Protein dynamics are often invoked in explanations of enzyme catalysis, but their design has proven elusive. Here we track the role of dynamics in evolution, starting from the evolvable and thermostable ancestral protein AncHLD-RLuc which catalyses both dehalogenase and luciferase reactions. Insertion-deletion (InDel) backbone mutagenesis of AncHLD-RLuc challenged the scaffold dynamics. Screening for both activities reveals InDel mutations localized in three distinct regions that lead to altered protein dynamics (based on crystallographic B-factors, hydrogen exchange, and molecular dynamics simulations). An anisotropic network model highlights the importance of the conformational flexibility of a loop-helix fragment of Renilla luciferases for ligand binding. Transplantation of this dynamic fragment leads to lower product inhibition and highly stable glow-type bioluminescence. The success of our approach suggests that a strategy comprising (i) constructing a stable and evolvable template, (ii) mapping functional regions by backbone mutagenesis, and (iii) transplantation of dynamic features, can lead to functionally innovative proteins.

A number of novel protein sequences dramatically increases in public databases, however, there are only very few functional annotations. This significantly limits the exploration of the available protein diversity obtained by next-generation sequencing. Conventional methods cannot keep up with current challenges and a development of a new generation of biochemical techniques is essential. The great potential of microfluidic technology with reduced sample requirements and powerful throughput can bring an adequate capacity for functional annotation of the rising protein diversity. However, even the most promising dropletbased systems still need to address a number of limitations. The leakage of hydrophobic compounds from water compartments to the carrier oil represents one of the major problems which limits the utilization of these systems to operate with hydrophilic reagents. Herein, we present an approach using a novel way of substrate delivery in droplet microfluidics applied to high-throughput functional characterization of enzymes converting hydrophobic substrates. The substrate delivery is based on the partitioning of hydrophobic chemicals between the oil and water phases. We applied a controlled distribution of hydrophobic haloalkanes from oil to reaction water droplets to perform activity screening of eight model enzymes from the haloalkane dehalogenase family. The droplet-on-demand microfluidic system reduces the reaction volume 65 000 times and increases the speed of the analysis almost 100 times, compared to the conventional method. Additionally, the microfluidic setup enables a convenient determination of temperature optima for a set of mesophilic and engineered hyper stable enzyme variants at the working range 5 to 90 °C. A quantitative comparison of the microfluidic data and the results from the the conventional method showed a high consistency with R 2 = 0.89 and 0.95 for the substrate specificity and temperature optima analysis, respectively. The microfluidic method demonstrated a high precision and an advanced analytical throughput >20,000 reactions per day. The presented substrate delivery approach extends the scope of microfluidics applications for high-throughput analysis of reactions including compounds with limited water solubility.

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