Effect of environmental conditions on aggregation and fibril formation of barstar.

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.