生物技术通报 ›› 2021, Vol. 37 ›› Issue (2): 236-245.doi: 10.13560/j.cnki.biotech.bull.1985.2020-0543

• 技术与方法 • 上一篇    下一篇

基于蛋白质二级结构热稳定性的正交方法

陈中(), 卢星宇   

  1. 1.中山大学化学学院,广州 510275
    2.西湖大学分子科学公共实验平台 浙江省功能分子精准合成重点实验室,杭州 310024
  • 收稿日期:2020-05-08 出版日期:2021-02-26 发布日期:2021-02-26
  • 作者简介:陈中,男,博士,研究方向:化学光谱;E-mail: chenzhong@westlake.edu.cn
  • 基金资助:
    国家自然科学基金青年科学基金项目(51703255)

An Orthogonal Method to Study the Thermal Stability of Secondary Structure of Protein

CHEN Zhong(), LU Xing-yu   

  1. 1. School of Chemistry,Sun Yat-sen University,Guangzhou 510275
    2. Key Laboratory of Precise Synthesis of Functional Molecules of Zhejiang Province,Instrumentation and Service Center for Molecular Sciences,Westlake University,Hangzhou 310024
  • Received:2020-05-08 Published:2021-02-26 Online:2021-02-26

摘要:

对蛋白质热稳定性的研究是解析蛋白高级结构,开发蛋白功能及新药物研发过程中的一个重要环节,是对其结构分析的一个重要关切点。观测蛋白质的圆二色光谱随温度程序变化而改变是研究其热稳定性的常用手段,传统的实验方法为选用某一单波长作为测试点,通过连续升温测试蛋白在单波长下的圆二色变温曲线,然后拟合出Tm值,此方法所得的信息有限,并且如何选取该单波长点是一个有争论的问题。本实验开发和优化了蛋白质二级结构热稳定性测试的一种正交方法,以牛血清蛋白及血红蛋白为研究对象,利用180-260 nm范围内的圆二色全光谱热变性测试来考察蛋白质构象随温度的变化而改变的过程。结果显示,当吸光度在合适的范围内,蛋白的热变性中点温度(Tm值)不受浓度的影响。作为对比,在180-260 nm范围内采用单波长法重复牛血清蛋白的热变性测试,结果表明,除了端点和交叉点外,其余波长的单点法的结果与全光谱正交法所测出的蛋白的Tm值是一致的,说明此两种方法在Tm值的测定上都是可靠的。以上通过对两种方法的全面对比发现,全光谱正交法不仅可以测定选定范围的每个波长的变温曲线,而且可以观测蛋白质的整体结构热变性过程,而用单波长法在同样的实验时间不能有效地记录蛋白构象在热变性过程中的变化过程。

关键词: 圆二色, 热变性, 程序升温, 蛋白

Abstract:

The study of protein thermal stabilization is a critical step in analyzing the higher structure of proteins,and developing protein function and new drugs,and thus is an important concern in the analysis of its structure. At present,the circular dichroism(CD)temperature ramping is a frequently used method to study the protein thermal stabilization. However,traditional method focuses on a single-wavelength temperature ramping CD curve to fit the Tm values,but the information by it is limited and how to select the single-wavelength point is still a controversial issue. This experiment developed and optimized an orthogonal method for the thermal stability analysis of protein secondary structure. We selected bovine serum protein and hemoglobin as research objects,and investigated the process of protein conformation changing with temperature by the temperature-ramping of full range CD spectrum in the range of 180-260 nm. The resules showed that the protein melting temperature(Tm value)of the thermal denaturation was not affected by the concentration when the absorbance was in the proper range. Also,we repeated the experiment by single-wavelength of 180-260 nm and the results showed that the Tm values measured by single-wavelength method(except the endpoint and intersection)and the full spectrum orthogonal method was consistent,indicating that the two methods on the determination of Tm values were reliable. In conclusion,the full spectrum orthogonal method can not only used to determine the temperature curve of each wavelength of the CD spectrum in the selected range,but also to observe the detailed protein thermal denaturation process by the CD spectrum analysis at every temperature points,while the single-wavelength method does not record the changes of protein conformation under the same experimental time.

Key words: circular dichroism, thermal denaturation, temperature-ramping, protein