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Photocontrolling Protein–Peptide Interactions: From Minimal Perturbation to Complete Unbinding


Jankovic, Brankica; Gulzar, Adnan; Zanobini, Claudio; Bozovic, Olga; Wolf, Steffen; Stock, Gerhard; Hamm, Peter (2019). Photocontrolling Protein–Peptide Interactions: From Minimal Perturbation to Complete Unbinding. Journal of the American Chemical Society, 141(27):10702-10710.

Abstract

An azobenzene-derived photoswitch has been covalently cross-linked to two sites of the S-peptide in the RNase S complex in a manner that the alpha-helical content of the S-peptide reduces upon cis-to-trans isomerization of the photoswitch. Three complementary experimental techniques have been employed, isothermal titration calorimetry, circular dichroism spectroscopy and intrinsic tyrosine fluorescence quenching, to determine the binding affinity of the S-peptide to the S-protein in the two states of the photoswitch. Five mutants with the photoswitch attached to different sites of the S-peptide have been explored, with the goal to maximize the change in binding affinity upon photoswitching, and to identify the mechanisms that determine the binding affinity. With regard to the first goal, one mutant has been identified, which binds with reasonable affinity in the one state of the photoswitch, while specific binding is completely switched off in the other state. With regard to the second goal, accompanying molecular dynamics simulations combined with a quantitative structure activity relationship revealed that the alpha-helicity of the S-peptide in the binding pocket correlates surprisingly well with measured dissociation constants. Moreover, the simulations show that both configurations of all S-peptides exhibit quite well-defined structures, even in apparently disordered states.

Abstract

An azobenzene-derived photoswitch has been covalently cross-linked to two sites of the S-peptide in the RNase S complex in a manner that the alpha-helical content of the S-peptide reduces upon cis-to-trans isomerization of the photoswitch. Three complementary experimental techniques have been employed, isothermal titration calorimetry, circular dichroism spectroscopy and intrinsic tyrosine fluorescence quenching, to determine the binding affinity of the S-peptide to the S-protein in the two states of the photoswitch. Five mutants with the photoswitch attached to different sites of the S-peptide have been explored, with the goal to maximize the change in binding affinity upon photoswitching, and to identify the mechanisms that determine the binding affinity. With regard to the first goal, one mutant has been identified, which binds with reasonable affinity in the one state of the photoswitch, while specific binding is completely switched off in the other state. With regard to the second goal, accompanying molecular dynamics simulations combined with a quantitative structure activity relationship revealed that the alpha-helicity of the S-peptide in the binding pocket correlates surprisingly well with measured dissociation constants. Moreover, the simulations show that both configurations of all S-peptides exhibit quite well-defined structures, even in apparently disordered states.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Scopus Subject Areas:Physical Sciences > Catalysis
Physical Sciences > General Chemistry
Life Sciences > Biochemistry
Physical Sciences > Colloid and Surface Chemistry
Language:English
Date:10 July 2019
Deposited On:07 Feb 2020 13:39
Last Modified:29 Jul 2020 13:54
Publisher:American Chemical Society (ACS)
ISSN:0002-7863
OA Status:Green
Publisher DOI:https://doi.org/10.1021/jacs.9b03222
Project Information:
  • : FunderSNSF
  • : Grant ID200021_165789
  • : Project TitleUltrafast Vibrational Spectroscopy of Allosteric Proteins
  • : FunderGerman Research Foundation (DFG)
  • : Grant IDSTO 247/10-2
  • : Project Title

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