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

doi:10.11938/cjmr20182637

Abstract

Abstract: 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 ¹H-¹⁵N 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.

Key words: denaturing-refolding, quick-dilution, nuclear magnetic resonance (NMR), AtGrp7 RNA recognition motif (RRM)domain, binding analysis

CLC Number:

O482.53

CHI Xiu-juan, QIAO Xiao-ya, LIU Ying, LIU Hui-li, CHEN Lei, WANG Ji-hui, AI Xuan-jun. Purification of the AtGrp7 RRM Domain from Arabidopsis thaliana and Its Preliminary Structure and Binding Analysis[J]. Chinese Journal of Magnetic Resonance, 2019, 36(1): 1-14.

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