Abstract：Metacaspases are cysteine-dependent proteases found in protozoa, fungi and plants and are distantly related to metazoan caspases. Most of MCPs activation are the calcium dependent, but the mechanisms are still unknown. Based on the techniques of CD spectroscopy, fluorescence spectroscopy, and Terbium Stains-all probe, we selected three purified recombinant proteins from key residues mutated in tomato metacaspase (LeMCA1), including conserved catalytic site (C139A) mutant, N-sequenced cleaved site (K223G) mutant and the predicted Ca2+ binding sites (D116A/D117A) mutant, to explore the interaction mechanism of LeMCA1 and Ca2+. CD spectroscopy and Stains-all probe results suggested that the intense binding does not exist between LeMCA1 and Ca2+ as well as Ca2+ has little effect on the secondary structure of LeMCA1. However, fluorescence spectroscopy and Tb3+ probe results showed that Ca2+-induced the changes occur in the tertiary structure of LeMCA1, which contributes to the activation of zymogen. In addition, predicted Ca2+ binding residues, Asp-116 and Asp117, are the key sites responsible for the Ca2+ interaction with LeMCA1, and the loss of these two residues resulted in decreased interaction. Our data firstly provided insight on the mechanism of the interaction between Ca2+ and recombinant purified Solanaceae type Ⅱ metacaspase by spectroscopy and molecular probe techniques. Combined the results we got before from sequence-alignment and sites-mutation, the key residues Asp-116 and Asp117 affect the Ca2+-induced the changes of LeMCA1 tertiary structure. Our data provided information for the further biochemical and crystal assays of LeMCA1.
温 帅，马营轩，吴坤胜，崔金田，罗云波，曲桂芹* . 光谱及分子探针方法研究番茄Metacaspase蛋白与Ca2+的相互作用 [J]. 光谱学与光谱分析, 2015, 35(06): 1643-1648.
WEN Shuai, MA Ying-xuan, WU Kun-sheng, CUI Jin-tian, LUO Yun-bo, QU Gui-qin* . Research on the Tomato Metacaspase Protein Interactions with Ca2+ by Spectroscopy and Molecular Probe . SPECTROSCOPY AND SPECTRAL ANALYSIS, 2015, 35(06): 1643-1648.
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