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

Titelangaben

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. Bd. 33 (März 2024) Heft 3 . - e4926.
ISSN 1469-896X
DOI: https://doi.org/10.1002/pro.4926

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Angaben zu Projekten

Projektfinanzierung: 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.

Weitere Angaben

Publikationsform: Artikel in einer Zeitschrift
Begutachteter Beitrag: Ja
Keywords: (β/α)8-barrel; DeNovoTIMs; TIM barrel; additive effects; asymmetry; de novo protein design; protein folding
Institutionen der Universität: Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie > Lehrstuhl Biochemie III - Proteindesign > Lehrstuhl Biochemie III - Proteindesign - Univ.-Prof. Dr. Birte Höcker
Fakultäten
Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften
Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie
Fakultäten > Fakultät für Biologie, Chemie und Geowissenschaften > Fachgruppe Chemie > Lehrstuhl Biochemie III - Proteindesign
Titel an der UBT entstanden: Ja
Themengebiete aus DDC: 500 Naturwissenschaften und Mathematik > 500 Naturwissenschaften
500 Naturwissenschaften und Mathematik > 540 Chemie
500 Naturwissenschaften und Mathematik > 570 Biowissenschaften; Biologie
Eingestellt am: 16 Mai 2024 08:08
Letzte Änderung: 02 Okt 2024 05:17
URI: https://eref.uni-bayreuth.de/id/eprint/89562