Purification of the AtGrp7 RRM Domain from Arabidopsis thaliana and Its Preliminary Structure and Binding Analysis

The glycine-rich RNA-binding protein, AtGrp7, is a component of a negative feedback loop in the circadian clock regulation of Arabidopsis thaliana . In our initial purification trial of the tobacco etch virus (TEV)-cleaved AtGrp7 RNA recognition motif (RRM) domain with the regular protocol, mixed ultraviolet signals of the target proteins and contaminants were observed. A two-step denaturing-refolding protocol was then tested, trying to solve the problem of impurities. The structure of the AtGrp7 1-90 RRM domain was fully recovered by quick-dilution refolding, evidenced by the fingerprint 1 H- 15 N HSQC spectrum and CS-Rosetta model structures. Isothermal titration calorimetry (ITC) and NMR titration experiments further confirmed that the RRM domain of AtGrp7 1-90 had proper functions with regards to RNA/DNA binding.