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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]
  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. https://doi.org/10.1039/C9SC02630D.
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
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.
@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}
}

Journal Article:
Free Publicly Available Full Text
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https://doi.org/10.1039/C9SC02630D

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Cited by: 3 works
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Works referenced in this record:

First-principles calculations of X-ray absorption spectra at the K-edge of 3d transition metals: an electronic structure analysis of the pre-edge
journal, January 2010

  • Cabaret, Delphine; Bordage, Amélie; Juhin, Amélie
  • Physical Chemistry Chemical Physics, Vol. 12, Issue 21
  • DOI: 10.1039/b926499j

ATHENA , ARTEMIS , HEPHAESTUS : data analysis for X-ray absorption spectroscopy using IFEFFIT
journal, June 2005


Probing Cytochrome c Folding Transitions upon Phototriggered Environmental Perturbations Using Time-Resolved X-ray Scattering
journal, April 2018

  • Rimmerman, Dolev; Leshchev, Denis; Hsu, Darren J.
  • The Journal of Physical Chemistry B, Vol. 122, Issue 20
  • DOI: 10.1021/acs.jpcb.8b03354

Heme Photolysis Occurs by Ultrafast Excited State Metal-to-Ring Charge Transfer
journal, May 2001


Ab initio electron density determination directly from solution scattering data
journal, January 2018


Spin-state/stereochemical relationships in iron porphyrins: implications for the hemoproteins
journal, December 1981

  • Scheidt, W. Robert; Reed, Christopher A.
  • Chemical Reviews, Vol. 81, Issue 6
  • DOI: 10.1021/cr00046a002

BioCARS: a synchrotron resource for time-resolved X-ray science
journal, May 2011


Insulin hexamer dissociation dynamics revealed by photoinduced T -jumps and time-resolved X-ray solution scattering
journal, January 2018

  • Rimmerman, Dolev; Leshchev, Denis; Hsu, Darren J.
  • Photochemical & Photobiological Sciences, Vol. 17, Issue 7
  • DOI: 10.1039/C8PP00034D

Role of Heme in Structural Organization of Cytochrome c Probed by Semisynthesis
journal, November 1999

  • Kang, Xinshan; Carey, Jannette
  • Biochemistry, Vol. 38, Issue 48
  • DOI: 10.1021/bi9919089

Fast events in protein folding initiated by nanosecond laser photolysis.
journal, December 1993

  • Jones, C. M.; Henry, E. R.; Hu, Y.
  • Proceedings of the National Academy of Sciences, Vol. 90, Issue 24
  • DOI: 10.1073/pnas.90.24.11860

Role of Ligand Substitution in Ferrocytochrome c Folding
journal, February 1999

  • Telford, Jason R.; Tezcan, F. Akif; Gray, Harry B.
  • Biochemistry, Vol. 38, Issue 6
  • DOI: 10.1021/bi981933z

Prediction of the association state of insulin using spectral parameters
journal, April 2003

  • Uversky, Vladimir N.; Garriques, Liza Nielsen; Millett, Ian S.
  • Journal of Pharmaceutical Sciences, Vol. 92, Issue 4
  • DOI: 10.1002/jps.10355

Resonant Inelastic X-ray Scattering on Ferrous and Ferric Bis-imidazole Porphyrin and Cytochrome c : Nature and Role of the Axial Methionine–Fe Bond
journal, December 2014

  • Kroll, Thomas; Hadt, Ryan G.; Wilson, Samuel A.
  • Journal of the American Chemical Society, Vol. 136, Issue 52
  • DOI: 10.1021/ja5100367

X-ray snapshots for metalloporphyrin axial ligation
journal, January 2010

  • Chen, Lin X.; Zhang, Xiaoyi; Wasinger, Erik C.
  • Chemical Science, Vol. 1, Issue 5
  • DOI: 10.1039/c0sc00323a

X-ray absorption spectroscopic characterization of a cytochrome P450 compound II derivative
journal, January 2008

  • Newcomb, M.; Halgrimson, J. A.; Horner, J. H.
  • Proceedings of the National Academy of Sciences, Vol. 105, Issue 24
  • DOI: 10.1073/pnas.0708299105

