Systematic characterization of indel variants using a yeast-based protein folding sensor
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Abstract
Gene variants resulting in insertions or deletions of amino acid residues (indels) have important consequences for evolution and are often linked to disease, yet, compared to missense variants, the effects of indels are poorly understood and predicted. We developed a sensitive protein folding sensor based on the complementation of uracil auxotrophy in yeast by circular permutated orotate phosphoribosyltransferase (CPOP). The sensor reports on the folding of disease-linked missense variants and de-novo-designed proteins. Applying the folding sensor to a saturated library of single-residue indels in human dihydrofolate reductase (DHFR) revealed that most regions that tolerate indels are confined to internal loops, the termini, and a central α helix. Several indels are temperature sensitive, and folding is rescued upon binding to methotrexate. Rosetta and AlphaFold2 predictions correlate with the observed effects, suggesting that most indels destabilize the native fold and that these computational tools are useful for the classification of indels observed in population sequencing.
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Uncovering differential tolerance to deletions versus substitutions with a protein language model
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- Authors
- Larsen-Ledet, S., Lindemose, S., Panfilova, A., Gersing, S., Suhr, C. H., Genzor, A. V., Lanters, H., Nielsen, S. V., Lindorff-Larsen, K., Winther, J. R., Stein, A., & Hartmann-Petersen, R.
- Affiliations
- University of Copenhagen (DK)
- Corresponding author
- Stein, Amelie
- ORCID
- Venue
- Structure
- Publisher
- Elsevier BV
- Published
- 2025-02
- Volume
- 33
- Issue
- 2
- Pages
- 262-273.e6
- ISSN
- 0969-2126 (ISSN-L)
- OpenAlex
- W4405567688
- Cited by
- 3
- References
- 67
- Citations by year
- 2026: 1 · 2025: 2
- FWCI
- 0.41
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- www.elsevier.com/tdm/userlicense/1.0/ (tdm)
- www.elsevier.com/legal/tdmrep-license (tdm)
- www.elsevier.com/open-access/userlicense/1.0/ (vor, 370 d)
- doi.org/10.15223/policy-017 (stm-asf)
- doi.org/10.15223/policy-037 (stm-asf)
- doi.org/10.15223/policy-012 (stm-asf)
- doi.org/10.15223/policy-029 (stm-asf)
- doi.org/10.15223/policy-004 (stm-asf)
- Locations
- Structure (publishedVersion)
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Research at the University of Copenhagen (University of Copenhagen) (submittedVersion)
- Funders
- Independent Research Fund Denmark
- Københavns Universitet
- Danmarks Frie Forskningsfond
- Novo Nordisk Foundation (NNF21OC0071057)
- Lundbeck Foundation (R272-2017-452)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Structure and Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
- Keywords
- indel variants, dihydrofolate reductase, protein stability, temperature sensitivity, protein design, structure prediction, missense mutations
- MeSH terms
- Humans, Methotrexate, Models, Molecular, Orotate Phosphoribosyltransferase, Saccharomyces cerevisiae, Tetrahydrofolate Dehydrogenase, Uracil, Protein Folding, Mutation, Missense, INDEL Mutation
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