Synthesis, Characterization, and Simulation of Four-Armed Megamolecules

Biomacromolecules. 2021 Jun 14;22(6):2363-2372. doi: 10.1021/acs.biomac.1c00118. Epub 2021 May 12.

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.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Catalytic Domain
  • Crystallography, X-Ray
  • Molecular Structure
  • Organophosphonates*
  • Protein Domains

Substances

  • Organophosphonates