TY - JOUR
T1 - Metal-responsive regulation of enzyme catalysis using genetically encoded chemical switches
AU - Zubi, Yasmine S.
AU - Seki, Kosuke
AU - Li, Ying
AU - Hunt, Andrew C.
AU - Liu, Bingqing
AU - Roux, Benoît
AU - Jewett, Michael C.
AU - Lewis, Jared C.
N1 - Funding Information:
This study was supported by the U.S. Army Research Laboratory and the U.S. Army Research Office under Contracts/Grants W911NF-18-1-0034 and W911NF-15-1-0334 (J.C.L.), W911NF-18-1-0181 (M.C.J.), and under Grant Number W911NF-18-1-0200 (J.C.L, M.C.J., and B.R.); the David and Lucile Packard Foundation (M.C.J.); and the Camille Dreyfus Teacher-Scholar Program (J.C.L. and M.C.J.). Y.S.Z gratefully acknowledges receipt of a predoctoral fellowship from the Graduate Training Program in Quantitative and Chemical Biology at Indiana University (T32 GM131994). K.S acknowledges support from a predoctoral fellowship from Chemistry of Life Processes Institute, supported by the National Institute of General Medical Sciences of the National Institutes of Health (T32 GM105538). A.H. acknowledges the Department of Defense National Defense Science and Engineering Graduate Fellowship Program (NDSEG-36373). We thank Mr. Matthew Jordan for performing ICP-MS measurements; Dr. Jonathan Trinidad for assistance with intact protein ESI-MS; Dr. Giovanni Gonzalez-Gutierrez for assistance with various instrumentation in the IU Physical Biochemistry Instrumentation Facility; Prof. Amar Flood for access to a UV-Vis spectrophotometer; Mr. Saman Shafaie and Dr. Benjamin Owen for training on ESI-MS instruments at IMSERC in NU; and Ashty Karim for helpful discussions on manuscript preparation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2022, The Author(s).
PY - 2022/12
Y1 - 2022/12
N2 - Dynamic control over protein function is a central challenge in synthetic biology. To address this challenge, we describe the development of an integrated computational and experimental workflow to incorporate a metal-responsive chemical switch into proteins. Pairs of bipyridinylalanine (BpyAla) residues are genetically encoded into two structurally distinct enzymes, a serine protease and firefly luciferase, so that metal coordination biases the conformations of these enzymes, leading to reversible control of activity. Computational analysis and molecular dynamics simulations are used to rationally guide BpyAla placement, significantly reducing experimental workload, and cell-free protein synthesis coupled with high-throughput experimentation enable rapid prototyping of variants. Ultimately, this strategy yields enzymes with a robust 20-fold dynamic range in response to divalent metal salts over 24 on/off switches, demonstrating the potential of this approach. We envision that this strategy of genetically encoding chemical switches into enzymes will complement other protein engineering and synthetic biology efforts, enabling new opportunities for applications where precise regulation of protein function is critical.
AB - Dynamic control over protein function is a central challenge in synthetic biology. To address this challenge, we describe the development of an integrated computational and experimental workflow to incorporate a metal-responsive chemical switch into proteins. Pairs of bipyridinylalanine (BpyAla) residues are genetically encoded into two structurally distinct enzymes, a serine protease and firefly luciferase, so that metal coordination biases the conformations of these enzymes, leading to reversible control of activity. Computational analysis and molecular dynamics simulations are used to rationally guide BpyAla placement, significantly reducing experimental workload, and cell-free protein synthesis coupled with high-throughput experimentation enable rapid prototyping of variants. Ultimately, this strategy yields enzymes with a robust 20-fold dynamic range in response to divalent metal salts over 24 on/off switches, demonstrating the potential of this approach. We envision that this strategy of genetically encoding chemical switches into enzymes will complement other protein engineering and synthetic biology efforts, enabling new opportunities for applications where precise regulation of protein function is critical.
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U2 - 10.1038/s41467-022-29239-y
DO - 10.1038/s41467-022-29239-y
M3 - Article
C2 - 35387988
AN - SCOPUS:85127702227
VL - 13
JO - Nature Communications
JF - Nature Communications
SN - 2041-1723
IS - 1
M1 - 1864
ER -