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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 similar to 2 mu s 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 misligationmore » to His26/His33 residues.« less

Authors:
ORCiD logo [1]; ORCiD logo [1];  [1];  [1];  [1];  [2];  [3]; ORCiD logo [1]
+ Show Author Affiliations
  1. Department of Chemistry, Northwestern University, Evanston, USA
  2. Center for Advanced Radiation Sources, The University of Chicago, USA
  3. X-ray Sciences Division of the Advanced Photon Source, Argonne National Laboratory, Argonne, USA
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States); Oak Ridge Associated Univ., Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC); National Institutes of Health (NIH) - National Institute of General Medical Sciences
OSTI Identifier:
1560756
Alternate Identifier(s):
OSTI ID: 1624979; OSTI ID: 1657824
Grant/Contract Number:  
AC02-06CH11357; SC0014664; R01-GM115761; R24GM111072
Resource Type:
Published Article
Journal Name:
Chemical Science
Additional Journal Information:
Journal Name: Chemical Science Journal Volume: 10 Journal Issue: 42; Journal ID: ISSN 2041-6520
Publisher:
Royal Society of Chemistry
Country of Publication:
United Kingdom
Language:
English
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; Chemistry

Citation Formats

Hsu, Darren J., Leshchev, Denis, Rimmerman, Dolev, Hong, Jiyun, Kelley, Matthew S., Kosheleva, Irina, Zhang, Xiaoyi, and Chen, Lin X.. X-ray snapshots reveal conformational influence on active site ligation during metalloprotein folding. United Kingdom: N. p., 2019. Web. doi:10.1039/C9SC02630D.
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Hsu, Darren J., Leshchev, Denis, Rimmerman, Dolev, Hong, Jiyun, Kelley, Matthew S., Kosheleva, Irina, Zhang, Xiaoyi, & Chen, Lin X.. X-ray snapshots reveal conformational influence on active site ligation during metalloprotein folding. United Kingdom. https://doi.org/10.1039/C9SC02630D
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Hsu, Darren J., Leshchev, Denis, Rimmerman, Dolev, Hong, Jiyun, Kelley, Matthew S., Kosheleva, Irina, Zhang, Xiaoyi, and Chen, Lin X.. Wed . "X-ray snapshots reveal conformational influence on active site ligation during metalloprotein folding". United Kingdom. https://doi.org/10.1039/C9SC02630D.
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@article{osti_1560756,
title = {X-ray snapshots reveal conformational influence on active site ligation during metalloprotein folding},
author = {Hsu, Darren J. and Leshchev, Denis and Rimmerman, Dolev and Hong, Jiyun and Kelley, Matthew S. and Kosheleva, Irina and Zhang, Xiaoyi and Chen, Lin X.},
abstractNote = {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 similar to 2 mu s 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.},
doi = {10.1039/C9SC02630D},
journal = {Chemical Science},
number = 42,
volume = 10,
place = {United Kingdom},
year = {2019},
month = {10}
}
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https://doi.org/10.1039/C9SC02630D
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