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The Stability Landscape of de novo TIM Barrels Explored by a Modular Design Approach.

ABSTRACT: The ability to design stable proteins with custom-made functions is a major goal in biochemistry with practical relevance for our environment and society. Understanding and manipulating protein stability provide crucial information on the molecular determinants that modulate structure and stability, and expand the applications of de novo proteins. Since the (β/⍺)₈-barrel or TIM-barrel fold is one of the most common functional scaffolds, in this work we designed a collection of stable de novo TIM barrels (DeNovoTIMs), using a computational fixed-backbone and modular approach based on improved hydrophobic packing of sTIM11, the first validated de novo TIM barrel, and subjected them to a thorough folding analysis. DeNovoTIMs navigate a region of the stability landscape previously uncharted by natural TIM barrels, with variations spanning 60 degrees in melting temperature and 22 kcal per mol in conformational stability throughout the designs. Significant non-additive or epistatic effects were observed when stabilizing mutations from different regions of the barrel were combined. The molecular basis of epistasis in DeNovoTIMs appears to be related to the extension of the hydrophobic cores. This study is an important step towards the fine-tuned modulation of protein stability by design.

SUBMITTER: Romero-Romero S

PROVIDER: S-EPMC8404036 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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The Stability Landscape of de novo TIM Barrels Explored by a Modular Design Approach.

Romero-Romero Sergio S Costas Miguel M Silva Manzano Daniel-Adriano DA Kordes Sina S Rojas-Ortega Erendira E Tapia Cinthya C Guerra Yasel Y Shanmugaratnam Sooruban S Rodríguez-Romero Adela A Baker David D Höcker Birte B Fernández-Velasco D Alejandro DA

Journal of molecular biology 20210714 18

The ability to design stable proteins with custom-made functions is a major goal in biochemistry with practical relevance for our environment and society. Understanding and manipulating protein stability provide crucial information on the molecular determinants that modulate structure and stability, and expand the applications of de novo proteins. Since the (β/⍺)<sub>8</sub>-barrel or TIM-barrel fold is one of the most common functional scaffolds, in this work we designed a collection of stable ...[more]

PMID: 34271011

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Project description: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.

Dataset Information

The Stability Landscape of de novo TIM Barrels Explored by a Modular Design Approach.

Publications

The Stability Landscape of de novo TIM Barrels Explored by a Modular Design Approach.

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