Protein misfolding is the result of amino acids incorrectly folding into their final quaternary structure. The process leads to the final protein structures that can not perform the original function of the native fold. Neurodegenerative diseases such as Alzhiemers can arise when these misfolded proteins aggregate, forming masses known as amyloid fibrils. One aspect of the protein unfolding research aims to better understand the mechanisms of unfolding reaction and aggregate formation. In this study we examined cytochrome c because of its small size, well-known structure, and most importantly the presence of the heme group that can serve as an internal chromophore. The cytochrome c can not be used in the emission studies of the unfolding process directly as the native heme with axially ligated centrally positioned iron does not have fluorescent properties. However, the substitution of the d5 metal Iron with a d10 metal such as Zinc yields the fluorescent protein without altering its structure. Our work demonstrates the safe, efficient and reproducible protocol for demetalation/remetalation of cytochrome c. The protocol is appropriate for the protein folding/unfolding studies in the undergraduate laboratories. The viability of the Zn substituted cytochrome c product for fluorescence based folding/unfolding studies was tested and confirmed using traditional unfolding methods and standard denaturants.

Demetallation and remetallation of cytochrome c for the fluorescence studies of protein unfolding process in undergraduate laboratory courses

ACS Spring 2022

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