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

Abstract

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 19F probe for determination of conformational ratio under various temperatures using 19F 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 19F-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 19F-labeled pUb thermosensor could be developed to detect intracellular temperature in-cell NMR assays.

Keywords

• ubiquitin
• phosphorylation
• nuclear magnetic resonance (nmr)
• thermosensor