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PreprintCited by 12Protein Structure and DynamicsProtein purification and stabilityProtein Interaction Studies and Fluorescence Analysis

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Diversity of hydrodynamic radii of intrinsically disordered proteins

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Białobrzewski, Michał K., Klepka, Barbara P., Michaś, Agnieszka, Cieplak-Rotowska, Maja K., Staszałek, Zuzanna, Niedźwiecka, Anna·2023·European Biophysics Journal
DOI 10.1101/2023.06.12.543592PMC PMC10618399

Preprint — published as 10.1007/s00249-023-01683-8 · Paper page

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Abstract

Intrinsically disordered proteins (IDPs) form an important class of biomolecules regulating biological processes in higher organisms. The lack of a fixed spatial structure facilitates them to perform their regulatory functions. Due to the possibility of large conformational changes of IDPs, the cellular milieu can also control productivity of biochemical reactions. From the biophysical point of view, IDPs are biopolymers with a broad configuration state space. The conformation of such a biopolymer depends on non-covalent interactions of its amino acid side chain groups at given temperature and chemical conditions. Thus, the hydrodynamic radius (R h ) of an IDP of a given polymer length (N) is a sequence- and environment-dependent variable. We have reviewed the literature values of hydrodynamic radii of IDPs determined experimentally by SEC, AUC, PFG NMR, DLS, and FCS, and complement them with our FCS results obtained for a series of protein fragments involved in regulation of human gene expression. The data collected herein show that the values of hydrodynamic radii of intrinsically disordered proteins can span the full space between the folded globular and denatured proteins in the R h (N) diagram.

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Fig. 1 Example putative conformations of the IDPs studied in this work (left panel) ordered top–down according to their molecular weights, CNOT1 M long, SUMO-GW182 SD, SUMO-GW182 SD10, PARN C-mCherry, GW182 SD10, and SUMO-GW182 SD peptide, predicted by AlphaFold 2 (Jumper et al. 2021; Mirdita et al. 2022) in comparison with the globular proteins (right panel), apoferritin (PDB id. code: 7vd8) (Fan et al. 2021), human serum albumin (PDB id. code: 1uor) (He and Carter 1992), α-chymotrypsinogen A (PDB id. code: 2cga) (Wang et al. 1985), CNOT1 M short (CNOT1(800-999), PDB id. code: 4j8s) (Fabian et al. 2013), and lysozyme (PDB id. code: 1e8l) (Schwalbe et al. 2001). The N-terminal fragment lacking in the crystal structure of CNOT1 M short was generated by AlphaFold 2. IDRs are shown in the CPK sphere atom representation colored according to partial charges, except for positively charged amino acid residues (Arg, Lys) marked in blue, negatively charged residues (Asp, Glu) in red, and hydrophobic aromatic residues (Trp, Phe) in dark yellow. Folded fragments are shown as ribbon colored according to secondary structural elements, α-helices in red, β-sheets in cyan, and β-turns in green
Fig. 1 Example putative conformations of the IDPs studied in this work (left panel) ordered top–down according to their molecular weights, CNOT1 M long, SUMO-GW182 SD, SUMO-GW182 SD10, PARN C-mCherry, GW182 SD10, and SUMO-GW182 SD peptide, predicted by AlphaFold 2 (Jumper et al. 2021; Mirdita et al. 2022) in comparison with the globular proteins (right panel), apoferritin (PDB id. code: 7vd8) (Fan et al. 2021), human serum albumin (PDB id. code: 1uor) (He and Carter 1992), α-chymotrypsinogen A (PDB id. code: 2cga) (Wang et al. 1985), CNOT1 M short (CNOT1(800-999), PDB id. code: 4j8s) (Fabian et al. 2013), and lysozyme (PDB id. code: 1e8l) (Schwalbe et al. 2001). The N-terminal fragment lacking in the crystal structure of CNOT1 M short was generated by AlphaFold 2. IDRs are shown in the CPK sphere atom representation colored according to partial charges, except for positively charged amino acid residues (Arg, Lys) marked in blue, negatively charged residues (Asp, Glu) in red, and hydrophobic aromatic residues (Trp, Phe) in dark yellow. Folded fragments are shown as ribbon colored according to secondary structural elements, α-helices in red, β-sheets in cyan, and β-turns in greenCC BY · PubMed Central
Fig. 2 Example normalized FCS autocorrelation curves with non-normalized fitting residuals for a folded protein, HSA (black), and an IDP, PARN C-mCherry (green), in 50 mM Tris/HCl buffer pH 8.0, 150 mM NaCl, 0.5 mM EDTA, and 1 mM TCEP, at 298 K. Dots, experimental points; lines, curves fitted according to Eq. 3
Fig. 2 Example normalized FCS autocorrelation curves with non-normalized fitting residuals for a folded protein, HSA (black), and an IDP, PARN C-mCherry (green), in 50 mM Tris/HCl buffer pH 8.0, 150 mM NaCl, 0.5 mM EDTA, and 1 mM TCEP, at 298 K. Dots, experimental points; lines, curves fitted according to Eq. 3CC BY · PubMed Central
Fig. 3 Hydrodynamic radii, Rh, of IDPs (black-bordered green squares), globular proteins (black circles), and chemical fluorescent probes (AF488 and Alexa Fluor 546, black diamonds) determined by FCS in this study (Table 1) (a) as a function of the molecular mass, M, compared to literature Rh values for IDPs (plain green squares, Table S1); and (b) as a function of the polymer residue number, N, compared to literature data (plain squares, Table S1), where the results for the same protein at different conditions are marked in shades of red–orange–magenta; open circles and diamonds denote folded globular and chemically denatured proteins from literature, respectively. Chemical fluorescent probes and apoferritin are not taken into account in (b). The analytical curve according to Eq. (2) was fitted for folded proteins to the literature data merged with the results of this study, and for denatured proteins to literature data. The gray shaded regions represent the 95% confidence interval bands
Fig. 3 Hydrodynamic radii, Rh, of IDPs (black-bordered green squares), globular proteins (black circles), and chemical fluorescent probes (AF488 and Alexa Fluor 546, black diamonds) determined by FCS in this study (Table 1) (a) as a function of the molecular mass, M, compared to literature Rh values for IDPs (plain green squares, Table S1); and (b) as a function of the polymer residue number, N, compared to literature data (plain squares, Table S1), where the results for the same protein at different conditions are marked in shades of red–orange–magenta; open circles and diamonds denote folded globular and chemically denatured proteins from literature, respectively. Chemical fluorescent probes and apoferritin are not taken into account in (b). The analytical curve according to Eq. (2) was fitted for folded proteins to the literature data merged with the results of this study, and for denatured proteins to literature data. The gray shaded regions represent the 95% confidence interval bandsCC BY · PubMed Central

