Scanning MscL Channels with Targeted Post-Translational Modifications for Functional Alterations
Scanning MscL Channels with Targeted Post-Translational Modifications for Functional Alterations
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DOI：
10.1371/journal.pone.0137994
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发表时间：
2015-09-14
期刊：
影响因子：
3.7
通讯作者：
Blount, Paul
中科院分区：
文献类型：
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作者：
Iscla, Irene;Wray, Robin;Blount, Paul
机械敏感通道存在于所有生物体中，被认为是触觉和听觉以及渗透调节和血管调节等各种重要生理功能的基础。来自大肠杆菌的大电导机械敏感通道（MscL）是第一个被证明编码机械敏感通道活性的蛋白质，并作为通道如何感知和响应机械刺激的范例。MscL在大肠杆菌中起渗透保护作用，作为一个紧急释放阀，在渗透下冲击后由于细胞肿胀而被膜张力激活。在渗透下冲击试验中使用渗透性脆弱的菌株，可以直接在体内确定通道功能。此外，使用硫醇试剂和单半胱氨酸替代表达的MscL蛋白，我们已经证明可以进行靶向翻译后修饰，并且任何导致功能失调蛋白的改变都可以通过体内检测来识别。在这里，我们展示了使用五种不同的巯基反应探针对113个msc半胱氨酸突变体进行扫描的结果，以赋予每个位点不同的电荷或疏水性。我们评估了哪些靶向修饰会影响通道功能，并使用膜片钳进一步研究了最佳候选，以直接确定通道活性如何受到影响。这个全面的筛选已经确定了许多对通道功能至关重要的残基，以及突出显示的MscL结构域和在门控时经历最剧烈的环境变化的残基。
Mechanosensitive channels are present in all living organisms and are thought to underlie the senses of touch and hearing as well as various important physiological functions like osmoregulation and vasoregulation. The mechanosensitive channel of large conductance (MscL) from Escherichia coli was the first protein shown to encode mechanosensitive channel activity and serves as a paradigm for how a channel senses and responds to mechanical stimuli. MscL plays a role in osmoprotection in E. coli, acting as an emergency release valve that is activated by membrane tension due to cell swelling after an osmotic down-shock. Using an osmotically fragile strain in an osmotic down-shock assay, channel functionality can be directly determined in vivo. In addition, using thiol reagents and expressed MscL proteins with a single cysteine substitution, we have shown that targeted post-translational modifications can be performed, and that any alterations that lead to dysfunctional proteins can be identified by this in vivo assay. Here, we present the results of such a scan performed on 113 MscL cysteine mutants using five different sulfhydryl-reacting probes to confer different charges or hydrophobicity to each site. We assessed which of these targeted modifications affected channel function and the top candidates were further studied using patch clamp to directly determine how channel activity was affected. This comprehensive screen has identified many residues that are critical for channel function as well as highlighted MscL domains and residues that undergo the most drastic environmental changes upon gating.