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Title: Unfolded states under folding conditions accommodate sequence-specific conformational preferences with random coil-like dimensions

Abstract

Proteins are marginally stable molecules that fluctuate between folded and unfolded states. Here, we provide a high-resolution description of unfolded states under refolding conditions for the N-terminal domain of the L9 protein (NTL9). We use a combination of time-resolved Förster resonance energy transfer (FRET) based on multiple pairs of minimally perturbing labels, time-resolved small-angle X-ray scattering (SAXS), all-atom simulations, and polymer theory. Upon dilution from high denaturant, the unfolded state undergoes rapid contraction. Although this contraction occurs before the folding transition, the unfolded state remains considerably more expanded than the folded state and accommodates a range of local and nonlocal contacts, including secondary structures and native and nonnative interactions. Paradoxically, despite discernible sequence-specific conformational preferences, the ensemble-averaged properties of unfolded states are consistent with those of canonical random coils, namely polymers in indifferent (theta) solvents. These findings are concordant with theoretical predictions based on coarse-grained models and inferences drawn from single-molecule experiments regarding the sequence-specific scaling behavior of unfolded proteins under folding conditions.

Authors:
 [1]; ORCiD logo [2];  [1]; ORCiD logo [2];  [3];  [4]
  1. Stony Brook Univ., NY (United States). Dept. of Chemistry
  2. Washington Univ., St. Louis, MO (United States)
  3. Univ. of Massachusetts, Worcester, MA (United States)
  4. Stony Brook Univ., NY (United States). Dept. of Chemistry; Univ. College London (United Kingdom)
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States)
Sponsoring Org.:
National Institutes of Health (NIH)
OSTI Identifier:
1545860
Grant/Contract Number:  
AC02-06CH11357
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Proceedings of the National Academy of Sciences of the United States of America
Additional Journal Information:
Journal Volume: 116; Journal Issue: 25; Journal ID: ISSN 0027-8424
Publisher:
National Academy of Sciences
Country of Publication:
United States
Language:
ENGLISH
Subject:
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 59 BASIC BIOLOGICAL SCIENCES; protein folding; unfolded state; FRET; compaction transition

Citation Formats

Peran, Ivan, Holehouse, Alex S., Carrico, Isaac S., Pappu, Rohit V., Bilsel, Osman, and Raleigh, Daniel P. Unfolded states under folding conditions accommodate sequence-specific conformational preferences with random coil-like dimensions. United States: N. p., 2019. Web. doi:10.1073/pnas.1818206116.
Peran, Ivan, Holehouse, Alex S., Carrico, Isaac S., Pappu, Rohit V., Bilsel, Osman, & Raleigh, Daniel P. Unfolded states under folding conditions accommodate sequence-specific conformational preferences with random coil-like dimensions. United States. https://doi.org/10.1073/pnas.1818206116
Peran, Ivan, Holehouse, Alex S., Carrico, Isaac S., Pappu, Rohit V., Bilsel, Osman, and Raleigh, Daniel P. Wed . "Unfolded states under folding conditions accommodate sequence-specific conformational preferences with random coil-like dimensions". United States. https://doi.org/10.1073/pnas.1818206116. https://www.osti.gov/servlets/purl/1545860.
@article{osti_1545860,
title = {Unfolded states under folding conditions accommodate sequence-specific conformational preferences with random coil-like dimensions},
author = {Peran, Ivan and Holehouse, Alex S. and Carrico, Isaac S. and Pappu, Rohit V. and Bilsel, Osman and Raleigh, Daniel P.},
abstractNote = {Proteins are marginally stable molecules that fluctuate between folded and unfolded states. Here, we provide a high-resolution description of unfolded states under refolding conditions for the N-terminal domain of the L9 protein (NTL9). We use a combination of time-resolved Förster resonance energy transfer (FRET) based on multiple pairs of minimally perturbing labels, time-resolved small-angle X-ray scattering (SAXS), all-atom simulations, and polymer theory. Upon dilution from high denaturant, the unfolded state undergoes rapid contraction. Although this contraction occurs before the folding transition, the unfolded state remains considerably more expanded than the folded state and accommodates a range of local and nonlocal contacts, including secondary structures and native and nonnative interactions. Paradoxically, despite discernible sequence-specific conformational preferences, the ensemble-averaged properties of unfolded states are consistent with those of canonical random coils, namely polymers in indifferent (theta) solvents. These findings are concordant with theoretical predictions based on coarse-grained models and inferences drawn from single-molecule experiments regarding the sequence-specific scaling behavior of unfolded proteins under folding conditions.},
doi = {10.1073/pnas.1818206116},
url = {https://www.osti.gov/biblio/1545860}, journal = {Proceedings of the National Academy of Sciences of the United States of America},
issn = {0027-8424},
number = 25,
volume = 116,
place = {United States},
year = {2019},
month = {6}
}

