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Expression and Solution NMR Study of Multi-site Phosphomimetic Mutant BCL-2 Protein

[ Vol. 26 , Issue. 6 ]

Author(s):

Ting Song, Keke Cao, Yu dan Fan, Zhichao Zhang*, Zong W. Guo, Min H. Zhang and Peng Liu   Pages 449 - 457 ( 9 )

Abstract:


Background: The significance of multi-site phosphorylation of BCL-2 protein in the flexible loop domain remains controversial, in part due to the lack of structural biology studies of phosphorylated BCL-2.

Objective: The purpose of the study is to explore the phosphorylation induced structural changes of BCL-2 protein.

Methods: We constructed a phosphomietic mutant BCL-2(62-206) (t69e, s70e and s87e) (EEEBCL- 2-EK (62-206)), in which the BH4 domain and the part of loop region was truncated (residues 2-61) to enable a backbone resonance assignment. The phosphorylation-induced structural change was visualized by overlapping a well dispersed 15N-1H heteronuclear single quantum coherence (HSQC) NMR spectroscopy between EEE-BCL-2-EK (62-206) and BCL-2.

Results: The EEE-BCL-2-EK (62-206) protein reproduced the biochemical and cellular activity of the native phosphorylated BCL-2 (pBCL-2), which was distinct from non-phosphorylated BCL-2 (npBCL-2) protein. Some residues in BH3 binding groove occurred chemical shift in the EEEBCL- 2-EK (62-206) spectrum, indicating that the phosphorylation in the loop region induces a structural change of active site.

Conclusion: The phosphorylation of BCL-2 induced structural change in BH3 binding groove.

Keywords:

Phosphorylated BCL-2, purification, NMR spectroscopy, structural change, chemical shift, BH3 binding groove.

Affiliation:

State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian, State Key Laboratory of Fine Chemicals, School of Chemistry, Dalian University of Technology, Dalian, School of Life Science and Technology, Dalian University of Technology, Dalian, School of Innovation Experiment, Dalian University of Technology, Dalian, School of Life Science and Technology, Dalian University of Technology, Dalian, School of Life Science and Technology, Dalian University of Technology, Dalian, School of Life Science and Technology, Dalian University of Technology, Dalian

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