Rate of Intrachain Diffusion of Unfolded Cytochrome c
journal, March 1997

  • Hagen, Stephen J.; Hofrichter, James; Eaton, William A.
  • The Journal of Physical Chemistry B, Vol. 101, Issue 13
  • DOI: 10.1021/jp9622997

Bayesian estimation of hyperparameters for indirect Fourier transformation in small-angle scattering
journal, December 2000


On the Role of Heme in the Formation of the Structure of Cytochrome c
journal, May 1973

  • Fisher, Waldo R.; Taniuchi, Hiroshi; Anfinsen, Christian B.
  • Journal of Biological Chemistry, Vol. 248, Issue 9
  • DOI: 10.1016/S0021-9258(19)44026-X

Detailed Transient Heme Structures of Mb-CO in Solution after CO Dissociation: An X-ray Transient Absorption Spectroscopic Study
journal, November 2012

  • Stickrath, Andrew B.; Mara, Michael W.; Lockard, Jenny V.
  • The Journal of Physical Chemistry B, Vol. 117, Issue 16
  • DOI: 10.1021/jp3086705

Role of cofactors in metalloprotein folding
journal, November 2004

  • Wilson, Corey J.; Apiyo, David; Wittung-Stafshede, Pernilla
  • Quarterly Reviews of Biophysics, Vol. 37, Issue 3-4
  • DOI: 10.1017/S003358350500404X

Protein Denaturation:  A Small-Angle X-ray Scattering Study of the Ensemble of Unfolded States of Cytochrome c
journal, September 1998

  • Segel, Daniel J.; Fink, Anthony L.; Hodgson, Keith O.
  • Biochemistry, Vol. 37, Issue 36
  • DOI: 10.1021/bi980535t

Parameter-free calculations of X-ray spectra with FEFF9
journal, January 2010

  • Rehr, John J.; Kas, Joshua J.; Vila, Fernando D.
  • Physical Chemistry Chemical Physics, Vol. 12, Issue 21
  • DOI: 10.1039/b926434e

Recent advances on ultrafast X-ray spectroscopy in the chemical sciences
journal, January 2014

  • Chen, L. X.; Zhang, X.; Shelby, M. L.
  • Chem. Sci., Vol. 5, Issue 11
  • DOI: 10.1039/C4SC01333F

Ligand binding processes in hemoglobin. Chemical reactivity of iron studied by XANES spectroscopy
journal, December 1985


Protein structural dynamics in solution unveiled via 100-ps time-resolved x-ray scattering
journal, April 2010

  • Cho, H. S.; Dashdorj, N.; Schotte, F.
  • Proceedings of the National Academy of Sciences, Vol. 107, Issue 16
  • DOI: 10.1073/pnas.1002951107

Femtosecond photolysis of CO-ligated protoheme and hemoproteins: appearance of deoxy species with a 350-fsec time constant.
journal, January 1983

  • Martin, J. L.; Migus, A.; Poyart, C.
  • Proceedings of the National Academy of Sciences, Vol. 80, Issue 1
  • DOI: 10.1073/pnas.80.1.173

Optical Triggers of Protein Folding
journal, October 1996


Cytochrome c/cardiolipin relations in mitochondria: a kiss of death
journal, June 2009


Structural Dynamics of Light-Driven Proton Pumps
journal, September 2009


Alzheimer's Disease and the Amyloid-β Peptide
journal, January 2010

  • Murphy, M. Paul; LeVine, Harry
  • Journal of Alzheimer's Disease, Vol. 19, Issue 1
  • DOI: 10.3233/JAD-2010-1221

Determination of Iron−Ligand Bond Lengths in Ferric and Ferrous Horse Heart Cytochrome c Using Multiple-Scattering Analyses of XAFS Data
journal, December 1999

  • Cheng, Ming-Chu; Rich, Anne M.; Armstrong, Robert S.
  • Inorganic Chemistry, Vol. 38, Issue 25
  • DOI: 10.1021/ic990395r

Spectroscopic Characterization of Nonnative Conformational States of Cytochrome c
journal, June 2002

  • Oellerich, Silke; Wackerbarth, Hainer; Hildebrandt, Peter
  • The Journal of Physical Chemistry B, Vol. 106, Issue 25
  • DOI: 10.1021/jp013841g

New insight into metalloporphyrin excited state structures and axial ligand binding from X-ray transient absorption spectroscopic studies
journal, October 2014

