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首页> 外文期刊>Journal of Molecular Biology >Design, production and molecular structure of a new family of artificial alpha-helicoidal repeat proteins (alphaRep) based on thermostable HEAT-like repeats.
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Design, production and molecular structure of a new family of artificial alpha-helicoidal repeat proteins (alphaRep) based on thermostable HEAT-like repeats.

机译：基于热稳定的类似HEAT的重复序列的新的人工α-螺旋重复蛋白家族（alphaRep）的设计，生产和分子结构。

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Repeat proteins have a modular organization and a regular architecture that make them attractive models for design and directed evolution experiments. HEAT repeat proteins, although very common, have not been used as a scaffold for artificial proteins, probably because they are made of long and irregular repeats. Here, we present and validate a consensus sequence for artificial HEAT repeat proteins. The sequence was defined from the structure-based sequence analysis of a thermostable HEAT-like repeat protein. Appropriate sequences were identified for the N- and C-caps. A library of genes coding for artificial proteins based on this sequence design, named alphaRep, was assembled using new and versatile methodology based on circular amplification. Proteins picked randomly from this library are expressed as soluble proteins. The biophysical properties of proteins with different numbers of repeats and different combinations of side chains in hypervariable positions were characterized. Circular dichroism and differential scanning calorimetry experiments showed that all these proteins are folded cooperatively and are very stable (T(m) >70 degrees C). Stability of these proteins increases with the number of repeats. Detailed gel filtration and small-angle X-ray scattering studies showed that the purified proteins form either monomers or dimers. The X-ray structure of a stable dimeric variant structure was solved. The protein is folded with a highly regular topology and the repeat structure is organized, as expected, as pairs of alpha helices. In this protein variant, the dimerization interface results directly from the variable surface enriched in aromatic residues located in the randomized positions of the repeats. The dimer was crystallized both in an apo and in a PEG-bound form, revealing a very well defined binding crevice and some structure flexibility at the interface. This fortuitous binding site could later prove to be a useful binding site for other low molecular mass partners.
机译：重复蛋白具有模块化的组织结构和规则的体系结构，使它们成为设计和定向进化实验的有吸引力的模型。 HEAT重复蛋白虽然很常见，但尚未用作人造蛋白的支架，可能是因为它们是由长且不规则的重复序列组成。在这里，我们提出并验证了人工HEAT重复蛋白的共有序列。该序列是从对热稳定的HEAT样重复蛋白的基于结构的序列分析中定义的。确定了N和C帽的适当序列。使用基于环形扩增的新型通用方法，组装了基于此序列设计的编码人工蛋白质的基因库，名为alphaRep。从该文库中随机选择的蛋白质表示为可溶性蛋白质。表征了在高变位置具有不同重复数和不同侧链组合的蛋白质的生物物理特性。圆二色性和差示扫描量热法实验表明，所有这些蛋白质可折叠在一起且非常稳定（T（m）> 70摄氏度）。这些蛋白质的稳定性随着重复次数的增加而增加。详细的凝胶过滤和小角度X射线散射研究表明，纯化的蛋白质形成单体或二聚体。解决了稳定的二聚体变体结构的X射线结构。蛋白质以高度规则的拓扑结构折叠，重复结构如预期般组织为成对的α螺旋。在这种蛋白质变体中，二聚化界面直接来自富集了位于重复序列随机位置的芳香族残基的可变表面。二聚体既以载脂蛋白形式也以PEG结合形式结晶，显示出非常明确的结合缝隙和界面处的某些结构柔性。该偶然的结合位点随后可被证明是其他低分子伴侣的有用结合位点。

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