Abstract
This paper describes the synthesis, characterization, and modeling of a series of molecules having four protein domains attached to a central core. The molecules were assembled with the "megamolecule"strategy, wherein enzymes react with their covalent inhibitors that are substituted on a linker. Three linkers were synthesized, where each had four oligo(ethylene glycol)-based arms terminated in a para-nitrophenyl phosphonate group that is a covalent inhibitor for cutinase. This enzyme is a serine hydrolase and reacts efficiently with the phosphonate to give a new ester linkage at the Ser-120 residue in the active site of the enzyme. Negative-stain transmission electron microscopy (TEM) images confirmed the architecture of the four-armed megamolecules. These cutinase tetramers were also characterized by X-ray crystallography, which confirmed the active-site serine-phosphonate linkage by electron-density maps. Molecular dynamics simulations of the tetracutinase megamolecules using three different force field setups were performed and compared with the TEM observations. Using the Amberff99SB-disp + pH7 force field, the two-dimensional projection distances of the megamolecules were found to agree with the measured dimensions from TEM. The study described here, which combines high-resolution characterization with molecular dynamics simulations, will lead to a comprehensive understanding of the molecular structures and dynamics for this new class of molecules.
Original language | English (US) |
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Pages (from-to) | 2363-2372 |
Number of pages | 10 |
Journal | Biomacromolecules |
Volume | 22 |
Issue number | 6 |
DOIs | |
State | Published - Jun 14 2021 |
Funding
This work was supported by the Army Research Office (ARO W911NF1810200 to M.M.) and the Air Force Office of Scientific Research (AFOSR FA9550-16-1-0150 to M.M.). K.P. gratefully 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. This work made use of the IMSERC at Northwestern University and the BioCryo Facility and EPIC of Northwestern University’s NUANCE Center, which has received support from the Soft and Hybrid Nanotechnology Experimental (SHyNE) Resource (NSF ECCS-1542205); the MRSEC program (NSF DMR-1720139) at the Materials Research Center; the International Institute for Nanotechnology (IIN); and the State of Illinois, through the IIN. LS-CAT/Sector 21 at the Advanced Photon Source, Argonne National Laboratory.
ASJC Scopus subject areas
- Bioengineering
- Biomaterials
- Polymers and Plastics
- Materials Chemistry