  • Shelby, Megan L.; Mara, Michael W.; Chen, Lin X.
  • Coordination Chemistry Reviews, Vol. 277-278
  • DOI: 10.1016/j.ccr.2014.05.025

Deciphering Solution Scattering Data with Experimentally Guided Molecular Dynamics Simulations
journal, January 2015

  • Björling, Alexander; Niebling, Stephan; Marcellini, Moreno
  • Journal of Chemical Theory and Computation, Vol. 11, Issue 2
  • DOI: 10.1021/ct5009735

The Protein-Folding Problem, 50 Years On
journal, November 2012


Time-Resolved Circular Dichroism Studies of Protein Folding Intermediates of Cytochrome c
journal, April 1998

  • Chen, Eefei; Wood, Matthew J.; Fink, Anthony L.
  • Biochemistry, Vol. 37, Issue 16
  • DOI: 10.1021/bi972369f

Molten Globule of Cytochrome c Studied by Small Angle X-ray Scattering
journal, February 1993

  • Kataoka, Mikio; Hagihara, Yoshihisa; Mihara, Ken'ichi
  • Journal of Molecular Biology, Vol. 229, Issue 3
  • DOI: 10.1006/jmbi.1993.1064

Folding of horse cytochrome c in the reduced state11Edited by C. R. Matthews
journal, October 2001

  • Bhuyan, Abani K.; Udgaonkar, Jayant B.
  • Journal of Molecular Biology, Vol. 312, Issue 5
  • DOI: 10.1006/jmbi.2001.4993

High-resolution three-dimensional structure of horse heart cytochrome c
journal, July 1990


Kinetic spectroscopy of heme hydration and ligand binding in myoglobin and isolated hemoglobin chains: an optical window into heme pocket water dynamics
journal, January 2010

  • Esquerra, Raymond M.; López-Peña, Ignacio; Tipgunlakant, Pooncharas
  • Physical Chemistry Chemical Physics, Vol. 12, Issue 35
  • DOI: 10.1039/c003606b

Electronic and Nuclear Structural Snapshots in Ligand Dissociation and Recombination Processes of Iron Porphyrin in Solution: A Combined Optical/X-ray Approach
journal, October 2013

  • Mara, Michael W.; Shelby, Megan; Stickrath, Andrew
  • The Journal of Physical Chemistry B, Vol. 117, Issue 45
  • DOI: 10.1021/jp407094u

Folding Mechanism of Reduced Cytochrome c: Equilibrium and Kinetic Properties in the Presence of Carbon Monoxide
journal, November 2008

  • Latypov, Ramil F.; Maki, Kosuke; Cheng, Hong
  • Journal of Molecular Biology, Vol. 383, Issue 2
  • DOI: 10.1016/j.jmb.2008.08.025

Molecular simulations of metal-coupled protein folding
journal, February 2015


Protein Folding Dynamics of Cytochrome c Seen by Transient Grating and Transient Absorption Spectroscopies
journal, March 2011

  • Choi, Jungkweon; Yang, Cheolhee; Kim, Jeongho
  • The Journal of Physical Chemistry B, Vol. 115, Issue 12
  • DOI: 10.1021/jp106588d

Structural Ensembles of Intrinsically Disordered Proteins Depend Strongly on Force Field: A Comparison to Experiment
journal, October 2015

  • Rauscher, Sarah; Gapsys, Vytautas; Gajda, Michal J.
  • Journal of Chemical Theory and Computation, Vol. 11, Issue 11
  • DOI: 10.1021/acs.jctc.5b00736

Metalloprotein entatic control of ligand-metal bonds quantified by ultrafast x-ray spectroscopy
journal, June 2017


Ultrafast myoglobin structural dynamics observed with an X-ray free-electron laser
journal, April 2015

  • Levantino, Matteo; Schirò, Giorgio; Lemke, Henrik Till
  • Nature Communications, Vol. 6, Issue 1
  • DOI: 10.1038/ncomms7772

Polarized X-ray absorption spectroscopy of the low-temperature photoproduct of carbonmonoxy-myoglobin
journal, November 1999

  • Della Longa, Stefano; Arcovito, Alessandro; Vallone, Beatrice
  • Journal of Synchrotron Radiation, Vol. 6, Issue 6
  • DOI: 10.1107/S0909049599010845