Journal Article:
Free Publicly Available Full Text
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Citation Metrics:
Cited by: 12 works
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Works referenced in this record:

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journal, January 2014


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Random-coil behavior and the dimensions of chemically unfolded proteins
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Natively unfolded proteins: A point where biology waits for physics
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Crystal structure of prokaryotic ribosomal protein L9: a bi-lobed RNA-binding protein.
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A Rigorous and Efficient Method To Reweight Very Large Conformational Ensembles Using Average Experimental Data and To Determine Their Relative Information Content
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SAXS versus FRET: A Matter of Heterogeneity?
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Conformational Heterogeneity and FRET Data Interpretation for Dimensions of Unfolded Proteins
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Collapse Precedes Folding in Denaturant-Dependent Assembly of Ubiquitin
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Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy
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Proline-Rich Salivary Proteins Have Extended Conformations
journal, July 2010


Early Events in Protein Folding Explored by Rapid Mixing Methods
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Specific collapse followed by slow hydrogen-bond formation of  -sheet in the folding of single-chain monellin
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The Protein-Folding Problem, 50 Years On
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How fast is protein hydrophobic collapse?
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Advanced ensemble modelling of flexible macromolecules using X-ray solution scattering
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Relating sequence encoded information to form and function of intrinsically disordered proteins
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Polypeptide chain collapse and protein folding
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The effects of nonnative interactions on protein folding rates: Theory and simulation
journal, July 2004


Coil-globule transition in the denatured state of a small protein
journal, July 2006


Conformational Distribution of a 14-Residue Peptide in Solution:  A Fluorescence Resonance Energy Transfer Study
journal, March 2005


Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS
journal, May 2014


Commonly used FRET fluorophores promote collapse of an otherwise disordered protein
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Non-native interactions play an effective role in protein folding dynamics: Non-Native Interactions in Protein Folding
journal, October 2010


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A practical guide to small angle X-ray scattering (SAXS) of flexible and intrinsically disordered proteins
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Direct observation of barrier-limited folding of BBL by single-molecule fluorescence resonance energy transfer
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Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy
journal, October 2002


Collapse Transitions of Proteins and the Interplay Among Backbone, Sidechain, and Solvent Interactions
journal, May 2018


Monomeric Huntingtin Exon 1 Has Similar Overall Structural Features for Wild-Type and Pathological Polyglutamine Lengths
journal, October 2017


Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein
journal, August 2008


Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods
journal, September 2016


Intrinsically unstructured proteins and their functions
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How accurate are polymer models in the analysis of Förster resonance energy transfer experiments on proteins?
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Early Collapse is not an Obligate Step in Protein Folding
journal, April 2004


Quantifying internal friction in unfolded and intrinsically disordered proteins with single-molecule spectroscopy
journal, April 2012


The folding of single domain proteins—have we reached a consensus?
journal, February 2011


Coordinate-dependent diffusion in protein folding
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Collapse of a polymer chain in poor solvents
journal, January 1975


Sequence Determinants of the Conformational Properties of an Intrinsically Disordered Protein Prior to and upon Multisite Phosphorylation
journal, November 2016


Comment on “Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water”
journal, August 2018


