Recombinant Expression, Biophysical Characterization, and Cardiolipin-Induced Changes of Two Caenorhabditis elegans Cytochrome c Proteins

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• 作者：Amber J. Vincelli ; Danielle S. Pottinger ; Fangfang Zhong ; Jonas Hanske ; St茅phane G. Rolland ; Barbara Conradt ; Ekaterina V. Pletneva
• 刊名：Biochemistry
• 出版年：2013
• 出版时间：January 29, 2013
• 年：2013
• 卷：52
• 期：4
• 页码：653-666
• 全文大小：651K
• 年卷期：v.52,no.4(January 29, 2013)
• ISSN：1520-4995

文摘

Cytochrome c (cyt c) is one of the most widely studied biomolecules, but not much is known about this protein from nematodes. Recombinant expression of Caenorhabditis elegans CYC-2.1 and CYC-2.2 allowed for detailed characterization of their structural features, redox properties, stabilities, and interactions with cardiolipin (CL)-containing liposomes. Using a variety of spectroscopic tools, we show that CYC-2.1 and CYC-2.2 adopt a globular 伪-helical fold with His/Met heme ligation. The longer CYC-2.2 has a lower thermodynamic stability than CYC-2.1 and lacks His residues to misligate to the heme in the protein鈥檚 denatured state. Both C. elegans proteins bind to CL-containing liposomes, and these interactions promote the proteins鈥?peroxidase activity but to a much greater degree for CYC-2.2. Dye-to-heme distance distributions from time-resolved fluorescence resonance energy transfer in bimane-labeled CYC-2.1 and CYC-2.2 revealed similar populations of extended and compact conformers for CL-bound proteins, suggesting that their distinct peroxidase activities in the presence of CL arise from differences in the local heme environments for the two polypeptide ensembles. Without inhibition from His misligation, a less stable and more prone to unfolding CYC-2.2 allows for better access of substrates to the heme and thus exhibits higher peroxidase activity. Similar features of the conformational ensembles of CYC-2.1 and CYC-2.2 to those of mammalian cyt c suggest that C. elegans proteins, particularly the former, could serve as useful models for examining the mechanism of cyt c鈥揅L interactions in live organisms.