Two fused proteins combining Stichopus japonicus arginine kinase and rabbit muscle creatine kinase.
Two fused proteins of dimeric arginine kinase (AK) from sea cucumber and dimeric creatine kinase (CK) from rabbit muscle, named AK-CK and CK-AK, were obtained through the expression of fused AK and CK genes. Both AK-CK and CK-AK had about 50% AK activity and about 2-fold K(m) values for arginine of native AK, as well as about 50% CK activity and about 2-fold K(m) values for creatine of native CK. This indicated that both AK and CK moieties are fully active in the two fused proteins. The structures of AK, CK, AK-CK, and CK-AK were compared by collecting data of far-UV circular dichroism, intrinsic fluorescence, 1-anilinonaphthalene-8-sulfonate binding fluorescence, and size-exclusion chromatography. The results indicated that dimeric AK and CK differed in the maximum emission wavelength, the exposure extent of hydrophobic surfaces, and molecular size, though they have a close evolutionary relationship. The structure and thermodynamic stability of AK, CK, AK-CK, and CK-AK were compared by guanidine hydrochloride (GdnHCl) titration. Dimeric AK was more dependent on the cooperation of two subunits than CK according to the analysis of residual AK or CK activity with GdnHCl concentration increase. Additionally, AK and CK had different denaturation curves induced by GdnHCl, but almost the same thermodynamic stability. The two fused proteins, AK-CK and CK-AK, had similar secondary structure, tertiary structure, molecular size, structure, and thermodynamic stability, which indicated that the expression order of AK and CK genes might have little effect on the characteristics of the fused proteins and might further verify the close relationship of dimeric AK and CK.
Zhang
Jian-Wei
JW
Department of Biological Sciences and Biotechnology, School of Life Science and Engineering, Tsinghua University, Beijing 100084, China.
Guo
Qin
Q
Zhao
Tong-Jin
TJ
Liu
Tao-Tao
TT
Wang
Xi-Cheng
XC
D000818:Animals; D001122:Arginine Kinase / Q000378:metabolism*; D002942:Circular Dichroism; D052283:Creatine Kinase, MM Form / Q000378:metabolism*; D019281:Dimerization; D019791:Guanidine / Q000494:pharmacology; D007700:Kinetics; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D012616:Sea Cucumbers / Q000201:enzymology*; D013050:Spectrometry, Fluorescence
D011993:Recombinant Fusion Proteins; D052283:Creatine Kinase, MM Form; D001122:Arginine Kinase; D019791:Guanidine