Folding versus aggregation: Polypeptide conformations on competing pathways
journal, January 2008


Molecular dimensions and their distributions in early folding intermediates
journal, February 2006


Energetically significant networks of coupled interactions within an unfolded protein
journal, August 2014


Describing sequence–ensemble relationships for intrinsically disordered proteins
journal, December 2012


Experiments and simulations show how long-range contacts can form in expanded unfolded proteins with negligible secondary structure
journal, January 2013


Site-specific time-resolved FRET reveals local variations in the unfolding mechanism in an apparently two-state protein unfolding transition
journal, January 2018


Protein collapse is encoded in the folded state architecture
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Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments
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Speeding protein folding beyond the G? model: How a little frustration sometimes helps
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Criterion that Determines the Foldability of Proteins
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Macromolecular clusters in poor-solvent polymer solutions
journal, October 1997


Rapid Collapse Precedes the Fast Two-state Folding of the Cold Shock Protein
journal, January 2004


An Adequate Account of Excluded Volume Is Necessary To Infer Compactness and Asphericity of Disordered Proteins by Förster Resonance Energy Transfer
journal, November 2015


Probing Protein Folding Using Site-Specifically Encoded Unnatural Amino Acids as FRET Donors with Tryptophan
journal, June 2009


Kinetics and thermodynamics of folding in model proteins.
journal, July 1993


Probing the Action of Chemical Denaturant on an Intrinsically Disordered Protein by Simulation and Experiment
journal, September 2016


Evidence for Initial Non-specific Polypeptide Chain Collapse During the Refolding of the SH3 Domain of PI3 Kinase
journal, October 2010


Comprehensive structural and dynamical view of an unfolded protein from the combination of single-molecule FRET, NMR, and SAXS
journal, August 2016


Microsecond Subdomain Folding in Dihydrofolate Reductase
journal, July 2011


Fine Structure Analysis of a Protein Folding Transition State; Distinguishing Between Hydrophobic Stabilization and Specific Packing
journal, December 2005


Molecular Simulation of ab Initio Protein Folding for a Millisecond Folder NTL9(1−39)
journal, February 2010


Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2
journal, May 2000


Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)
journal, July 2009


The Limited Role of Nonnative Contacts in the Folding Pathways of a Lattice Protein
journal, October 2009


Urea-Induced Unfolding of the Immunity Protein Im9 Monitored by spFRET
journal, September 2006


Single-Molecule Measurement of Protein Folding Kinetics
journal, August 2003


Cryptic sequence features within the disordered protein p27 Kip1 regulate cell cycle signaling
journal, May 2016


Saxs Confirms that FRET Dyes Promote Collapse of an Otherwise Fully Disordered Protein
journal, February 2018


Role of Backbone−Solvent Interactions in Determining Conformational Equilibria of Intrinsically Disordered Proteins
journal, June 2008


Small-Angle X-ray Scattering and Single-Molecule FRET Spectroscopy Produce Highly Divergent Views of the Low-Denaturant Unfolded State
journal, May 2012


Denatured State Ensembles with the Same Radii of Gyration Can Form Significantly Different Long-Range Contacts
journal, December 2013


Are there pathways for protein folding?
journal, January 1968


    Works referencing / citing this record:

    Experiments and simulations show how long-range contacts can form in expanded unfolded proteins with negligible secondary structure
    journal, January 2013


    Fine Structure Analysis of a Protein Folding Transition State; Distinguishing Between Hydrophobic Stabilization and Specific Packing
    journal, December 2005


    Probing the Action of Chemical Denaturant on an Intrinsically Disordered Protein by Simulation and Experiment
    journal, September 2016


    Collapse Precedes Folding in Denaturant-Dependent Assembly of Ubiquitin
    journal, January 2017


    Proline-Rich Salivary Proteins Have Extended Conformations
    journal, July 2010


    Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS)
    journal, July 2009


    Energetically significant networks of coupled interactions within an unfolded protein
    journal, August 2014