X-ray absorption spectroscopy of carbonyl basket handle Fe(II) porphyrins: the distortion of the tetrapyrrolic macrocycle
journal, February 1992

  • Cartier, Christophe; Momenteau, Michel; Dartyge, Elisabeth
  • Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, Vol. 1119, Issue 2
  • DOI: 10.1016/0167-4838(92)90387-S

Induction of Apoptotic Program in Cell-Free Extracts: Requirement for dATP and Cytochrome c
journal, July 1996


A Multiplet Analysis of Fe K-Edge 1s → 3d Pre-Edge Features of Iron Complexes
journal, July 1997

  • Westre, Tami E.; Kennepohl, Pierre; DeWitt, Jane G.
  • Journal of the American Chemical Society, Vol. 119, Issue 27
  • DOI: 10.1021/ja964352a

Structural characterization of proteins and complexes using small-angle X-ray solution scattering
journal, October 2010


X-ray absorption spectroscopy of chloroperoxidase compound I: Insight into the reactive intermediate of P450 chemistry
journal, November 2005

  • Stone, K. L.; Behan, R. K.; Green, M. T.
  • Proceedings of the National Academy of Sciences, Vol. 102, Issue 46
  • DOI: 10.1073/pnas.0507069102

A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins
journal, August 2015


Direct Observation of Insulin Association Dynamics with Time-Resolved X-ray Scattering
journal, September 2017

  • Rimmerman, Dolev; Leshchev, Denis; Hsu, Darren J.
  • The Journal of Physical Chemistry Letters, Vol. 8, Issue 18
  • DOI: 10.1021/acs.jpclett.7b01720

Analysis of small-angle scattering data using model fitting and Bayesian regularization
journal, July 2018

  • Larsen, Andreas Haahr; Arleth, Lise; Hansen, Steen
  • Journal of Applied Crystallography, Vol. 51, Issue 4
  • DOI: 10.1107/S1600576718008956

Heme Coordination States of Unfolded Ferrous Cytochrome c
journal, October 2006


Rapid Intrachain Binding of Histidine-26 and Histidine-33 to Heme in Unfolded Ferrocytochrome c
journal, January 2002

  • Hagen, Stephen J.; Latypov, Ramil F.; Dolgikh, Dimitry A.
  • Biochemistry, Vol. 41, Issue 4
  • DOI: 10.1021/bi011371a

Tracking the structural dynamics of proteins in solution using time-resolved wide-angle X-ray scattering
journal, September 2008

  • Cammarata, Marco; Levantino, Matteo; Schotte, Friedrich
  • Nature Methods, Vol. 5, Issue 10
  • DOI: 10.1038/nmeth.1255

XAFS study of CO binding to denatured cytochrome c
journal, March 1995


Interpretation of Solution X-Ray Scattering by Explicit-Solvent Molecular Dynamics
journal, May 2015


Pump-Probe X-ray Solution Scattering Reveals Accelerated Folding of Cytochrome c Upon Suppression of Misligation
journal, March 2014

  • Kim, Tae Wu; Kim, Jong Goo; Yang, Cheolhee
  • Bulletin of the Korean Chemical Society, Vol. 35, Issue 3
  • DOI: 10.5012/bkcs.2014.35.3.697

Protein Tertiary Structural Changes Visualized by Time-Resolved X-ray Solution Scattering
journal, October 2009

  • Ahn, Sena; Kim, Kyung Hwan; Kim, Youngmin
  • The Journal of Physical Chemistry B, Vol. 113, Issue 40
  • DOI: 10.1021/jp906983v

Application of Bayesian analysis to indirect Fourier transformation in small-angle scattering
journal, November 2006


Time Scales for the Formation of the Most Probable Tertiary Contacts in Proteins with Applications to Cytochrome c
journal, January 1999

  • Thirumalai, D.
  • The Journal of Physical Chemistry B, Vol. 103, Issue 4
  • DOI: 10.1021/jp982362n

Direct Observation of Node-to-Node Communication in Zeolitic Imidazolate Frameworks
journal, September 2018

  • Pattengale, Brian; SantaLucia, Daniel J.; Yang, Sizhuo
  • Journal of the American Chemical Society, Vol. 140, Issue 37
  • DOI: 10.1021/jacs.8b06727

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