Abstract
Human cytochrome c is a multi-functional protein with key roles in both the mitochondrial electron transfer chain and in apoptosis. In the latter, a complex formed between the mitochondrial phospholipid cardiolipin and cytochrome c is crucial for instigating the release of pro-apoptotic factors, including cytochrome c, from the mitochondrion into the cytosol. The G41S mutant of human cytochrome c is the only known disease-related variant of cytochrome c and causes increased apoptotic activity in patients with autosomal dominant thrombocytopenia. NMR spectroscopy can be used to investigate the interaction of human cytochrome c with cardiolipin and the structural and dynamic factors, which may contribute to enhanced apoptotic activity for the G41S mutant. We present here essentially full backbone amide resonance assignments for ferric human cytochrome c (98 %) as well as assignments of both the ferric (92 %) and ferrous (95 %) forms of the G41S mutant. Backbone amide chemical shift differences between the wild type and G41S mutant in the ferric state reveals significant changes around the mutation site, with many other amides also affected. This suggests the possibility of increased dynamics and/or a change in the paramagnetic susceptibility tensor of the G41S mutant relative to the wild type protein.
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Acknowledgments
We acknowledge the open access use of the Wellcome Trust-funded (grants 083796/Z/07/Z and 099185/Z/12/Z) 900 MHz spectrometer at the HWB-NMR facility at the University of Birmingham and thank Dr Sara Whittaker for assistance with the acquisition of the triple resonance experiments at this facility. This work was supported by a Leverhulme Trust project grant (RPG-2013-164) to JARW and a Leverhulme Trust emeritus fellowship (EM-2014-088) to GRM.
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Karsisiotis, A.I., Deacon, O.M., Rajagopal, B.S. et al. Backbone resonance assignments of ferric human cytochrome c and the pro-apoptotic G41S mutant in the ferric and ferrous states. Biomol NMR Assign 9, 415–419 (2015). https://doi.org/10.1007/s12104-015-9621-3
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DOI: https://doi.org/10.1007/s12104-015-9621-3