%0 Journal Article
%T Enzyme Engineering by Force: DNA Springs for the Modulation of Biocatalytic Trajectories.
%A Gokulu IS
%A Banta S
%J ACS Synth Biol
%V 13
%N 8
%D 2024 Aug 16
%M 39110689
%F 5.249
%R 10.1021/acssynbio.4c00431
%X The engineering of enzymatic activity generally involves alteration of the protein primary sequences, which introduce structural changes that give rise to functional improvements. Mechanical forces have been used to interrogate protein biophysics, leading to deep mechanistic insights in single-molecule studies. Here, we use simple DNA springs to apply small pulling forces to perturb the active site of a thermostable alcohol dehydrogenase. Methods were developed to enable the study of different spring lengths and spring orientations under bulk catalysis conditions. Tension applied across the active site expanded the binding pocket volume and shifted the preference of the enzyme for longer chain-length substrates, which could be tuned by altering the spring length and the resultant applied force. The substrate specificity changes did not occur when the DNA spring was either severed or rotated by ∼90°. These findings demonstrate an alternative approach in protein engineering, where active site architectures can be dynamically and reversibly remodeled using applied mechanical forces.