Browsing by Author "Goulet, Danica"

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    Studies on the unfolding and zinc status of thermolysin and carboxypeptidase A, and the design of a fret-quenched peptide for assaying thermolysin-like proteases
    (2020-09-16) Goulet, Danica
    The chromophoric chelator 4-(2-pyridylazo)resorcinol (PAR) has previously been employed as a probe to determine structural transitions during protein (un)folding in the zincdependent anthrax lethal factor protease (LF). Whether PAR can be employed more widely as a zinc accessibility probe (ZAP) to study the unfolding of other zinc proteins remains to be established. Using a combination of intrinsic tryptophan fluorescence spectroscopy and chelator studies, the unfolding pathways and the influence of the protein fold on the metal status were investigated for the zinc proteases thermolysin (TL) and carboxypeptidase A (CPA). These studies revealed considerable differences in the unfolding pathways and the resistance to metal dissociation for these enzymes. In addition, the observations reported herein demonstrate that ZAPs might be of value in future investigations to gain further insight into the mechanisms underlying the metal-mediated misfolding of proteins involved in a variety of neurodegenerative diseases. Finally, this study describes a new fluorescence-quenched heptapeptide (Dabcyl-FKFLGKE-EDANS), which shows the largest specificity constant documented for any TL substrate.