SUNi mutagenesis: Scalable and uniform nicking for efficient generation of variant libraries

Mighell, Taylor L.; Toledano, Ignasi; Lehner, Burkhard

doi:10.1371/journal.pone.0288158

Cited by 4 publications

(1 citation statement)

References 20 publications

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“…In future work it will be important to extend the approach to longer domains and to full length proteins. Advances in DNA synthesis 40 , assembly 41 and mutagenesis 42,43 should facilitate this. In this study we have reported the effects of Domainome 1.0 variants on cellular protein abundance.…”

Section: Discussionmentioning

confidence: 99%

Site saturation mutagenesis of 500 human protein domains reveals the contribution of protein destabilization to genetic disease

Beltran,

Jiang,

Shen

et al. 2024

Preprint

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Missense variants that change the amino acid sequences of proteins cause one third of human genetic diseases1. Tens of millions of missense variants exist in the current human population, with the vast majority having unknown functional consequences. Here we present the first large-scale experimental analysis of human missense variants across many different proteins. Using DNA synthesis and cellular selection experiments we quantify the impact of >500,000 variants on the abundance of >500 human protein domains. This dataset - Human Domainome 1.0 - reveals that >60% of pathogenic missense variants reduce protein stability. The contribution of stability to protein fitness varies across proteins and diseases, and is particularly important in recessive disorders. Mutational effects on stability are largely conserved in homologous domains, allowing accurate stability prediction across entire protein families using energy models. Combining stability measurements with protein language models annotates functional sites. Domainome 1.0 demonstrates the feasibility of assaying human protein variants at scale and provides a large consistent reference dataset for clinical variant interpretation and the training and benchmarking of computational methods.

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Section: Discussionmentioning

confidence: 99%

Site saturation mutagenesis of 500 human protein domains reveals the contribution of protein destabilization to genetic disease

Beltran,

Jiang,

Shen

et al. 2024

Preprint

Self Cite

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Saturation mutagenesis-reinforced functional assays for disease-related genes

Ma,

Huang,

et al. 2024

Cell

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Massively parallel genetic perturbation reveals the energetic architecture of an amyloid beta nucleation reaction

Arutyunyan,

Seuma,

Faure

et al. 2024

Preprint

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Amyloid protein aggregates are pathological hallmarks of more than fifty human diseases including the most common neurodegenerative disorders. The atomic structures of amyloid fibrils have now been determined, but the process by which soluble proteins nucleate to form amyloids remains poorly characterised and difficult to study, even though this is the key step to understand to prevent the formation and spread of aggregates. Here we use massively parallel combinatorial mutagenesis, a kinetic selection assay, and machine learning to reveal the transition state of the nucleation reaction of amyloid beta, the protein that aggregates in Alzheimer's disease. By quantifying the nucleation of >140,000 proteins we infer the changes in activation energy for all 798 amino acid substitutions in amyloid beta and the energetic couplings between >600 pairs of mutations. This unprecedented dataset provides the first comprehensive view of the energy landscape and the first large-scale measurement of energetic couplings for a protein transition state. The energy landscape reveals that the amyloid beta nucleation transition state contains a short structured C-terminal hydrophobic core with a subset of interactions similar to mature fibrils. This study demonstrates the feasibility of using mutation-selection-sequencing experiments to study transition states and identifies the key molecular species that initiates amyloid beta aggregation and, potentially, Alzheimer's disease.

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SUNi mutagenesis: Scalable and uniform nicking for efficient generation of variant libraries

Cited by 4 publications

References 20 publications

Site saturation mutagenesis of 500 human protein domains reveals the contribution of protein destabilization to genetic disease

Site saturation mutagenesis of 500 human protein domains reveals the contribution of protein destabilization to genetic disease

Saturation mutagenesis-reinforced functional assays for disease-related genes

Massively parallel genetic perturbation reveals the energetic architecture of an amyloid beta nucleation reaction

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