基于^(19)F化学标记磷酸化泛素的温度传感器开发

Development of a Temperature Senor Based on^(19) F-labeled Phosphorylated Ubiquitin

泛素是一种真核细胞信号分子,主要参与蛋白质降解和DNA修复等生命活动.泛素Ser65位被磷酸化之后,在溶液中呈现两个稳定的溶液构象,这两种构象的比例能够被pH调控,本研究利用NMR进一步发现它还受到温度影响.基于该发现,对磷酸化泛素进行了^(19)F化学标记,利用^(19)F NMR方法表征了不同温度下磷酸化泛素两种构象的比例,发现两者比例变化与温度之间的关系可以通过线性方程来描述,利用该方程可以通过构象比例计算样品内部温度,因此可以作为一种基于NMR检测的温度传感器.本文所开发的基于^(19)F化学标记磷酸化泛素的温度传感器不仅能够作为体外样品温度检测的有力工具,还有望用于检测细胞内部的温度.从而有助于揭示生物学特性和功能.

Ubiquitin(Ub)is a signaling protein in eukaryotic cells,and it functions in the processes of protein degradation and DNA repairing.The published data indicated that the phosphorylation of Ub on Ser65 leads to two stable solution conformations,which can be regulated by pH.In the present project,it was found that the conformational ratio could also be modulated by temperature.Based on this feature,pUb was chemically labeled with a^(19)F probe for determination of conformational ratio under various temperatures using^(19)F NMR.We showed that the conformation ratio of pUb was linearly proportional to temperature gradient.Consequently,the temperature of a biological sample can be calculated with a given conformational ratio of pUb,and^(19)F-labeled pUb can function as a thermosensor.This thermosensor could be used to monitor temperature of in vitro experiments,and it is also able to be taken to explore regulation of pUb function by temperature.Furthermore,the^(19)F-labeled pUb thermosensor could be developed to detect intracellular temperature in-cell NMR assays.