Studying the Protein Organization of the Postsynaptic Density by a Novel Solid Phase- and Chemical Cross-linking-based Technology

Abstract

Cells contain numerous supramolecules, such as the nuclear pore complex, proteosome, chlorosome, ribosome, and flagellar motor complex, and these are used to carry out a wide variety of complicated functions. A supramolecule usually consists of a population of diverse biomolecules, and these biomolecules are organized in such a way that their contributions to the function of the supramolecule are coordinated spatially and temporally. Understanding the protein organization of a supramolecule is thus essential for fully understanding the molecular mechanism by which the supramolecule functions. Several powerful techniques, such as nuclear magnetic resonance, cryoelectron microscopy, x-ray crystallography, and chemical cross-linking, have been used to investigate the protein organization of supramolecules. Here we report a novel methodology that was designed to distinguish proteins lining the surface from those embedded in the interior of supramolecules. After verification with a synthetic multilayered protein complex of known structure, this methodology was used to investigate the protein organization of a landmark structure of the asymmetric synapses in the mammalian central nervous system, the postsynaptic density (PSD).

A Study of the Spatial Protein Organization of the Postsynaptic Density Isolated from Porcine Cerebral Cortex and Cerebellum

“…1B). These results were consistent with our earlier findings that NDT1 and NDT2 could interact with proteins lining the surface and with proteins residing within 8.0 nm deep from the surface in a protein complex, respectively (44,46). The results also indicated that the azido group of NDT3 could indeed penetrate into a region between 7.30 and 10.99 nm deep in a protein complex and form covalent links with proteins residing in this depth range.…”

“…Isolation of Cerebral and Cerebellar PSD-PSDs were isolated from porcine cerebral cortex and cerebellum according to the procedures reported previously (44). Briefly, fresh pig brains were obtained from a local slaughterhouse.…”

“…Chemical Syntheses of NDT1, NDT2, and NDT3 Reagents-The NDT1 and NDT2 reagents were synthesized according to the procedures reported earlier (44,46). For the synthesis of NDT3 (Fig.…”

“…An NDT reagent, NDT1 (originally called as NDT3. 5), has been used to identify proteins lining the surface of the PSD (44), and a second NDT reagent, NDT2 (originally called as NDT7.0), has been found to be a useful tool to identify proteins residing in regions within 7.2 nm deep from the surface of the PSD (46). Here, we report the synthesis of a third NDT reagent, called NDT3 (Fig.…”

“…The majority of actin molecules in the PSD have been proposed to stay in the polymerized state (43). By using a solid-phase and chemical crosslinking-based methodology (see below), the proteins lining the surface of the PSD have been identified (44). An earlier study has indicated that the PSD isolated from dog cerebral cortex differs from the PSD isolated from dog cerebellum in protein composition, protein phosphorylation, and morphology (17).…”