fibrils, instead amorphous aggregation is observed at high ionic strength. Further destabilization of the structure is required to transform the polypeptide chain into an ensemble of conformations capable of forming amyloid fibrils. At moderate ionic strength (75 mM NaCl), the onset and the rate of fibril formation can be sensitively tuned by increasing the temperature. Two types of fibrils can be detected differing in their morphology, length distribution and characteristic far UV CD spectrum. The formation of the different types depends on the particular environmental conditions. The sequence of conversion: A-state-->fibril type I-->fibril type II appears to be irreversible. The transition into fibrils is most effective when the protein chain fulfills particular requirements concerning secondary structure, structural flexibility and tendency to cluster.

Principles, approaches, and challenges for predicting protein aggregation rates and shelf life

“…This is an important mechanism of aggregate growth in some polypeptide systems that readily form large fibrils (lengths $microns). 45,50,52,53 Independent of whether aggregates are fibrillar, if they are insoluble this often results in visually apparent precipitate, particulate, or haze formation. 9,54-56…”

“…33,48,49,67,68 Aggregation With Condensation Condensation effects manifest in primarily two qualitatively distinct ways: (i) precipitate or particulate formation; 9,36,54-56 (ii) aggregateaggregate association creating high-MW, soluble aggregates and gels. 45,50,52,53,69 In the former situation, aggregate concentrations in solution are either saturated or remain low and at local steady state because the rate of creation of new soluble aggregates is balanced by the rate at which aggregates precipitate from solution. 21,36,59 As a result, the kinetics of growth and condensation do not significantly affect the rate of conversion of monomer to aggregate, and the kinetics of monomer loss under native-favoring conditions are well described by Eqs.…”

“…is the characteristic time scale, or the inverse rate coefficient, for the self-association of two preformed aggregates 18,37,50,52 As with all of the preceding situations except unfolding-limited aggregation, k obs and t shelf depend directly on DG 0 un and c 0 . Unlike the earlier situations, the effects of protein-protein interactions extend beyond monomer-monomer and monomer-aggregate interactions, as t ð0Þ c depends on aggregate-aggregate interactions and whether the condensation mechanism better resembles polymerization or fractal, colloidal aggregation.…”

“…10,45,74,75,82 Both techniques have the advantage of potentially being applicable for in situ monitoring, however practical constraints may limit this depending on the time scale(s), protein concentration, and sample conditions of interest. 10,45,50,52 Neither dynamic nor static light scattering reports directly on the extent of reaction. The former provides information about the distribution of diffusive relaxation times, which can be related to aggregate size distributions by assuming or knowing characteristics of the aggregate morphology.…”

“…84 On its own, M w is not a direct measure of m and can be used to determine m(t) and/or k obs only by assuming an aggregation model; and in some cases also by restricting the parameter space. 10,18,45,50,52 However, M w data can be fit in combination with other data such as DLS or SEC to provide robust values for k obs . 10,18,45,48 Combining SLS with SEC or another direct method for m(t) is particularly appealing and potentially powerful, in that the former is most sensitive to high-MW species while the strength of the latter lies in resolving monomer and low-MW species.…”