👤 Daniel J Deredge

Ran-binding domain-containing protein 2 (ZRANB2) is a zinc finger (ZF) protein that plays a key role in alternative splicing. ZRANB2 is composed of two ZF domains that contain four invariant cysteine residues per domain. ZRANB2 binds RNA targets that contain AGGUAA sequence motifs. Three constructs of ZRANB2, ZRANB2-ZF1 (first ZF domain), ZRANB2-ZF2 (second ZF domain), and ZRANB2-2D (both ZF domains), were isolated in the apo form and shown to bind Zn(II) via UV-visible-monitored competitive titrations with Co(II) as a spectroscopic probe. Zn binding to each construct led to the adoption of a limited secondary structure of each domain, as measured by circular dichroism (CD). Hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS) of the two-domain construct, ZRANB2-2D, revealed that both ZF domains adopt a more rigid structure upon Zn binding. Zn binding to the first ZF domain resulted in a greater decrease in the conformational dynamics than Zn binding to the second ZF domain. RNA binding to TRA2B pre-mRNA, a physiological splicing target, was measured by fluorescence anisotropy (FA), and high-affinity RNA binding was found to require Zn coordination to both domains. HDX-MS of ZRANB2-2D with TRA2B RNA as well as two optimized RNA sequences that contain a single and double AGGUAA hexamer revealed additional protection from H/D exchange for ZRANB2 in the presence of RNA. Here, greater protection was observed for the second ZF of ZRANB2-2D, suggesting a larger effect on conformational dynamics. A model for zinc-mediated RNA binding of ZRANB2 is proposed. Show less

Cytochrome c (cyt c) is known for its role in the electron transport chain but transitions to a peroxidase-active state upon exposure to oxidative species. The peroxidase activity ultimately results in the release of cyt c into the cytosol for the engagement of apoptosis. The accumulation of oxidative modifications that accompany the onset of the peroxidase function are well-characterized. However, the concurrent structural and conformational transitions of cyt c remain undercharacterized. Fast photochemical oxidation of proteins (FPOP) coupled with mass spectrometry is a protein footprinting technique used to structurally characterize proteins. FPOP coupled with native ion mobility separation shows that exposure to H2O2 results in the accumulation of a compact state of cyt c. Subsequent top-down fragmentation to localize FPOP modifications reveals changes in heme coordination between conformers. A time-resolved functional assay suggests that this compact conformer is peroxidase active. Altogether, combining FPOP, ion mobility separation, and top-down and bottom-up mass spectrometry allows us to discern individual conformations in solution and obtain a better understanding of the conformational ensemble and structural transitions of cyt c as it transitions from a respiratory role to a proapoptotic role. Show less