Department of Biochemistry and ‡Center for Structural Biology, Vanderbilt University School of Medicine , Nashville, Tennessee 37232, United States.
Biochemistry. 2013 May 14;52(19):3229-41. doi: 10.1021/bi301635f. Epub 2013 May 2.
Misfolding of the α-helical membrane protein peripheral myelin protein 22 (PMP22) has been implicated in the pathogenesis of the common neurodegenerative disease known as Charcot-Marie-Tooth disease (CMTD) and also several other related peripheral neuropathies. Emerging evidence suggests that the propensity of PMP22 to misfold in the cell may be due to an intrinsic lack of conformational stability. Therefore, quantitative studies of the conformational equilibrium of PMP22 are needed to gain insight into the molecular basis of CMTD. In this work, we have investigated the folding and unfolding of wild type (WT) human PMP22 in mixed micelles. Both kinetic and thermodynamic measurements demonstrate that the denaturation of PMP22 by n-lauroyl sarcosine (LS) in dodecylphosphocholine (DPC) micelles is reversible. Assessment of the conformational equilibrium indicates that a significant fraction of unfolded PMP22 persists even in the absence of the denaturing detergent. However, we find the stability of PMP22 is increased by glycerol, which facilitates quantitation of thermodynamic parameters. To our knowledge, this work represents the first report of reversible unfolding of a eukaryotic multispan membrane protein. The results indicate that WT PMP22 possesses minimal conformational stability in micelles, which parallels its poor folding efficiency in the endoplasmic reticulum. Folding equilibrium measurements for PMP22 in micelles may provide an approach to assess the effects of cellular metabolites or potential therapeutic agents on its stability. Furthermore, these results pave the way for future investigation of the effects of pathogenic mutations on the conformational equilibrium of PMP22.
α-螺旋膜蛋白外周髓鞘蛋白 22（PMP22）的错误折叠与一种常见的神经退行性疾病——遗传性运动感觉神经病（CMTD）以及其他几种相关的周围神经病的发病机制有关。新出现的证据表明，PMP22 在细胞中易于错误折叠可能是由于其内在的构象稳定性不足。因此，需要对 PMP22 的构象平衡进行定量研究，以深入了解 CMTD 的分子基础。在这项工作中，我们研究了野生型（WT）人 PMP22 在混合胶束中的折叠和展开。动力学和热力学测量都表明，PMP22 在十二烷基磷酸胆碱（DPC）胶束中由月桂酰肌氨酸（LS）变性是可逆的。构象平衡的评估表明，即使在没有变性洗涤剂的情况下，仍有很大一部分未折叠的 PMP22 存在。然而，我们发现甘油能增加 PMP22 的稳定性，这有利于热力学参数的定量。据我们所知，这是首次报道真核多跨膜蛋白的可逆展开。结果表明，WT PMP22 在胶束中具有最小的构象稳定性，这与其在内质网中较差的折叠效率相平行。在胶束中对 PMP22 的折叠平衡测量可能提供一种方法来评估细胞代谢物或潜在治疗剂对其稳定性的影响。此外，这些结果为进一步研究致病性突变对 PMP22 构象平衡的影响铺平了道路。
