Nanopore analysis of protein structure and folding

在健康的细胞中，大多数蛋白质在合成后不久就会折叠成特定的结构或构象。当不再需要时，蛋白质分解，氨基酸构建块被重新使用以制造新的蛋白质。在某些病理条件下，正常的合成和更新过程发生故障，错误折叠的蛋白质可以开始积累。错误折叠的蛋白质是不寻常的，因为它们很难降解，它们的存在最终会干扰关键的细胞功能，导致细胞死亡。错误折叠的蛋白质通常聚集成“淀粉样斑块”，这可以在显微镜下识别。大脑中淀粉样蛋白斑块的存在是许多神经退行性疾病的标志，如阿尔茨海默氏症和帕金森氏症。虽然引起这些疾病的蛋白质是已知的，但它们错误折叠的原因还不清楚。我的NSERC研究计划使用纳米孔分析来研究蛋白质的折叠和错误折叠。在这种技术中，纳米尺寸的小孔被插入到膜中。当在孔上施加电压时，电流将流动;如果小蛋白质进入孔，电流将被阻挡长达一微秒，并且该事件可以被电子检测到。我们已经发现，由蛋白质引起的信号在其进入孔时对其精确构象非常敏感。例如，我们已经发现，参与帕金森病的α-突触核蛋白在结合尼古丁时折叠成稳定的构象。这可以解释为什么吸烟者患这种疾病的风险降低。在接下来的5年里，我的短期目标集中在使用三种不同的研究方法（开发一种新型的孔，使用交流电场研究蛋白质构象，以及分析模型蛋白质）来收集详细的结构信息。我的长期目标是了解影响这些构象变化的因素，因为这些知识可能会导致开发可用于治疗神经退行性疾病和其他蛋白质错误折叠疾病的药物。

In a healthy cell, most proteins fold into a specific structure or conformation soon after they are synthesized. When no longer needed, the proteins break down and the amino acid building blocks are reused to make new proteins. Under certain pathological conditions, the normal process of synthesis and renewal malfunctions and misfolded proteins can begin to accumulate. Misfolded proteins are unusual because they are difficult to degrade and their presence eventually interferes with critical cell functions and the cell dies. The misfolded proteins often aggregate into "amyloid plaques", which can be identified under a microscope. The presence of amyloid plaques in the brain is the hallmark of many neurodegenerative diseases, such as Alzheimer's and Parkinson's. Although the proteins that cause these diseases are known, the reasons why they misfold are not well understood. My NSERC research program uses nanopore analysis to study the folding and misfolding of proteins. In this technique, a small pore of nanometre dimensions is inserted into a membrane. A current will flow when a voltage is applied across the pore; if a small protein enters the pore, the current will be blocked for as long as a microsecond and this event can be detected electronically. We have found that the signal caused by the protein is very sensitive to its precise conformation as it enters the pore. For example, we have discovered that a-synuclein, which is involved in Parkinson's disease, folds into a stable conformation upon binding nicotine. This may explain why cigarette smokers have a reduced risk for developing the disease. Over the next 5 years, my short-term objectives focus on the use of three different research approaches (development of a new type of pore, use of alternating current fields to study protein conformation, and analysis of model proteins) to gather detailed structural information. My long-term goal is to understand the factors that influence these conformational changes, as this knowledge may lead to the development of drugs that could be used to treat neurodegenerative and other protein misfolding diseases.