Protein folding on biosensor tips: folding of maltodextrin glucosidase monitored by its interactions with GroEL.
Protein folding on biosensor tips: folding of maltodextrin glucosidase monitored by its interactions with GroEL.
复制标题
生物传感器尖端上的蛋白质折叠：通过其与 GroEL 的相互作用监测麦芽糖糊精葡萄糖苷酶的折叠。
DOI：
10.1111/febs.13796
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发表时间：
2016
期刊：
影响因子：
--
通讯作者：
Chaudhuri,TapanK
中科院分区：
文献类型：
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作者：
Pastor,Ashutosh;Singh,AmitK;Fisher,MarkT;Chaudhuri,TapanK
在过去的60年里，蛋白质折叠已经被广泛地研究，通过采用基于溶液的方法，如溶解度、酶活性、二级结构分析和分析方法，如FRET、NMR和HD交换。然而，对于折叠过程的快速分析，基于溶液的方法经常受到聚集副反应的困扰，导致收率低。在这项工作中，我们证明了一种生物层干涉（BLI）伴侣蛋白检测系统可以识别变性蛋白的优越再折叠条件。固定蛋白折叠的程度作为时间的函数可以通过监测高亲和力的无核苷酸伴侣蛋白GroEL的结合来检测。GroEL优先与疏水表面暴露在未折叠或部分折叠形式的蛋白质相互作用，因此随着折叠的进行，GroEL结合的减少可能与疏水表面的埋藏有关。在生物层干涉生物传感器上固定的蛋白质上，GroEL结合的大小与蛋白质折叠和疏水残留物掩埋的程度成反比。我们展示了加速折叠的条件，可以观察到易于聚集的蛋白麦芽糊精葡萄糖苷酶（MalZ）。在生物层干涉法生物传感器表面发现的优越的固定折叠条件在Ni - NTA sepharose bead表面重现，与在溶液中大量折叠条件相比，释放的MalZ的折叠率显著提高（通过酶活性测量）。
Protein folding has been extensively studied for the past six decades by employing solution‐based methods such as solubility, enzymatic activity, secondary structure analysis, and analytical methods like FRET, NMR, and HD exchange. However, for rapid analysis of the folding process, solution‐based approaches are often plagued with aggregation side reactions resulting in poor yields. In this work, we demonstrate that a bio‐layer interferometry (BLI) chaperonin detection system can identify superior refolding conditions for denatured proteins. The degree of immobilized protein folding as a function of time can be detected by monitoring the binding of the high‐affinity nucleotide‐free form of the chaperonin GroEL. GroEL preferentially interacts with proteins that have hydrophobic surfaces exposed in their unfolded or partially folded form, so a decrease in GroEL binding can be correlated with burial of hydrophobic surfaces as folding progresses. The magnitude of GroEL binding to the protein immobilized on bio‐layer interferometry biosensor inversely reflects the extent of protein folding and hydrophobic residue burial. We demonstrate conditions where accelerated folding can be observed for the aggregation‐prone protein maltodextrin glucosidase (MalZ). Superior immobilized folding conditions identified on the bio‐layer interferometry biosensor surface were reproduced on Ni‐NTA sepharose bead surfaces and resulted in significant improvement in folding yields of released MalZ (measured by enzymatic activity) compared to bulk refolding conditions in solution.