O'Neil K T, Hoess R H, Raleigh D P, DeGrado W F
DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19880-0328, USA.
Proteins. 1995 Jan;21(1):11-21. doi: 10.1002/prot.340210103.
A method has been developed to select proteins that are thermodynamically destabilized yet still folded and functional. The DNA encoding the B1 IgG-binding domain from Group G Streptococcus (Strp G) has been fused to gene III of bacteriophage M13. The resulting fusion protein is displayed on the surface of the phage thus enabling the phage to bind to IgG molecules. In addition, these phage exhibit a small plaque phenotype that is reversed by mutations that destabilize the Strp G domain. By selecting phage with large plaque morphology that retain their IgG-binding function, it is possible to identify mutants that are folded but destabilized compared with wild-type Strp G. Such mutants can be divided into three general categories: 1) those that disrupt packing of hydrophobic side chains in the protein interior; 2) those that destabilize secondary structure; and 3) those that alter specific hydrogen bonds involving amino acid side chains. A number of the mutants have been physically characterized by circular dichroism and nuclear magnetic resonance and have been shown to have structures similar to wild-type Strp G but stabilities that were decreased by 2-5 kcal/mol.
已开发出一种方法来筛选那些在热力学上不稳定但仍能折叠并保持功能的蛋白质。编码来自G组链球菌（Strp G）的B1 IgG结合结构域的DNA已与噬菌体M13的基因III融合。由此产生的融合蛋白展示在噬菌体表面，从而使噬菌体能够结合IgG分子。此外，这些噬菌体表现出小噬菌斑表型，而这种表型会因使Strp G结构域不稳定的突变而逆转。通过选择具有大噬菌斑形态且保留其IgG结合功能的噬菌体，就有可能鉴定出与野生型Strp G相比已折叠但不稳定的突变体。这类突变体可大致分为三类：1）那些破坏蛋白质内部疏水侧链堆积的突变体；2）那些使二级结构不稳定的突变体；3）那些改变涉及氨基酸侧链的特定氢键的突变体。许多突变体已通过圆二色性和核磁共振进行了物理表征，结果表明它们具有与野生型Strp G相似的结构，但稳定性降低了2 - 5千卡/摩尔。