    Single-molecule spectroscopy reveals polymer effects of disordered proteins in crowded environments
    journal, March 2014


    The effects of nonnative interactions on protein folding rates: Theory and simulation
    journal, July 2004


    Conformational Distribution of a 14-Residue Peptide in Solution:  A Fluorescence Resonance Energy Transfer Study
    journal, March 2005


    Crystal structure of prokaryotic ribosomal protein L9: a bi-lobed RNA-binding protein.
    journal, January 1994


    Non-native interactions play an effective role in protein folding dynamics: Non-Native Interactions in Protein Folding
    journal, October 2010


    Evidence for Initial Non-specific Polypeptide Chain Collapse During the Refolding of the SH3 Domain of PI3 Kinase
    journal, October 2010


    Microsecond acquisition of heterogeneous structure in the folding of a TIM barrel protein
    journal, August 2008


    Coordinate-dependent diffusion in protein folding
    journal, December 2009


    How accurate are polymer models in the analysis of Förster resonance energy transfer experiments on proteins?
    journal, March 2009


    Sequence Determinants of the Conformational Properties of an Intrinsically Disordered Protein Prior to and upon Multisite Phosphorylation
    journal, November 2016


    Saxs Confirms that FRET Dyes Promote Collapse of an Otherwise Fully Disordered Protein
    journal, February 2018


    Collapse Transitions of Proteins and the Interplay Among Backbone, Sidechain, and Solvent Interactions
    journal, May 2018


    Protein-folding dynamics
    journal, April 1976


    Early Collapse is not an Obligate Step in Protein Folding
    journal, April 2004


    Role of Backbone−Solvent Interactions in Determining Conformational Equilibria of Intrinsically Disordered Proteins
    journal, June 2008


    Decoupling of size and shape fluctuations in heteropolymeric sequences reconciles discrepancies in SAXS vs. FRET measurements
    journal, July 2017


    Urea-Induced Unfolding of the Immunity Protein Im9 Monitored by spFRET
    journal, September 2006


    Consistent View of Polypeptide Chain Expansion in Chemical Denaturants from Multiple Experimental Methods
    journal, September 2016


    How Fast-Folding Proteins Fold
    journal, October 2011


    Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water
    journal, October 2017


    Natively unfolded proteins: A point where biology waits for physics
    journal, April 2002


    Denatured State Ensembles with the Same Radii of Gyration Can Form Significantly Different Long-Range Contacts
    journal, December 2013


    An Adequate Account of Excluded Volume Is Necessary To Infer Compactness and Asphericity of Disordered Proteins by Förster Resonance Energy Transfer
    journal, November 2015


    Molecular Simulation of ab Initio Protein Folding for a Millisecond Folder NTL9(1−39)
    journal, February 2010


    Semiflexible chains under tension
    journal, March 1997


    Intrinsically unstructured proteins and their functions
    journal, March 2005


    Probing the free-energy surface for protein folding with single-molecule fluorescence spectroscopy
    journal, October 2002


    Rapid Collapse Precedes the Fast Two-state Folding of the Cold Shock Protein
    journal, January 2004


    Probing Protein Folding Using Site-Specifically Encoded Unnatural Amino Acids as FRET Donors with Tryptophan
    journal, June 2009


    Speeding protein folding beyond the G? model: How a little frustration sometimes helps
    journal, January 2001


    Comprehensive structural and dynamical view of an unfolded protein from the combination of single-molecule FRET, NMR, and SAXS
    journal, August 2016


    Quantitative Assessments of the Distinct Contributions of Polypeptide Backbone Amides versus Side Chain Groups to Chain Expansion via Chemical Denaturation
    journal, February 2015


    Monomeric Huntingtin Exon 1 Has Similar Overall Structural Features for Wild-Type and Pathological Polyglutamine Lengths
    journal, October 2017


    Single-molecule protein folding: Diffusion fluorescence resonance energy transfer studies of the denaturation of chymotrypsin inhibitor 2
    journal, May 2000


    Quantifying internal friction in unfolded and intrinsically disordered proteins with single-molecule spectroscopy
    journal, April 2012


