Structural transition of human calcitonin and molecular mechanism of fibrillization

通过各种生物物理方法研究了人体降钙素（hCT）的结构和聚集与溶液中酒精，特别是六氟-2-丙醇（HFIP）的pH和浓度的关系。在浓度为20 μg/ml的溶液中考察了hCT的结构对pH和HFIP浓度的影响。可识别出四种结构状态：无规线圈、α-螺旋单体、α-螺旋低聚物和淀粉样聚集体。加入适量的HFIP对hCT的聚集有显著的影响。在pH（大于或等于）4.8时，聚集体在中等浓度的HFIP溶液中迅速形成；在pH 7时，hCT溶解在5-15% HFIP溶液中仅几分钟就形成。这是由于(1)由于hCT上净电荷的减少，分子间库仑排斥力降低，(2)加入中等浓度的HFIP稳定了hCT的α-螺旋结构，这是引发聚集所必需的，但(3)得到的α-螺旋低聚物没有过度稳定，因此它们很容易转化为更大的聚集体。相反，在不含HFIP的中性溶液中，即使在非常高的hCT浓度为10mg/ml时，hCT也需要一个多小时才能形成纤维聚集体。

Dependence of the structure and aggregation of human calcitonin(hCT) on pH and concentration of alcohol, especially of hexafluoro-2-propanol (HFIP), in solution was examined by various biophysical methods. The structure of hCT in solution with a concentration of 20 μg/ml was surveyed against pH and HFIP concentration. Four structural states could be identified as the random coil, the α-helical monomer, the α-helical oligomer, and the amyloid-like aggregate. Addition of a moderate amount of HFIP was found to have remarkable effects on the aggregation of hCT. At pH【greater than or equal】4.8, aggregates are formed very rapidly in solution with a medium concentration of HFIP : At pH 7, they are formed within only several minutes after dissolving hCT in solution with 5-15% HFIP. This results from the combined effects that (1)intermolecular Coulombic repulsive forces are reduced due to decrease in the net charge on hCT, (2)addition ofa moderate concentration of HFIP stabilizes the α-helical structure of hCT necessary to initiate aggregation, but (3)resultant α-helical oligomers are not stabilized excessively so that they can be easily converted to larger aggregates. Conversely, in a neutral solution not containing HFIP, it takes more than an hour for hCT to form fibrillar aggregates even at a very high hCT concentration of 10mg/ml.