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首页> 外文学位 >The functional incorporation of the mechanosensitive channel of large conductance within a tethered lipid bilayer and the future reconstitution of future designer channels.
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The functional incorporation of the mechanosensitive channel of large conductance within a tethered lipid bilayer and the future reconstitution of future designer channels.

机译：在束缚的脂质双层内大电导的机械敏感通道的功能性整合以及未来设计者通道的未来重构。

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摘要

When creating a biosensor based on single ion channel activity, the conformational changes within a protein as well as the protein's ability to reconstitute successfully into non-native lipid environment and remain active must be understood. Therefore, the proximity of key residues in the C-terminal region of the Mechanosensitive Channel of Large Conductance (MscL) was quantified via disulfide bridging of cysteine mutations made to conserved hydrophobic residues in the linker and S3-bundle of this region. The biochemical assays utilizing disulfide bridging provided insight into which residues were dynamic and interactive, enabling them to be studied further via patch clamping. The channel's C-terminal region during gating was under debate and our results support the theory of the S3-bundle remaining closed during gating. Results from this study also enabled the generation of two new mutant channels that could coordinate heavy metals and the recognition response was a decrease in conductance, and slowed channel kinetics.;After the correct configuration of the protein was established, the ideal lipid environment for studying single ion channels in a tethered device for biosensor applications was investigated. The tip-dip electrophysiology method was used to determine an electrically stable lipid environment between different diphytanoyl compositions. Diphytanoyl lipids were vital components of this research due to their increased durability and stability when tethering to solid supports such as the gold surface on the device. Results indicated all of the lipid-ratios suffered from pore formation due to electrical breakdown, both reversible and irreversible. This was the first time that electroporation was reported at such low potentials as 125 mV and 40 mV which may be characteristic of using the tip-dip method with diphtanoyl lipids. The pore formation was random; however distinguishable from single ion channel conductance by configuring I-V curves and evaluating the kinetics of the pores formed in comparison to channel activity. The lipid ratio of 70PC/30PE was chosen as the most stable lipid ratio and was integrated as the synthetic lipid environment for both Gramicidin and MscL on the tBLM device. Results for Gramicidin, indicated that single channel activity within the tBLM were characteristic of the channel. On the other hand, a high applied voltage was required to gate MscL and the conductance response was lower for this channel in comparison to when it is in its natural environment. This lead to the incorporation of a voltage sensitive MscL mutant, K31E, which sensed tension when voltages as low as 85 mV were applied. Preliminary results indicate that this mutant is very active within the tBLM and single channel activity is attainable.
机译：当基于单离子通道活性创建生物传感器时，必须了解蛋白质内的构象变化以及蛋白质成功重构为非天然脂质环境并保持活性的能力。因此，通过对在该区域的接头和S3-束中的保守的疏水残基进行半胱氨酸突变的二硫键桥接，对大电导的机械敏感通道（MscL）C端区域中关键残基的接近程度进行了定量。利用二硫键桥接的生化测定可洞悉哪些残基是动态且相互作用的，从而可通过膜片钳位对其进行进一步研究。门控期间通道的C端区域尚有争议，我们的结果支持S3束在门控期间保持闭合的理论。这项研究的结果还使得能够生成两个新的突变通道，这些通道可以协调重金属，并且识别响应是电导的降低和通道动力学的减慢。；在蛋白质的正确配置建立之后，理想的脂质环境成为了研究的理想环境研究了用于生物传感器的系留设备中的单个离子通道。尖端浸没电生理方法用于确定不同的双植烷酰基成分之间的电稳定脂质环境。双植烷脂类脂质是这项研究的重要组成部分，因为它们在与固体支持物（例如设备的金表面）栓系时具有更高的耐用性和稳定性。结果表明，由于电击穿，所有脂质比率都遭受孔形成，可逆和不可逆。这是首次报道电穿孔的低电势，如125 mV和40 mV，这可能是使用尖端浸渍法与二戊酰基脂质一起使用的特征。孔的形成是随机的。但是，通过配置I-V曲线并评估与通道活性相比形成的孔的动力学，可以区别于单个离子通道电导。选择70PC / 30PE的脂质比率为最稳定的脂质比率，并将其整合为tBLM装置上的Gramicidin和MscL的合成脂质环境。 Gramicidin的结果表明，tBLM中的单通道活性是该通道的特征。另一方面，与处于自然环境中的通道相比，需要较高的施加电压来控制MscL，并且该通道的电导响应较低。这导致了电压敏感的MscL突变体K31E的结合，当施加低至85 mV的电压时，该突变体会感应到张力。初步结果表明，该突变体在tBLM内非常活跃，可以实现单通道活性。

著录项

  • 作者

    Wilson, Danyell S.;

  • 作者单位

    University of Florida.;

  • 授予单位 University of Florida.;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 159 p.
  • 总页数 159
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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