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Cited by 124Protein Structure and DynamicsChemical Synthesis and AnalysisProtein purification and stability

Protein destabilization by electrostatic repulsions in the two‐stranded α‐helical coiled‐coil/leucine zipper

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Kohn, Wayne D., Kay, Cyril M., Hodges, Robert S.·1995·Protein Science
DOI 10.1002/pro.5560040210PMC PMC2143052
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Abstract

The destabilizing effect of electrostatic repulsions on protein stability has been studied by using synthetic two‐stranded α‐helical coiled‐coils as a model system. The native coiled‐coil consists of two identical 35‐residue polypeptide chains with a heptad repeat QgVaGbAcLdQeKf and a Cys residue at position 2 to allow formation of an interchain disulfide bridge. This peptide, designed to contain no intrahelical or interhelical electrostatic interactions, forms a stable coiled‐coil structure at 20 °C in benign medium (50 mM KCl, 25 mM PO 4, pH 7) with a [urea]1/2 value of 6.1 M. Four mutant coiled‐coils were designed to contain one or two Glu substitutions for Gln per polypeptide chain. The resulting coiled‐coils contained potential i to i' +5 Glu‐Glu interchain repulsions (denoted as peptide E 2(15,20)), i to i' + 2 Glu‐Glu interchain repulsions (denoted E 2(20,22)), or no interchain ionic interactions (denoted E 2(13,22) and E 1(20)). The stabilities of the coiled‐coils were determined by measuring the ellipticities at 222 nm as a function of urea or guanidine hydrochloride concentration at 20 °C in the presence and absence of an interchain disulfide bridge. At pH 7, in the presence of urea, the stabilities of E 2(13,22) and E 2(20,22) were identical suggesting that the potential i to i' + 2 interchain Glu‐Glu repulsion in the E 2(20,22) coiled‐coil does not occur. In contrast, the mutant E 2(15,20) is substantially less stable than E 2(13,22) or E 2(15,20) by 0.9 kcal/mol due to the presence of two i to i' +5 interchain Glu‐Glu repulsions, which destabilize the coiled‐coil by 0.45 kcal/mol each. At pH 3 the coiled‐coils were found to increase in stability as the number of Glu substitutions were increased. This, combined with reversed‐phase HPLC results at pH 7 and pH 2, supports the conclusion that the protonated Glu side chains present at low pH are significantly more hydrophobic than Gln side chains which are in turn more hydrophobic than the ionized Glu side chains present at neutral pH. The protonated Glu residues increase the hydrophobicity of the coiled‐coil interface leading to higher coiled‐coil stability. The guanidine hydrochloride results at pH 7 show similar stabilities between the native and mutant coiled‐coils indicating that guanidine hydrochloride masks electrostatic repulsions due to its ionic nature and that Glu and Gln in the e and g positions of the heptad repeat have very similar effects on coiled‐coil stability in the presence of GdnHCl.

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  • Thermodynamic cycle analysis of mutant coiled coils indicated that a charged residue at a g position at residue i can interact favorably or unfavorably with a charged residue at the e' position at i+5.

    “Thermodynamic cycle analysis of mutant coiled coils showed that charged residues at a g position at residue i can interact favorably or unfavorably with a charged residue at the e' position at i + 5 (Krylov et al., 1994; Zhou et al., 1994a, 1994b; Kohn et al., 1995), consistent with the orientation of ion pairs observed in the crystal structures.”

    From Oligomerization properties of GCN4 leucine zipper <i>e</i> and <i>g</i> position mutants (1997) · cites this paper as Kohn et al., 1995

    The Conclusions report that an i-to-i′+5 Glu–Glu interaction destabilized the mutant coiled coil, supporting the unfavorable-interaction part of the claim. They explicitly say that double-mutant analysis was not necessary for this study; the passages provided do not show a favorable interaction demonstrated by Kohn et al. (LLM, conf=0.88)

    Experimental — accuracy not yet measured

  • Salt dependence of leucine zipper stability indicates that ionic effects are involved in stabilizing and/or destabilizing leucine zippers.

