Deep mutational scanning: assessing protein function on a massive scale

By Carlos L. Araya, Douglas M. Fowler. 2011

217 citations recorded by the source corpus.

Analysis of protein mutants is an effective means to understand their function. Protein display is an approach that allows large numbers of mutants of a protein to be selected based on their activity, but only a handful with maximal activity have been traditionally identified for subsequent functional analysis. However, the recent application of high-throughput DNA sequencing to protein display and selection has enabled simultaneous assessment of the function of hundreds of thousands of mutants that span the activity range from high to low. Such deep mutational scanning approaches are rapid and inexpensive, with the potential for broad utility. Here, we discuss the emergence of deep mutational scanning, the challenges associated with its use, and some of its exciting applications.

Subfields: Molecular Biology, Radiology, Nuclear Medicine and Imaging

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Citations in the corpus

Improved Modeling of Side-Chain–Base Interactions and Plasticity in Protein–DNA Interface Design (2012, result): “…mis-predicted due to the limited size of our experimental data set, we expect that recent work combining next-generation sequencing technology with protein selection 43 will revolutionize studies of this sort that attempt to correlate protein mutations with functional characteristics.”
Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis (2013, methodology): “Such limitations prompted us to use a highthroughput method, known as “deep mutational scanning” (12, 13), that can assess the effects of over 10(5) sequence variants of a single protein simultaneously.”
Enrich: software for analysis of protein function by enrichment and depletion of variants (2011, background): “Such ‘deep mutational scanning’ (Araya and Fowler, 2011) experiments engender significant analysis challenges.”
Measuring the activity of protein variants on a large scale using deep mutational scanning (2014, methodology): “More complex experimental designs, including the use of subassembly to sequence longer regions 22 and the collection of multiple time points, have subsequently been published 5,23 .”
High-throughput Analysis of in vivo Protein Stability* (2013, background): “(4, 5), in which a physical association between each protein variant and the DNA that encodes it allows DNA sequencing”
The inherent mutational tolerance and antigenic evolvability of influenza hemagglutinin (2014, background): “Epidemic influenza poses an annual threat to human health largely because the virus rapidly evolves to escape the immunity elicited by previous infections or vaccinations.”
Comprehensive mutational scanning of a kinase in vivo reveals substrate-dependent fitness landscapes (2014, background): “Engineering families of biomolecules with orthogonal activities is a central challenge in biotechnology and synthetic biology (2,17) due to (i) the difficulty of performing direct selection for the absence of activity in one or more conditions and (ii) the apparently limited overlap between mutation”
A fundamental protein property, thermodynamic stability, revealed solely from large-scale measurements of protein function (2012, methodology): “We used deep mutational scanning to measure the ability of 47,000 unique variants of the hYAP65 WW domain to bind to their polyproline peptide ligand (2, 4).”
Deep mutational scanning of an antibody against epidermal growth factor receptor using mammalian cell display and massively parallel pyrosequencing (2013, methodology): “In particular, the use of NGS to analyze the results of sorted protein display libraries in deep mutational scanning approaches promises to be the method of choice for generation of very large sequence-function fitness landscapes.(4) We devised a deep mutational scanning method in which NGS is used ”
Combining Natural Sequence Variation with High Throughput Mutational Data to Reveal Protein Interaction Sites (2015, methodology): “Recently, a method known as deep mutational scanning was developed to assess the functional consequences of up to hundreds of thousands of variants of a protein in a single experiment [14,15].”