DOI: 10.5281/zenodo.17517360
title: Sequencing data for the manuscript "Contrasting dynamic regimes reveal flexibility as a driver of enzyme evolvability"

description: Sequencing data for the manuscript "Contrasting dynamic regimes reveal flexibility as a driver of enzyme evolvability"

Context:

This is the preliminary sequencing data deposition for the manuscript

J. David Schnettler, Alexander Klug, Chiara Longhi, Lisa Freund, and Macarena Toll-Riera: Contrasting dynamic regimes reveal flexibility as a driver of enzyme evolvability, manuscript in preparation

Abstract [preliminary]:Enzymes vary widely in their ability to evolve new catalytic function, but the molecular properties that influence this evolvability remain unknown. While stability is thought to promote mutational robustness and conformational flexibility to enhance promiscuity, how these features together shape adaptive potential remains unclear. Here we test whether increased conformational flexibility promotes adaptation to a new catalytic function by comparing the evolutionary trajectories of two homologous class C beta-lactamases with distinct intrinsic dynamics, the psychrophilic enzyme pAmpC and the mesophilic enzyme mAmpC. We subjected both enzymes to six rounds of directed evolution towards improved cefepime resistance under contrasting dynamic regimes: a “flexibilising” condition (pAmpC evolved at 37 °C) and a “rigidifying” condition (mAmpC evolved at 20 °C). The flexibilising regime led to faster adaptation and higher resistance, with mutations accumulating more readily than under rigidifying conditions. Stability predictions and network analyses both pointed to the same trend: evolution under flexibilising conditions followed broader, more varied paths, whereas the rigidifying regime produced narrower, founder-dominated trajectories. These findings show that the dynamic context in which proteins evolve profoundly shapes its their pace and trajectories. Conformational flexibility, even at the expense of reduced stability, appears to expand the accessible sequence space and may represent a key determinant of enzyme evolvability.

Structure of the dataset:

This PacBio HiFi sequencing dataset was acquired on a PacBio Revio instrument in the NGS Platform of the University of Bern on 10th May 2024. Further technical information, including barcoding for demultiplexing and primary quality control is detailed in the report of the sequencing run

There is one fastq file per sequencing sample, containing all demultiplexed HiFi reads for one gene/temperature/replicate/round combination. In total, there are 128 fast files, covering two genes (pAmpC/mAmpC) x two temperatures (20/37 °C) x six rounds x four replicates and controls.

Further reference files are contained in references.zip:- list of treatments (including sample labels matched to gene/temperature/round/replicate combination- gene sequences for variant calling- probe sequences for frame detection during variant calling- list of pre-selection barcodes (bc1)- report of the sequencing run

Sequencing data will additionally be made available on the European Nucleotide Archive (ENA) upon publication of the manuscript. The data analysis pipeline will be made available on Github.