Development of an Enzyme-Inhibitor Reaction Using Cellular Retinoic Acid Binding Protein II for One-Pot Megamolecule Assembly

Blaise R. Kimmel; Milan Mrksich

doi:10.1002/chem.202103059

Development of an Enzyme-Inhibitor Reaction Using Cellular Retinoic Acid Binding Protein II for One-Pot Megamolecule Assembly

Blaise R. Kimmel, Milan Mrksich^*

^*Corresponding author for this work

Biomedical Engineering

Research output: Contribution to journal › Article › peer-review

6 Scopus citations

Abstract

This paper presents an enzyme building block for the assembly of megamolecules. The system is based on the inhibition of the human-derived cellular retinoic acid binding protein II (CRABP2) domain. We synthesized a synthetic retinoid bearing an arylfluorosulfate group, which uses sulfur fluoride exchange click chemistry to covalently inhibit CRABP2. We conjugated both the inhibitor and a fluorescein tag to an oligo(ethylene glycol) backbone and measured a second-order rate constant for the protein inhibition reaction of approximately 3,600 M⁻¹s⁻¹. We used this new enzyme-inhibitor pair to assemble multi-protein structures in one-pot reactions using three orthogonal assembly chemistries to demonstrate exact control over the placement of protein domains within a single, homogeneous molecule. This work enables a new dimension of control over specificity, orientation, and stoichiometry of protein domains within atomically precise nanostructures.

Original language	English (US)
Pages (from-to)	17843-17848
Number of pages	6
Journal	Chemistry - A European Journal
Volume	27
Issue number	71
DOIs	https://doi.org/10.1002/chem.202103059
State	Published - Dec 20 2021

Funding

B.R.K. acknowledges support from the Ryan Fellowship, the International Institute for Nanotechnology at Northwestern University, and the National Science Foundation Graduate Research Fellowship under Grant DGE-1842165. Research reported in this paper was supported by ARO MURI FA9550-16-1-0150. The authors thank Justin Modica and Bethel Shekour for help in collecting NMR spectra and Zhaoyi Gu in the purification of chemical linkers. The authors thank Valerie Tokars and Pamela Focia for performing TEM imaging and X-ray Crystallographic structure determination and refinement. This research used resources of the Advanced Photon Source; a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357. Use of beamlines of the Life Sciences Collaborative Access Team (LS-CAT) located at Sector 21 of the APS was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). This work also used resources of the Northwestern University Structural Biology Facility, which is supported by the NCI CCSG P30 CA060553 grant awarded to the Robert H. Lurie Comprehensive Cancer Center.

Keywords

bioorganic chemistry
enzymes
inhibitors
kinetics
protein structures

ASJC Scopus subject areas

General Chemistry
Catalysis
Organic Chemistry

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10.1002/chem.202103059

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title = "Development of an Enzyme-Inhibitor Reaction Using Cellular Retinoic Acid Binding Protein II for One-Pot Megamolecule Assembly",

abstract = "This paper presents an enzyme building block for the assembly of megamolecules. The system is based on the inhibition of the human-derived cellular retinoic acid binding protein II (CRABP2) domain. We synthesized a synthetic retinoid bearing an arylfluorosulfate group, which uses sulfur fluoride exchange click chemistry to covalently inhibit CRABP2. We conjugated both the inhibitor and a fluorescein tag to an oligo(ethylene glycol) backbone and measured a second-order rate constant for the protein inhibition reaction of approximately 3,600 M−1s−1. We used this new enzyme-inhibitor pair to assemble multi-protein structures in one-pot reactions using three orthogonal assembly chemistries to demonstrate exact control over the placement of protein domains within a single, homogeneous molecule. This work enables a new dimension of control over specificity, orientation, and stoichiometry of protein domains within atomically precise nanostructures.",

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author = "Kimmel, {Blaise R.} and Milan Mrksich",

note = "Funding Information: B.R.K. acknowledges support from the Ryan Fellowship, the International Institute for Nanotechnology at Northwestern University, and the National Science Foundation Graduate Research Fellowship under Grant DGE-1842165. Research reported in this paper was supported by ARO MURI FA9550-16-1-0150. The authors thank Justin Modica and Bethel Shekour for help in collecting NMR spectra and Zhaoyi Gu in the purification of chemical linkers. The authors thank Valerie Tokars and Pamela Focia for performing TEM imaging and X-ray Crystallographic structure determination and refinement. This research used resources of the Advanced Photon Source; a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract DE-AC02-06CH11357. Use of beamlines of the Life Sciences Collaborative Access Team (LS-CAT) located at Sector 21 of the APS was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P1000817). This work also used resources of the Northwestern University Structural Biology Facility, which is supported by the NCI CCSG P30 CA060553 grant awarded to the Robert H. Lurie Comprehensive Cancer Center. Publisher Copyright: {\textcopyright} 2021 Wiley-VCH GmbH",

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N2 - This paper presents an enzyme building block for the assembly of megamolecules. The system is based on the inhibition of the human-derived cellular retinoic acid binding protein II (CRABP2) domain. We synthesized a synthetic retinoid bearing an arylfluorosulfate group, which uses sulfur fluoride exchange click chemistry to covalently inhibit CRABP2. We conjugated both the inhibitor and a fluorescein tag to an oligo(ethylene glycol) backbone and measured a second-order rate constant for the protein inhibition reaction of approximately 3,600 M−1s−1. We used this new enzyme-inhibitor pair to assemble multi-protein structures in one-pot reactions using three orthogonal assembly chemistries to demonstrate exact control over the placement of protein domains within a single, homogeneous molecule. This work enables a new dimension of control over specificity, orientation, and stoichiometry of protein domains within atomically precise nanostructures.

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ER -

Development of an Enzyme-Inhibitor Reaction Using Cellular Retinoic Acid Binding Protein II for One-Pot Megamolecule Assembly

Abstract

Funding

Keywords

ASJC Scopus subject areas

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Cellular Retinoic Acid Binding Protein II with Bound Inhibitor 4-[6-({4-[(fluorosulfonyl)oxy]phenyl}ethynyl)-4,4-dimethyl-3,4-dihydroquinolin-1(2H)-yl]-4-oxobutanoic acid

Cite this

Development of an Enzyme-Inhibitor Reaction Using Cellular Retinoic Acid Binding Protein II for One-Pot Megamolecule Assembly

Abstract

Funding

Keywords

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Datasets

Cellular Retinoic Acid Binding Protein II with Bound Inhibitor 4-[6-({4-[(fluorosulfonyl)oxy]phenyl}ethynyl)-4,4-dimethyl-3,4-dihydroquinolin-1(2H)-yl]-4-oxobutanoic acid

Cite this