Circular permutation profiling by deep sequencing libraries created using transposon mutagenesis.

Area of Science:

• Molecular Biology
• Protein Engineering
• Biophysics

Background:

• Deep mutational scanning maps functional consequences of point mutations.
• Circular permutation engineering alters protein contact order but lacks high-resolution mapping.
• Previous methods did not assess protein tolerance to circular permutation comprehensively.

Purpose of the Study:

• To develop and validate a new method, circular permutation profiling with DNA sequencing (CPP-seq), for analyzing protein tolerance to circular permutation.
• To apply CPP-seq to adenylate kinase to understand its behavior under permutation.
• To relate protein structure and biophysical characteristics to functional activity after permutation.

Main Methods:

• Developed CPP-seq, combining one-step transposon mutagenesis, functional selection, deep sequencing, and computational analysis.
• Created libraries of circularly permuted adenylate kinase genes.
• Analyzed sequence enrichment and mapped functional data onto protein structure.

Main Results:

• CPP-seq generates vectors with differential protein expression capabilities.
• >65% of expressing vectors were enriched over non-expressing ones after functional selection.
• The mobile AMP binding and rigid core domains showed higher tolerance to backbone fragmentation than the mobile lid domain.

Conclusions:

• CPP-seq provides unbiased insight into protein tolerance to circular permutation.
• The method effectively links protein biophysical characteristics to activity retention upon permutation.
• CPP-seq is a valuable tool for protein engineering and understanding structure-function relationships.