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Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae
Published on: July 9, 2013
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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.
Structure (London, England : 1993)
|December 20, 2024
Summary
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.
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.