    Response to Comment on “Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water”
    journal, August 2018


    The folding of single domain proteins—have we reached a consensus?
    journal, February 2011


    Microsecond Subdomain Folding in Dihydrofolate Reductase
    journal, July 2011


    Commonly used FRET fluorophores promote collapse of an otherwise disordered protein
    journal, April 2019


    Modeling the role of disulfide bonds in protein folding: Entropic barriers and pathways
    journal, May 1995


    Kinetics and thermodynamics of folding in model proteins.
    journal, July 1993


    Early Events in Protein Folding Explored by Rapid Mixing Methods
    journal, May 2006


    Cryptic sequence features within the disordered protein p27 Kip1 regulate cell cycle signaling
    journal, May 2016


    The Limited Role of Nonnative Contacts in the Folding Pathways of a Lattice Protein
    journal, October 2009


    Coil-globule transition in the denatured state of a small protein
    journal, July 2006


    Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy
    journal, September 2012


    Specific collapse followed by slow hydrogen-bond formation of  -sheet in the folding of single-chain monellin
    journal, February 2005


    Are there pathways for protein folding?
    journal, January 1968


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


    Dominant forces in protein folding
    journal, August 1990


    A quantitative measure for protein conformational heterogeneity
    journal, September 2013


    The Amyloid State of Proteins in Human Diseases
    journal, March 2012


    Charge interactions can dominate the dimensions of intrinsically disordered proteins
    journal, July 2010


    Comment on “Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water”
    journal, August 2018


    Macromolecular clusters in poor-solvent polymer solutions
    journal, October 1997


    Structural origin of slow diffusion in protein folding
    journal, September 2015


    Small-Angle X-ray Scattering and Single-Molecule FRET Spectroscopy Produce Highly Divergent Views of the Low-Denaturant Unfolded State
    journal, May 2012


    Relating sequence encoded information to form and function of intrinsically disordered proteins
    journal, June 2015


    Conformational Heterogeneity and FRET Data Interpretation for Dimensions of Unfolded Proteins
    journal, September 2017


    SAXS versus FRET: A Matter of Heterogeneity?
    journal, September 2017


    Expanding the Genetic code
    journal, June 2006


    Single-Molecule Measurement of Protein Folding Kinetics
    journal, August 2003


    Molecular dimensions and their distributions in early folding intermediates
    journal, February 2006


    Comment on “Innovative scattering analysis shows that hydrophobic disordered proteins are expanded in water”
    journal, August 2018


    Single-Molecule FRET Spectroscopy and the Polymer Physics of Unfolded and Intrinsically Disordered Proteins
    journal, July 2016


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


    Polymer Principles in Protein Structure and Stability
    journal, June 1991


    Direct observation of barrier-limited folding of BBL by single-molecule fluorescence resonance energy transfer
    journal, September 2009


    Polypeptide chain collapse and protein folding
    journal, March 2013


    Microsecond Barrier-Limited Chain Collapse Observed by Time-Resolved FRET and SAXS
    journal, May 2014


    How fast is protein hydrophobic collapse?
    journal, October 2003


    Single-Molecule Studies of Intrinsically Disordered Proteins
    journal, January 2014


    How, when and why proteins collapse: the relation to folding
    journal, February 2012


    Folding versus aggregation: Polypeptide conformations on competing pathways
    journal, January 2008


    Asymmetry in the Shapes of Folded and Denatured States of Proteins
    journal, May 2004


    A Rigorous and Efficient Method To Reweight Very Large Conformational Ensembles Using Average Experimental Data and To Determine Their Relative Information Content
    journal, December 2015


    Random-coil behavior and the dimensions of chemically unfolded proteins
    journal, August 2004


    Collapse of a polymer chain in poor solvents
    journal, January 1975


    Protein folding while chaperone bound is dependent on weak interactions
    journal, October 2019


    Information theoretic measures for quantifying sequence–ensemble relationships of intrinsically disordered proteins
    journal, April 2019