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Metadata

Authors
Białobrzewski, M. K., Klepka, B. P., Michaś, A., Cieplak-Rotowska, M. K., Staszałek, Z., & Niedźwiecka, A.
Affiliations
Institute of Physics (PL)
ORCID
Venue
European Biophysics Journal
Publisher
openRxiv
Published
2023-06-12
Volume
52
Issue
6-7
Pages
607-618
ISSN
0175-7571 (ISSN-L)
DOI
10.1101/2023.06.12.543592
PMC
PMC10618399
OpenAlex
W4387599617
Cited by
12
References
1
Citations by year
2026: 2 · 2025: 7 · 2024: 2 · 2023: 1
FWCI
1.06
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • European Biophysics Journal (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • Narodowe Centrum Nauki (UMO-2016/22/E/NZ1/00656)
  • European Regional Development Fund (POIG.02.02.00-00- 025/09)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Interaction Studies and Fluorescence Analysis
Keywords
intrinsically disordered proteins, hydrodynamic radius, protein conformation, sequence-dependent conformation, temperature dependence, non-covalent interactions, protein fragments, gene expression regulation
MeSH terms
Humans, Magnetic Resonance Spectroscopy, Protein Conformation, Radius, Temperature, Hydrodynamics, Intrinsically Disordered Proteins

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Białobrzewski, M. K., Klepka, B. P., Michaś, A., Cieplak-Rotowska, M. K., Staszałek, Z., & Niedźwiecka, A. (2023). Diversity of hydrodynamic radii of intrinsically disordered proteins. European Biophysics Journal. https://doi.org/10.1101/2023.06.12.543592