| CPC G16B 15/00 (2019.02) [C12N 9/003 (2013.01); C12N 9/18 (2013.01); C12N 9/90 (2013.01); C12Y 105/01003 (2013.01); C12Y 301/01008 (2013.01); C12Y 502/01008 (2013.01); G16B 5/00 (2019.02)] | 27 Claims |
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1. A method of engineering a target enzyme to modify its catalytic activity, the method comprising:
accessing biophysical and dynamics data for the target enzyme;
analyzing, using a computing device, the biophysical and dynamics data to identify at least one energy transfer network within the target enzyme, the at least one energy transfer network comprising a series of residues spanning from a surface of the target enzyme to a catalytic site of the target enzyme;
identifying at least one surface loop or neighboring region within the at least one energy transfer network, wherein the at least one surface loop or neighboring region comprises one or more residues showing promoting dynamical motions coupled to the catalytic activity of the target enzyme;
iteratively modifying at least one residue of the one or more residues of the at least one surface loop or neighboring region to produce a plurality of candidate engineered enzyme sequences such that each of the plurality of candidate engineered enzyme sequences comprises a different residue at the location of the at least one residue, wherein the at least one surface loop or neighboring region has a longer side chain as a result of the modifying;
for each candidate engineered enzyme sequence of the plurality of candidate engineered enzyme sequences:
determining a modified network conductance of the candidate engineered enzyme sequence exceeds a baseline network conductance of the target enzyme and provides an increase in energy flow directed into the catalytic site via the at least one energy transfer network by:
calculating a residue energy conductance for each residue of the at least one energy transfer network for each of the target enzyme and the candidate engineered enzyme sequence; and
combining the residue energy conductance for each residue of the at least one energy transfer network for each of the baseline network conductance and the modified network conductance;
calculating the change in thermo-dynamical coupling with an external environment or solvent by calculating the surface area of interaction due to the modified residue;
calculating the changes in conformations in the functionally important sub-states related to the enzyme activity due to the changes in energy conductance through the network;
assigning a score or ranking of each candidate engineered enzyme sequence of the plurality of candidate engineered enzyme sequences using the modified network conductance, the change in thermo-dynamical coupling, and the changes in conformations;
selecting a subset of top ranking candidates of the plurality of candidate engineered enzyme sequences; and
constructing an engineered enzyme with increased catalytic activity by transfecting cells with nucleic acids encoding the engineered enzyme sequence selected from the subset of top ranking candidates and purifying the engineered enzyme.
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