@article{f4850617012c4f5292069a1f46039a2f,
title = "X-ray snapshots reveal conformational influence on active site ligation during metalloprotein folding",
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.",
author = "Hsu, {Darren J.} and Denis Leshchev and Dolev Rimmerman and Jiyun Hong and Kelley, {Matthew S.} and Irina Kosheleva and Xiaoyi Zhang and Chen, {Lin X.}",
note = "Funding Information: The authors thank Dr Michael Mara and Dr Brian Phelan for fruitful discussions. This work was supported by the National Institute of Health (NIH), under contract no. R01-GM115761. D. J. H. acknowledges support from the National Institute of General Medical Sciences (NIGMS) of NIH for a training grant (5T32GM008382) as well as the U.S. Department of Energy (DOE), Office of Science Graduate Student Research program, administered by the Oak Ridge Institute for Science and Education, managed by ORAU under contract number DE-SC0014664. This research used resources of the APS, a U.S. DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. Use of BioCARS was also supported by the NIGMS of the NIH under grant number R24GM111072. Time-resolved setup at Sector 14 was funded in part through collaboration with Philip Anfinrud (NIH/NIDDK). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We would also like to acknowledge Guy Macha (BioCARS) for his assistance in designing the sample holder. Portions of this work were performed at the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) located at Sector 5 of the APS. DND-CAT is supported by Northwestern University, E.I. DuPont de Nemours & Co., and The Dow Chemical Company. Data was collected using an instrument funded by the National Science Foundation under Award Number 0960140. Funding Information: The authors thank Dr Michael Mara and Dr Brian Phelan for fruitful discussions. This work was supported by the National Institute of Health (NIH), under contract no. R01-GM115761. D. J. H. acknowledges support from the National Institute of General Medical Sciences (NIGMS) of NIH for a training grant (5T32GM008382) as well as the U.S. Department of Energy (DOE), Office of Science Graduate Student Research program, administered by the Oak Ridge Institute for Science and Education, managed by ORAU under contract number DESC0014664. This research used resources of the APS, a U.S. DOE Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357. Use of BioCARS was also supported by the NIGMS of the NIH under grant number R24GM111072. Time-resolved setup at Sector 14 was funded in part through collaboration with Philip Annrud (NIH/NIDDK). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. We would also like to acknowledge Guy Macha (BioCARS) for his assistance in designing the sample holder. Portions of this work were performed at the DuPont-Northwestern-Dow Collaborative Access Team (DND-CAT) located at Sector 5 of the APS. DND-CAT is supported by Northwestern University, E.I. DuPont de Nemours & Co., and The Dow Chemical Company. Data was collected using an instrument funded by the National Science Foundation under Award Number 0960140. Publisher Copyright: This journal is {\textcopyright} The Royal Society of Chemistry.",
year = "2019",
doi = "10.1039/c9sc02630d",
language = "English (US)",
volume = "10",
pages = "9788--9800",
journal = "Chemical Science",
issn = "2041-6520",
publisher = "Royal Society of Chemistry",
number = "42",
}