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Stepwise introduction of stabilizing mutations reveals nonlinear additive effects in de novo TIM barrels

DOI zum Zitieren der Version auf EPub Bayreuth: https://doi.org/10.15495/EPub_UBT_00007944
URN to cite this document: urn:nbn:de:bvb:703-epub-7944-5

Title data

Koch, Johanna-Sophie ; Romero-Romero, Sergio ; Höcker, Birte:
Stepwise introduction of stabilizing mutations reveals nonlinear additive effects in de novo TIM barrels.
In: Protein Science. Vol. 33 (March 2024) Issue 3 . - e4926.
ISSN 1469-896X
DOI der Verlagsversion: https://doi.org/10.1002/pro.4926

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Project information

Project financing: Alexander von Humboldt-Stiftung
VolkswagenStiftung
European Research Council
Bayer Science & Education Foundations

Abstract

Over the past decades, the TIM-barrel fold has served as a model system for the exploration of how changes in protein sequences affect their structural, stability, and functional characteristics, and moreover, how this information can be leveraged to design proteins from the ground up. After numerous attempts to design de novo proteins with this specific fold, sTIM11 was the first validated de novo design of an idealized four-fold symmetric TIM barrel. Subsequent efforts to enhance the stability of this initial design resulted in the development of DeNovoTIMs, a family of de novo TIM barrels with various stabilizing mutations. In this study, we present an investigation into the biophysical and thermodynamic effects upon introducing a varying number of stabilizing mutations per quarter along the sequence of a four-fold symmetric TIM barrel. We compared the base design DeNovoTIM0 without any stabilizing mutations with variants containing mutations in one, two, three, and all four quarters-designated TIM1q, TIM2q, TIM3q, and DeNovoTIM6, respectively. This analysis revealed a stepwise and nonlinear change in the thermodynamic properties that correlated with the number of mutated quarters, suggesting positive nonadditive effects. To shed light on the significance of the location of stabilized quarters, we engineered two variants of TIM2q which contain the same number of mutations but positioned in different quarter locations. Characterization of these TIM2q variants revealed that the mutations exhibit varying effects on the overall protein stability, contingent upon the specific region in which they are introduced. These findings emphasize that the amount and location of stabilized interfaces among the four quarters play a crucial role in shaping the conformational stability of these four-fold symmetric TIM barrels. Analysis of de novo proteins, as described in this study, enhances our understanding of how sequence variations can finely modulate stability in both naturally occurring and computationally designed proteins.

Further data

Item Type: Article in a journal
Keywords: (β/α)8-barrel; DeNovoTIMs; TIM barrel; additive effects; asymmetry; de novo protein design; protein folding
DDC Subjects: 500 Science > 500 Natural sciences
500 Science > 540 Chemistry
500 Science > 570 Life sciences, biology
Institutions of the University: Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry III - Protein Design > Chair Biochemistry III - Protein Design - Univ.-Prof. Dr. Birte Höcker
Faculties
Faculties > Faculty of Biology, Chemistry and Earth Sciences
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry
Faculties > Faculty of Biology, Chemistry and Earth Sciences > Department of Chemistry > Chair Biochemistry III - Protein Design
Language: English
Originates at UBT: Yes
URN: urn:nbn:de:bvb:703-epub-7944-5
Date Deposited: 02 Oct 2024 05:16
Last Modified: 02 Oct 2024 05:17
URI: https://epub.uni-bayreuth.de/id/eprint/7944

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