High-throughput Analysis of in vivo Protein Stability*

2013

71 citations recorded by the source corpus.

Determining the half-life of proteins is critical for an understanding of virtually all cellular processes. Current methods for measuring in vivo protein stability, including large-scale approaches, are limited in their throughput or in their ability to discriminate among small differences in stability. We developed a new method, Stable-seq, which uses a simple genetic selection combined with high-throughput DNA sequencing to assess the in vivo stability of a large number of variants of a protein. The variants are fused to a metabolic enzyme, which here is the yeast Leu2 protein. Plasmids encoding these Leu2 fusion proteins are transformed into yeast, with the resultant fusion proteins accumulating to different levels based on their stability and leading to different doubling times when the yeast are grown in the absence of leucine. Sequencing of an input population of variants of a protein and the population of variants after leucine selection allows the stability of tens of thousands of variants to be scored in parallel. By applying the Stable-seq method to variants of the protein degradation signal Deg1 from the yeast Matα2 protein, we generated a high-resolution map that reveals the effect of ∼30,000 mutations on protein stability. We identified mutations that likely affect stability by changing the activity of the degron, by leading to translation from new start codons, or by affecting N-terminal processing. Stable-seq should be applicable to other organisms via the use of suitable reporter proteins, as well as to the analysis of complex mixtures of fusion proteins.

Subfields: Molecular Biology, Oncology

Citations in the corpus

• Functional Constraint Profiling of a Viral Protein Reveals Discordance of Evolutionary Conservation and Functionality (2015, background): “To obtain a more accurate measurement of protein stability, high-throughput experimental analysis on protein stability may provide an alternative [76, 77].”
• Large-scale de novo DNA synthesis: technologies and applications (2014, background): “Finally, the development of deep mutational scanning techniques to measure structure-function relationships in multiplex will enable rapid characterization of large designed synthetic gene libraries as synthesis methods improve [90] [91] [92] [93] [94] [95] .”
• Multiplex Assessment of Protein Variant Abundance by Massively Parallel Sequencing (2018, methodology): “Inspired by earlier methods to assess the stability of protein variants in yeast 14 and bacteria 15 , and by a microarray-based assay that globally profiled mammalian protein stability 16 , we developed VAMP-seq.”
• Elements of the ERAD ubiquitin ligase Doa10 regulating sequential poly-ubiquitylation of its targets (2022, unknown): “…characteristics of Doa10-dependent degrons have been defined (Chua et al., 2017; Furth et al., 2011; Johnson et al., 1998; Kats et al., 2018; Kim et al., 2013), the functional domains in Doa10 involved in substrate recognition remain completely unknown and no direct substrate interaction…”
• Measuring the activity of protein variants on a large scale using deep mutational scanning (2014, methodology): “In one example, a deep mutational scan of a yeast degron was conducted using a cell-based reporter consisting of a fusion of the degron to a metabolic enzyme 16 .”