Rational Construction of Compact de Novo-Designed Biliverdin-Binding Proteins.
Rational Construction of Compact de Novo-Designed Biliverdin-Binding Proteins.
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紧凑的从头设计的胆绿素结合蛋白的合理构建。
DOI：
10.1021/acs.biochem.8b01076
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发表时间：
2018
期刊：
影响因子：
2.9
通讯作者：
Chow,BrianY
中科院分区：
文献类型：
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作者：
Sheehan,MollyM;Magaraci,MichaelS;Kuznetsov,IvanA;Mancini,JoshuaA;Kodali,Goutham;Moser,ChristopherC;Dutton,PLeslie;Chow,BrianY
根据蛋白质设计的第一原理，结合Rosetta的能量最小化模型，我们报道了新设计的胆绿素结合蛋白的合理结构。自组装的四螺旋结构束在体外与胆绿素IXa(BV)辅助因子结合，就像光敏感蛋白一样，结合BV(以及相关的胆蛋白或线性四联吡咯)，尽管与天然同源蛋白缺乏序列和结构上的同源性。在确定了将辅因子连接到蛋白质支架的合适位置后，在疏水核心内逐步放置稳定的BV残基。在没有高分辨率结构的情况下，使用Rosetta建模来告知辅因子结合口袋的结构-功能关系。全息蛋白的形成稳定了BV，导致远红BV荧光增强。通过去除与辅因子稳定或束稳定性无关的片段，最初的15kDade novo设计的荧光激活蛋白被截断，而其光学性质没有任何变化，下降到一个微型的10 kDa“迷你”，其中蛋白质支架仅延伸到Blinin D-环假设位置之外的半个七肽重复序列。这项工作展示了如何利用新蛋白设计技术和天然辅因子合理地构建高度致密的全蛋白荧光染料。
We report the rational construction ofde novo-designed biliverdin-binding proteins by first principles of protein design, informed by energy minimization modeling in Rosetta. The self-assembling tetrahelical bundles bind biliverdin IXa (BV) cofactor autocatalyticallyin vitro, like photosensory proteins that bind BV (and related bilins or linear tetrapyrroles) despite lacking sequence and structural homology to the natural counterparts. Upon identification of a suitable site for ligation of the cofactor to the protein scaffold, stepwise placement of residues stabilized BV within the hydrophobic core. Rosetta modeling was used in the absence of a high-resolution structure to inform the structure-function relationships of the cofactor binding pocket. Holoprotein formation stabilized BV, resulting in increased far-red BV fluorescence. Via removal of segments extraneous to cofactor stabilization or bundle stability, the initial 15 kDade novo-designed fluorescence-activating protein was truncated without any change to its optical properties, down to a miniature 10 kDa “mini”, in which the protein scaffold extends only a half-heptad repeat beyond the hypothetical position of the bilin D-ring. This work demonstrates how highly compact holoprotein fluorochromes can be rationally constructed usingde novoprotein design technology and natural cofactors.