Harmonizing Experimental Data with Modeling to Predict Membrane Protein Insertion in Yeast
Harmonizing Experimental Data with Modeling to Predict Membrane Protein Insertion in Yeast
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DOI：
10.1016/j.bpj.2019.07.013
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发表时间：
2019-08-20
影响因子：
3.4
通讯作者：
Brodsky, Jeffrey L.
中科院分区：
文献类型：
--
作者：
Guerriero, Christopher J.;Gomez, Yessica K.;Brodsky, Jeffrey L.
膜蛋白必须在生物膜内采用其适当的拓扑结构，但实现正确的拓扑结构受到边缘疏水性跨膜螺旋（TMH）的存在的影响。在这项研究中，我们报告了一种新的模式膜蛋白在酵母中，窝藏两个TMH融合到一个不稳定的核苷酸结合域。由于该报告基因中的第二螺旋（TMH2）具有不利的预测插入自由能，我们采用已建立的方法来产生改变TMH2插入自由能的变体。我们首先发现改变TMH 2并不会显着影响细胞质量控制机制的蛋白质降解程度。接下来，我们从基于知识的能量尺度与测量的TMH2插入的表观自由能相关的预测插入自由能。虽然预测和表观插入能量显示出类似的趋势，预测的自由能变化跨越一个意想不到的狭窄范围。相反，通过使用基于物理的模型，我们获得了更广泛的自由能，与实验得出的值的大小相当吻合。尽管如此，一些变体仍然比基于能量的尺度预测的更好地插入酵母中。因此，进行分子动力学模拟，并表明相应的突变引起TMH2内的构象变化，这改变了稳定氢键的数量。总之，我们的研究结果提供了深入了解细胞质量控制机制的能力，以识别构象不同的错误折叠的拓扑异构体，提供了一个模型来评估TMH插入体内，并表明TMH插入能量尺度可能是有限的，这取决于特定的蛋白质和突变的存在。
Membrane proteins must adopt their proper topologies within biological membranes, but achieving the correct topology is compromised by the presence of marginally hydrophobic transmembrane helices (TMHs). In this study, we report on a new model membrane protein in yeast that harbors two TMHs fused to an unstable nucleotide-binding domain. Because the second helix (TMH2) in this reporter has an unfavorable predicted free energy of insertion, we employed established methods to generate variants that alter TMH2 insertion free energy. We first found that altering TMH2 did not significantly affect the extent of protein degradation by the cellular quality control machinery. Next, we correlated predicted insertion free energies from a knowledge-based energy scale with the measured apparent free energies of TMH2 insertion. Although the predicted and apparent insertion energies showed a similar trend, the predicted free-energy changes spanned an unanticipated narrow range. By instead using a physics-based model, we obtained a broader range of free energies that agreed considerably better with the magnitude of the experimentally derived values. Nevertheless, some variants still inserted better in yeast than predicted from energy-based scales. Therefore, molecular dynamics simulations were performed and indicated that the corresponding mutations induced conformational changes within TMH2, which altered the number of stabilizing hydrogen bonds. Together, our results offer insight into the ability of the cellular quality control machinery to recognize conformationally distinct misfolded topomers, provide a model to assess TMH insertion in vivo, and indicate that TMH insertion energy scales may be limited depending on the specific protein and the mutation present.