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JKMRS Volume 20, No 2, pp 56; NMR Signal Assignments of Human ...
2016년 06월 20일 / 조회수: 582

NMR Signal Assignments of Human Adenylate Kinase 1 (hAK1) and

its R138A Mutant (hAK1R138A)

 

 

Gilhoon Kim1, Hwanbong Chang2*, and Hoshik Won1

 

 

1 Department of Applied Chemistry, Hanyang University, 55, Hanyang Daehak-Ro,

Sangrok-Gu, Ansan City, 426-791, Korea

2Dong-Wha Pharm. Co., 71, Tapsil-ro 35beon-gil, Giheung-Gu, Yongin City, 17084, Korea

 

 

 

 

Received Apr 10, 2016; Revised May 12, 2016; Accepted June 2, 2016

 


Abstract Adenylate kinase (AK) enzyme which acts as the catalyst of reversible high energy phosphorylation reaction between ATP and AMP which associate with energetic metabolism and nucleic acid synthesis and signal transmission. This enzyme has three distinct domains: Core, AMP binding domain (AMPbd) and Lid domain (LID). The primary role of AMPbd and LID is associated with conformational changes due to flexibility of two domains. Three dimensional structure of human AK1 has not been confirmed and various mutation experiments have been done to determine the active sites. In this study, AK1R138A which is changed arginine[138] of LID domain with alanine[138] was made and conducted with NMR experiments, backbone dynamics analysis and mo-lecular docking dynamic simulation to find the cause of structural change and substrate binding site. Synthetic human muscle type adenylate kinase 1 (hAK1) and its mutant (AK1R138A) were re-combinded with E. coli and expressed in M9 cell. Expressed proteins were purified and finally gained at 0.520 mM hAK1 and 0.252 mM AK1R138A. Multinuclear multidimensional NMR experiments including HNCA, HN(CO)CA, were conducted for amino acid sequence analysis and signal assignments of 1H-15N HSQC spectrum. Our chemical shift perturbation data is shown LID domain residues and around alanine[138] and per-turbation value(0.22ppm) of valine[179] is consid-ered as inter-communication effect with LID domain and the structural change between hAK1 and AK1R138A.

 

Keywords HNCA, HN(CO)CA, HSQC, NMR, AK1
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