Minard P, Scalley-Kim M, Watters A, Baker D
Department of Biochemistry, Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA.
Protein Sci. 2001 Jan;10(1):129-34. doi: 10.1110/ps.32401.
As a step toward selecting folded proteins from libraries of randomized sequences, we have designed a 'loop entropy reduction'-based phage-display method. The basic premise is that insertion of a long disordered sequence into a loop of a host protein will substantially destabilize the host because of the entropic cost of closing a loop in a disordered chain. If the inserted sequence spontaneously folds into a stable structure with the N and C termini close in space, however, this entropic cost is diminished. The host protein function can, therefore, be used to select folded inserted sequences without relying on specific properties of the inserted sequence. This principle is tested using the IgG binding domain of protein L and the lck SH2 domain as host proteins. The results indicate that the loop entropy reduction screen is capable of discriminating folded from unfolded sequences when the proper host protein and insertion point are chosen.
作为从随机序列文库中筛选折叠蛋白的一个步骤，我们设计了一种基于“环熵降低”的噬菌体展示方法。基本前提是，由于在无序链中闭合环的熵成本，将长的无序序列插入宿主蛋白的环中会使宿主蛋白显著不稳定。然而，如果插入序列自发折叠成N端和C端在空间上接近的稳定结构，这种熵成本就会降低。因此，宿主蛋白功能可用于选择折叠的插入序列，而无需依赖插入序列的特定特性。使用蛋白L的IgG结合结构域和lck SH2结构域作为宿主蛋白来测试这一原理。结果表明，当选择合适的宿主蛋白和插入点时，环熵降低筛选能够区分折叠序列和未折叠序列。
