Facile Method for High-throughput Identification of Stabilizing Mutations

Area of Science:

• Biophysics
• Molecular Biology
• Protein Engineering

Background:

• Understanding protein mutation effects on stability is crucial for protein function and evolution.
• High-throughput assays exist for reduced stability mutations, but screening for increased stability is challenging.
• Increased thermodynamic stability mutations are rare and their effects are subtle, complicating screening.

Purpose of the Study:

• To develop a multiplex assay for high-throughput screening of protein folding and identify stabilizing mutations.
• To demonstrate the assay's correlation with stability and its ability to detect subtle increases.
• To analyze substitution patterns associated with increased protein stability.

Main Methods:

• Combined deep mutational scanning, fluorescence-activated cell sorting, and deep sequencing.
• Developed a multiplex assay for high-throughput screening of protein folding.
• Analyzed a library of 2000 Adenylate kinase variants.

Main Results:

• The assay readout correlated with protein stability.
• Identified mutants with up to a 13°C increase in thermal melting temperature.
• Achieved a low false positive rate in identifying stabilizing mutations.
• Enabled analysis of substitution patterns linked to increased stability.

Conclusions:

• The developed high-throughput method effectively identifies stabilizing mutations in proteins.
• This assay can be combined with functional screens to improve both protein stability and activity.
• Facilitates discovery of mutations enhancing thermodynamic stability.