Structural transition of human calcitonin and molecular mechanism of fibrillization

用生物物理学方法研究了人降钙素（hCT）的结构和聚集对溶液中乙醇，特别是六氟异丙醇（HFIP）的pH和浓度的依赖性。研究了浓度为20 μg/ml的hCT在溶液中的结构与pH和HFIP浓度的关系。四种结构状态可以被鉴定为无规卷曲、α-螺旋单体、α-螺旋寡聚体和淀粉样聚集体。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，聚集体在具有中等浓度HFIP的溶液中非常迅速地形成：在pH 7时，它们在将hCT溶解在具有5- 15%HFIP的溶液中后仅几分钟内形成。这是由以下综合效应导致的：（1）由于hCT上的净电荷减少，分子间库仑排斥力降低，（2）加入中等浓度的HFIP稳定了引发聚集所需的hCT的α-螺旋结构，但（3）所得α-螺旋低聚物不会过度稳定，因此它们可以容易地转化为更大的聚集体。相反，在不含HFIP的中性溶液中，即使在10 mg/ml的非常高的hCT浓度下，hCT形成纤维状聚集体也需要超过1小时。

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.