Concerted evolution of structure and function in a miniature protein
Concerted evolution of structure and function in a miniature protein
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DOI：
10.1021/ja0056668
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发表时间：
2001-03-28
影响因子：
15
通讯作者：
Schepartz, A
中科院分区：
文献类型：
--
作者：
Chin, JW;Schepartz, A
非结构化的肽仅仅用用于识别的功能基团装饰，很少（如果有的话）具有功能。相比之下，由球状蛋白质呈递相同的官能团可以产生高亲和力配体。最近，我们描述了一种设计微型功能蛋白的策略，其中来自bZIP蛋白GCN 4的DNA结合残基被接枝到溶剂暴露的禽胰多肽（aPP）的R-螺旋上。4 aPP含有紧密堆积的疏水核心，由来自R-螺旋的6个残基（L17、F20、L24、Y27、L28、V30和V31）和来自II型聚脯氨酸（PPII）螺旋的4个残基（G1、P2、Q4、P5和Y 7）组成（图1）。4在我们的第一个接枝肽（PPBR 4）中，1 Y27、L28和V30被改变为精氨酸以保持DNA亲和力，并且V31被改变为丙氨酸以提高螺旋倾向。虽然PPBR 4在4 ℃下很好地结合DNA，但这些突变破坏了aPP核心。结果，PPBR 4在4 ℃下仅表现出新生螺旋性，在环境温度下没有DNA结合的证据。27、28、30和31位的突变不能产生既是螺旋DNA又是结合DNA的分子。5我们推断，PPBR 4的1、2、4、5和7位氨基酸的某些组合可能会重建受损的核心，并将R-螺旋预组织成更接近DNA结合所需的构象。如果是这样的话，含有这种序列的肽应该以高亲和力结合特异性DNA。我们设想通过亲和纯化N-末端PPII螺旋中含有突变的PPBR 4类似物的文库来定位适当的预组织的微型蛋白质。创建两个噬菌体文库以鉴定适当折叠的PPBR 4类似物（图1）。文库A和B的成员在PPII螺旋上的3个（文库A）或4个（文库B）位置不同于PPBR 4。保留在文库A的位置2和5处的脯氨酸残基在PP折叠蛋白中是高度保守的。8我们预期保留这两个脯氨酸将有效地限制文库A成员可用的构象空间9，并且大多数将包含N-末端PPII螺旋。这样的构象限制在文库B中不存在，承认可能有许多方法来稳定DNA结合的R-螺旋。由于文库B的位置2和5处的氨基酸不限于脯氨酸，我们预期该文库将取样较大部分的可用脯氨酸空间。
Unstructured peptides merely garnished with functional groups used for recognition rarely, if ever, possess function. By contrast, presentation of the same functional groups by a globular protein can result in a high affinity ligand. Recently we described1 a strategy for the design of miniature functional proteins2, 3 in which DNA-binding residues from the bZIP protein GCN4 were grafted onto the solvent-exposed R-helix of avian pancreatic polypeptide (aPP). 4 aPP contains a close-packed hydrophobic core comprised of six residues from the R-helix (L17, F20, L24, Y27, L28, V30, and V31) and four residues from a type II polyproline (PPII) helix (G1, P2, Q4, P5, and Y7)(Figure 1). 4 In our first grafted peptide (PPBR4), 1 Y27, L28, and V30 were changed to arginine to preserve DNA affinity, and V31 was changed to alanine to improve helical propensity. Although PPBR4 bound DNA well at 4 C, these mutations destroyed the aPP core. As a result, PPBR4 exhibited only nascent helicity at 4 C and no evidence of DNA binding at ambient temperature. Mutations at positions 27, 28, 30, and 31 failed to produce molecules that were both helical and bound DNA. 5We reasoned that some combinations of amino acids at positions 1, 2, 4, 5, and 7 of PPBR4 might rebuild the damaged core and pre-organize the R-helix into a conformation closer to that required for DNA binding. If so, peptides containing such sequences should bind specific DNA with high affinity. We conceived to locate appropriately pre-organized miniature proteins by affinity purification of a library of PPBR4 analogues containing mutations in the N-terminal PPII helix. Two phage libraries were created to identify appropriately folded PPBR4 analogues (Figure 1). The members of libraries A and B differ from PPBR4 at 3 (library A) or 4 (library B) positions on the PPII helix. The proline residues retained at positions 2 and 5 of library A are highly conserved among PP-fold proteins. 8 We anticipated that retention of these two prolines would effectively constrain the conformational space available to library A members9 and that most would contain N-terminal PPII helices. Such conformational constraints are absent in library B, acknowledging that there may be many ways to stabilize DNA-bound R-helices. 10 Since the amino acids at positions 2 and 5 of library B are not restricted to proline, we anticipated that this library would sample a larger fraction of available-ψ space.