Investigation of factors that modulate amyloid formation of proteins is important to understand and mitigate amyloid-related diseases. To understand the role of electrostatic interactions and the effect of ionic cosolutes, especially anions, on amyloid formation, we have investigated the effect of salts such as NaCl, NaI, NaClO(4), and Na(2)SO(4) on the amyloid fibril growth of beta(2)-microglobulin, the protein involved in dialysis-related amyloidosis. Under acidic conditions, these salts exhibit characteristic optimal concentrations where the fibril growth is favored. The presence of salts leads to an increase in hydrophobicity of the protein as reported by 8-anilinonaphthalene-1-sulfonic acid, indicating that the anion interaction leads to the necessary electrostatic and hydrophobic balance critical for amyloid formation. However, high concentrations of salts tilt the balance to high hydrophobicity, leading to partitioning of the protein to amorphous aggregates. Such amorphous aggregates are not competent for fibril growth. The order of anions based on the lowest concentration at which fibril formation is favored is SO(4)(2)(-) > ClO(4)(-) > I(-) > Cl(-), consistent with the order of their electroselectivity series, suggesting that preferential anion binding, rather than general ionic strength effect, plays an important role in the amyloid fibril growth. Anion binding is also found to stabilize the amyloid fibrils under acidic condition. Interestingly, sulfate promotes amyloid growth of beta(2)-microglobulin at pH between 5 and 6, closer to its isoelectric point. Considering the earlier studies on the role of glycosaminoglycans and proteoglycans (i.e., sulfated polyanions) on amyloid formation, our study suggests that preferential interaction of sulfate ions with amyloidogenic proteins may have biological significance.

To obtain insight into the mechanism of fibril formation, we examined the effects of ultrasonication, a strong agitator, on ␤ 2 -microglobulin (␤2-m), a protein responsible for dialysis-related amyloidosis. Upon sonication of an acid-unfolded ␤2-m solution at pH 2.5, thioflavin T fluorescence increased markedly after a lag time of 1-2 h with a simultaneous increase of light scattering. Atomic force microscopy images showed the formation of a large number of short fibrils 3 nm in diameter. When the sonication-induced fibrils were used as seeds in the next seeding experiment at pH 2.5, a rapid and intense formation of long fibrils 3 nm in diameter was observed demonstrating seed-dependent fibril growth. We then examined the effects of sonication on the native ␤2-m at neutral pH, conditions under which amyloid deposits occur in patients. In the presence of 0.5 mM sodium dodecyl sulfate, a model compound of potential trigger and stabilizer of amyloid fibrils in patients, a marked increase of thioflavin T fluorescence was observed after 1 day of sonication at pH 7.0. The products of sonication caused the accelerated fibril formation at pH 7.0. Atomic force microscopy images showed that the fibrils formed at pH 7.0 have a diameter of more than 7 nm, thicker than those prepared at pH 2.5. These results indicate that ultrasonication is one form of agitation triggering the formation of amyloid fibrils of ␤2-m, producing fibrils adapted to the respective pH.Amyloidosis results from the deposition of normally soluble proteins into insoluble amyloid fibrils: long, unbranched, and often twisted fibrillar structures a few nanometers in diameter and predominantly composed of cross ␤-sheets (1-4). Among various amyloidogenic proteins,3 is a target of extensive study because of its clinical importance and suitable size for examining the relation between protein folding and amyloid fibril formation (5-12). Dialysis-related amyloidosis is a common and serious complication in patients receiving hemodialysis for more than 10 years (5, 6). ␤2-m, a typical immunoglobulin domain made of 99 residues, is present as the non-polymorphic light chain of the class I major histocompatibility complex (13). As part of its normal catabolic cycle, ␤2-m dissociates from the class I major histocompatibility complex and is transported in serum to the kidneys where the majority (95%) of it is degraded (6). Renal failure disrupts the clearance of ␤2-m from the serum, and moreover the ␤2-m does not pass through the dialysis membrane, resulting in an increase in the ␤2-m concentration by up to 50-fold in the blood circulation (6). By a mechanism that is currently not well understood, ␤2-m then self-associates to form amyloid fibrils under physiological conditions.The incubation of ␤2-m in vitro under acidic conditions in the presence or absence of seed fibrils results in the formation of high yields of amyloid fibrils with a range of different morphologies (7-11). In contrast, the generation of a substantial amount of amyloid fibrils at neutral pH has be...