Transient local secondary structure in the intrinsically disordered C-term of the Albino3 insertase
Transient local secondary structure in the intrinsically disordered C-term of the Albino3 insertase
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DOI：
10.1016/j.bpj.2021.10.013
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发表时间：
2021-11-16
影响因子：
3.4
通讯作者：
Heyes, Colin D.
中科院分区：
文献类型：
--
作者：
Baucom, Dustin R.;Furr, Mercede;Heyes, Colin D.
Albino 3（Alb 3）是一种膜蛋白，是捕光复合物（LHC）蛋白插入类囊体膜的基础。Alb 3含有一个暴露于基质的C末端（Alb 3-Cterm），负责在LHC膜插入之前结合LHC装载的转运复合物。据报道，Alb 3-Cterm本质上是无序的，但缺乏其识别和结合转运复合物的精确机制细节，其四个不同基序的功能作用一直存在争议。使用一种新的实验和计算技术，如单分子荧光共振能量转移，圆二色性与去卷积分析，定点诱变，胰蛋白酶消化分析，并结合增强采样技术的全原子分子动力学模拟相结合，我们表明，Alb 3-Cterm包含在基序I和II的瞬时二级结构。实验数据和计算数据之间的良好一致性提供了定量一致的图像，并使我们能够识别一个异质结构系综，突出了二级结构的局部和瞬态性质。用该结构系综预测了单分子残基间距离分布和瞬时二级结构的表观解折叠自由能，结果与实验值吻合良好，我们推测这种瞬时局部二级结构可能在LHC转运复合物识别Alb 3-Cterm中起重要作用。这些结果应该提供一个框架，以更好地了解蛋白质靶向的Alb 3-Oxa 1-YidC家族的插入酶。
Albino3 (Alb3) is an integral membrane protein fundamental to the targeting and insertion of light-harvesting com-plex (LHC) proteins into the thylakoid membrane. Alb3 contains a stroma-exposed C-terminus (Alb3-Cterm) that is responsiblefor binding the LHC-loaded transit complex before LHC membrane insertion. Alb3-Cterm has been reported to be intrinsicallydisordered, but precise mechanistic details underlying how it recognizes and binds to the transit complex are lacking, andthe functional roles of its four different motifs have been debated. Using a novel combination of experimental and computationaltechniques such as single-molecule fluorescence resonance energy transfer, circular dichroism with deconvolution analysis,site-directed mutagenesis, trypsin digestion assays, and all-atom molecular dynamics simulations in conjunction with enhancedsampling techniques, we show that Alb3-Cterm contains transient secondary structure in motifs I and II. The excellent agree-ment between the experimental and computational data provides a quantitatively consistent picture and allows us to identifya heterogeneous structural ensemble that highlights the local and transient nature of the secondary structure. This structuralensemble was used to predict both the inter-residue distance distributions of single molecules and the apparent unfoldingfree energy of the transient secondary structure, which were both in excellent agreement with those determined experimentally.We hypothesize that this transient local secondary structure may play an important role in the recognition of Alb3-Cterm for theLHC-loaded transit complex, and these results should provide a framework to better understand protein targeting by the Alb3-Oxa1-YidC family of insertases.