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Issue 42, 2019
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X-ray snapshots reveal conformational influence on active site ligation during metalloprotein folding

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Abstract

Cytochrome c (cyt c) has long been utilized as a model system to study metalloprotein folding dynamics and the interplay between active site ligation and tertiary structure. However, recent reports regarding the weakness of the native Fe(II)–S bond (Fe–Met80) call into question the role of the active site ligation in the protein folding process. In order to investigate the interplay between protein conformation and active site structures, we directly tracked the evolution of both during a photolysis-induced folding reaction using X-ray transient absorption spectroscopy and time-resolved X-ray solution scattering techniques. We observe an intermediate Fe–Met80 species appearing on ∼2 μs timescale, which should not be sustained without stabilization from the folded protein structure. We also observe the appearance of a new active site intermediate: a weakly interacting Fe–H2O state. As both intermediates require stabilization of weak metal–ligand interactions, we surmise the existence of a local structure within the unfolded protein that protects and limits the movement of the ligands, similar to the entatic state found in the native cyt c fold. Furthermore, we observe that in some of the unfolded ensemble, the local stabilizing structure is lost, leading to expansion of the unfolded protein structure and misligation to His26/His33 residues.

Graphical abstract: X-ray snapshots reveal conformational influence on active site ligation during metalloprotein folding

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Supplementary files

Article information


Submitted
30 May 2019
Accepted
01 Sep 2019
First published
03 Sep 2019

This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry

Chem. Sci., 2019,10, 9788-9800
Article type
Edge Article

X-ray snapshots reveal conformational influence on active site ligation during metalloprotein folding

D. J. Hsu, D. Leshchev, D. Rimmerman, J. Hong, M. S. Kelley, I. Kosheleva, X. Zhang and L. X. Chen, Chem. Sci., 2019, 10, 9788
DOI: 10.1039/C9SC02630D

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