    “The salt dependence of leucine zipper stability also indicates that ionic effects are involved in stabilizing and/or destabilizing leucine zippers (Monera et al., 1994; Zhou et al., 1994b; Kohn et al., 1995; Yu et al., 1996).”

    From Oligomerization properties of GCN4 leucine zipper <i>e</i> and <i>g</i> position mutants (1997) · cites this paper as Kohn et al., 1995

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Research works printed but not linked: 6

  1. 1469896x, 1995, 2, 30:657-676. · DOI
  2. Goto Y, Nishikiori S. 1991. Role of electrostatic repulsion in the acidic mol- Press. pp 255-527. ten globule of cytochrome c. J Mol Biol 222:11908-11914.
  3. Sereda TJ, Mant CT, Sonnichsen FD, Hodges RS. 1994. Reversed-phase chro- matography of synthetic amphipathic a-helical peptides as a model for ligand-receptor interactions: Effect of changing hydrophobic environ- 1469896x, 1995, 2, menton the relative hydrophilicity/hydrophobicity of amino acid side- chains. J Chromatogr 676:139-153. · DOI
  4. Stone D, Sodek J, Johnson P, Smillie LB. 1974. Tropomyosin: Correlation of amino acid sequence and structure. In: Biro ENA, ed. Proceedings of the IX Federation of European Biochemical Societies Meeting, Pro- teins of Contractile Systems. Amsterdam: North Holland Publishing. pp 125-136.
  5. Zhou NE, Zhu BY, Kay CM, Hodges RS. 1992c. Importance of intra-chain ionic interactions in stabilizing a-helical peptides in proteins. In: Du YC, Tam JP, Zhang YS, eds. Peptides, biology andrhemistry: Proceedings of the 1992 Chinese Peptide Symposium. Leiden: ESCOM. pp 217-220.
  6. Zhu BY, Zhou NE, Semchuk PD, Kay C, Hodges RS. 1992. Design, syn- thesis and structural characterization of model heterodimeric coiled-coil proteins. In1 JPept Protein Res 40:171-179.

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Authors
Kohn, W. D., Kay, C. M., & Hodges, R. S.
Affiliations
University of Alberta (CA)
Corresponding author
Hodges, Robert S.
ORCID
Hodges, Robert S.
Venue
Protein Science
Publisher
Wiley
Published
1995-02
Volume
4
Issue
2
Pages
237-250
ISSN
  • 0961-8368 (ISSN-L)
  • 1469-896X
DOI
10.1002/pro.5560040210
PMC
PMC2143052
OpenAlex
W2009528324
Cited by
124
References
0
Citations by year
2026: 2 · 2025: 2 · 2023: 2 · 2022: 1 · 2021: 2 · 2020: 2 · 2019: 5 · 2018: 1 · 2017: 1 · 2016: 3 · 2014: 3 · 2013: 8 · 2012: 3
FWCI
3.72
Type
journal article
Language
en
Open access
Licence
onlinelibrary.wiley.com/termsAndConditions#vor (vor, 5082 d)
Locations
  • Protein Science (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
Natural Sciences and Engineering Research Council of Canada
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Chemical Synthesis and Analysis
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein purification and stability
Keywords
protein destabilization, electrostatic repulsion, coiled coil, leucine zipper, protein stability, heptad repeat, pH dependence, urea denaturation, guanidine hydrochloride denaturation, ellipticity, synthetic peptides, electrostatic interactions
MeSH terms
Amino Acid Sequence, Circular Dichroism, Disulfides, Hydrogen-Ion Concentration, Molecular Sequence Data, Oxidation-Reduction, Peptides, Protein Denaturation, Repetitive Sequences, Nucleic Acid, Structure-Activity Relationship, Thermodynamics, Molecular Structure, Leucine Zippers, Point Mutation, Protein Structure, Secondary, Protein Structure, Tertiary, Protein Folding, Glutamic Acid

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