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

Sergio Romero-Romero; Miguel Costas; Daniel-Adriano Silva Manzano; Sina Kordes; Erendira Rojas-Ortega; Cinthya Tapia; Yasel Guerra; Sooruban Shanmugaratnam; Adela Rodríguez-Romero; David Baker; Birte Höcker; D Alejandro Fernández-Velasco

doi:10.1016/j.jmb.2021.167153

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

J Mol Biol. 2021 Sep 3;433(18):167153. doi: 10.1016/j.jmb.2021.167153. Epub 2021 Jul 14.

Affiliations

¹ Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, 04510 Mexico City, Mexico; Department of Biochemistry, University of Bayreuth, 95447 Bayreuth, Germany.
² Laboratorio de Biofisicoquímica, Departamento de Fisicoquímica, Facultad de Química, Universidad Nacional Autónoma de México, 04510 Mexico City, Mexico.
³ Department of Biochemistry, University of Washington, 98195 Seattle, USA; Institute for Protein Design, University of Washington, 98195 Seattle, USA.
⁴ Department of Biochemistry, University of Bayreuth, 95447 Bayreuth, Germany.
⁵ Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, 04510 Mexico City, Mexico.
⁶ Instituto de Química, Universidad Nacional Autónoma de México, 04510 Mexico City, Mexico.
⁷ Department of Biochemistry, University of Washington, 98195 Seattle, USA; Institute for Protein Design, University of Washington, 98195 Seattle, USA. Electronic address: dabaker@uw.edu.
⁸ Department of Biochemistry, University of Bayreuth, 95447 Bayreuth, Germany. Electronic address: birte.hoecker@uni-bayreuth.de.
⁹ Laboratorio de Fisicoquímica e Ingeniería de Proteínas, Departamento de Bioquímica, Facultad de Medicina, Universidad Nacional Autónoma de México, 04510 Mexico City, Mexico. Electronic address: fdaniel@unam.mx.

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.

Keywords: (β/α)(8)-barrel; de novo protein design; non-additive effects; protein folding and stability; stability landscape.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Evolution, Molecular
Hydrophobic and Hydrophilic Interactions
Models, Molecular
Protein Conformation, alpha-Helical*
Protein Conformation, beta-Strand*
Protein Folding*
Protein Stability*
Proteins / chemistry*
Temperature

Substances

Proteins