Koide A, Bailey C W, Huang X, Koide S
Department of Biochemistry and Biophysics, University of Rochester Medical Center, Rochester, NY, 14642, USA.
J Mol Biol. 1998 Dec 11;284(4):1141-51. doi: 10.1006/jmbi.1998.2238.
The fibronectin type III domain (FN3) is a small autonomous folding unit which occurs in many animal proteins involving in ligand binding. The beta-sandwich structure of FN3 closely resembles that of immunoglobulin domains. We have prepared a phage display library of FN3 in which residues in two surface loops were randomized. We have selected mutant FN3s which bind to a test ligand, ubiquitin, with significant affinities, while the wild-type FN3 shows no measurable affinity. A dominant clone was expressed as a soluble protein and its properties were investigated in detail. Heteronuclear NMR characterization revealed that the selected mutant protein retains the global fold of FN3. It also has a modest conformational stability despite mutations at 12 out of 94 residues. These results clearly show the potential of FN3 as a scaffold for engineering novel binding proteins.
纤连蛋白III型结构域（FN3）是一种小的自主折叠单元，存在于许多参与配体结合的动物蛋白中。FN3的β-三明治结构与免疫球蛋白结构域的结构非常相似。我们制备了一个FN3噬菌体展示文库，其中两个表面环中的残基是随机的。我们筛选出了与测试配体泛素具有显著亲和力的突变型FN3，而野生型FN3则没有可测量的亲和力。一个优势克隆被表达为可溶性蛋白，并对其性质进行了详细研究。异核核磁共振表征表明，所选突变蛋白保留了FN3的整体折叠结构。尽管94个残基中有12个发生了突变，但它仍具有适度的构象稳定性。这些结果清楚地表明了FN3作为构建新型结合蛋白支架的潜力。
