基于蛋白质二级结构热稳定性的正交方法

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,...

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

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.