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Systematic characterization of indel variants using a yeast-based protein folding sensor.

Sven Larsen-Ledet1, Søren Lindemose1, Aleksandra Panfilova1

  • 1Department of Biology, University of Copenhagen, Ole Maaløes Vej 5, 2200 Copenhagen, Denmark.

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Gene variants called indels impact evolution and disease but are poorly understood. A new yeast-based sensor reveals most indels destabilize protein folding, with computational tools aiding prediction.

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AlphaFold2DMSMAVERosettadeep mutational scanningmultiplexed assays of variant effectspredictionsprotein foldingprotein stabilityproteostasis

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Genetics

Background:

  • Gene variants, specifically insertions/deletions (indels), significantly influence evolution and disease.
  • The functional impact of indels on protein structure and function is less understood than missense variants.
  • Predicting the effects of indels remains a challenge in genetic and disease research.

Purpose of the Study:

  • To develop a sensitive assay for predicting the effects of indels on protein folding.
  • To investigate the impact of single-residue indels on the folding stability of human dihydrofolate reductase (DHFR).
  • To evaluate the utility of computational tools like Rosetta and AlphaFold2 in predicting indel effects.

Main Methods:

  • Development of a yeast-based protein folding sensor utilizing circular permutated orotate phosphoribosyltransferase (CPOP).
  • Application of the sensor to a comprehensive library of single-residue indels in human DHFR.
  • Analysis of indel effects on protein folding, temperature sensitivity, and rescue by methotrexate binding.
  • Comparison of experimental folding data with predictions from Rosetta and AlphaFold2.

Main Results:

  • Most tolerated indels in DHFR were located in protein termini, internal loops, and a central alpha helix.
  • Several indels exhibited temperature-sensitive folding phenotypes, which could be rescued by methotrexate.
  • Computational predictions from Rosetta and AlphaFold2 generally correlated with experimentally observed folding effects.
  • The study suggests that the majority of indels tend to destabilize the native protein fold.

Conclusions:

  • The developed yeast folding sensor is effective for assessing the impact of indels on protein stability.
  • Indel tolerance is spatially restricted in proteins, primarily occurring in flexible regions.
  • Computational tools show promise for classifying the functional consequences of indels found in population sequencing data.
  • Understanding indel effects is crucial for interpreting genetic variation in health and disease contexts.