Bates Alison, Williams Kathryn M, Hagerman Ann E
Department of Chemistry and Biochemistry, Miami University, Oxford, Ohio, USA.
Department of Biology, Saint Mary's University of Minnesota, Winona, Minnesota, USA.
Biochem Mol Biol Educ. 2025 Mar-Apr;53(2):209-217. doi: 10.1002/bmb.21880. Epub 2025 Jan 11.
We created a novel laboratory experience where undergraduate students explore the techniques used to study protein misfolding, unfolding, and aggregation. Despite the importance of protein misfolding and aggregation diseases, protein unfolding is not typically explored in undergraduate biochemistry laboratory classes. Yeast alcohol dehydrogenase (YADH) is used in the undergraduate biochemistry laboratory course at Miami University as the model system to explore protein overexpression and purification, bioinformatics, and enzyme characterization. Using one model protein across the entire semester allows the students to independently link topics introduced in the individual experiments; for example, students might draw connections between the thermal denaturation experiment and the requirement to keep the enzyme cold during a kinetics experiment. Students quantitated changes in secondary structure resulting from thermal denaturation by analyzing circular dichroism data. Monitoring the turbidity of a YADH solution with a temperature-controlled UV-Vis spectrometer was a reliable and easy method for undergraduate students to observe the thermally-induced aggregation of YADH. Together these experiments provide undergraduate students with first-hand experience in techniques to study protein unfolding and aggregation.
我们创建了一种新颖的实验室体验，让本科生探索用于研究蛋白质错误折叠、去折叠和聚集的技术。尽管蛋白质错误折叠和聚集疾病很重要，但在本科生物化学实验课中通常不会探讨蛋白质去折叠。迈阿密大学的本科生物化学实验课程使用酵母乙醇脱氢酶（YADH）作为模型系统，来探索蛋白质的过表达和纯化、生物信息学以及酶的特性。在整个学期中使用一种模型蛋白，能让学生独立地将各个实验中引入的主题联系起来；例如，学生可能会在热变性实验和动力学实验中保持酶低温的要求之间建立联系。学生通过分析圆二色性数据来定量热变性导致的二级结构变化。使用温度可控的紫外可见光谱仪监测YADH溶液的浊度，是本科生观察YADH热诱导聚集的一种可靠且简便的方法。这些实验共同为本科生提供了研究蛋白质去折叠和聚集技术的第一手经验。
