Synthesis, Characterization, and Simulation of Four-Armed Megamolecules

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.

Scanning Transmission Electron Microscopy in a Scanning Electron Microscope for the High-Throughput Imaging of Biological Assemblies

“…Plasmid construction, protein expression, protein purification, and four-armed linker synthesis for the construction of the tetracutinase megamolecule are previously described. 17 The megamolecule assembly reaction was performed in phosphate buffered saline (PBS) (1× PBS; 2.7 mM KCl, 138 mM NaCl, pH 7.4). To 44 nmol of cutinase protein (44 μM, 1.1 equivalents) was added 10 nmol (10 μM, 1 equivalent) of the fourarmed PEG11 linker with terminal p-nitrophenyl phosphonate covalent inhibitors.…”

“…Plasmid construction, protein expression, protein purification, and four-armed linker synthesis for the construction of the tetracutinase megamolecule are previously described . The megamolecule assembly reaction was performed in phosphate buffered saline (PBS) (1× PBS; 2.7 mM KCl, 138 mM NaCl, pH 7.4).…”

“…The cell size is 250 Å × 250 Å × 70 Å and contains 236,034 total atoms. The tetracutinase coordinates were adapted from Zhou et al, and the amorphous carbon structure was adapted from Ricolleau et al We modified the original PRISM code to introduce chromatic aberrations ( C c ) via relationship with beam spreading d c in: where Δ E is the energy spread of the electron beam, E 0 is the energy of incident electron beam, and α is the convergence semiangle. Bright field (BF) and dark field (DF) images were plotted using collection angles of 0–6 mrad and greater than 80 mrad, respectively.…”

“…This work focuses on the characterization of a class of biological materials referred to as megamolecules. , Linear, cyclic, and branched megamolecules are assembled through covalent inhibition reactions between a monomeric protein domain and a targeted covalent inhibitor on a poly­(ethylene glycol) (PEG) backbone. − Here, we use a previously described protein scaffold, known as tetracutinase, as a model system . The linker used to form the tetracutinase megamolecule has four equivalents of a p -nitrophenyl phosphonate covalent inhibitor connected by 11 unit PEG arms to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid core.…”

“…14−16 Here, we use a previously described protein scaffold, known as tetracutinase, as a model system. 17 The linker used to form the tetracutinase megamolecule has four equivalents of a p-nitrophenyl phosphonate covalent inhibitor connected by 11 unit PEG arms to a 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid core. Reaction of the nucleophilic serine residue (Ser 120) in the active site of cutinase with the electrophilic phosphonate group yields a covalent linkage, allowing for the atomically precise synthesis of the tetracutinase megamolecule in one-pot.…”