Abstract
Agarose beads carrying a cleavable, fluorescent, and photoreactive cross-linking reagent on the surface were synthesized and used to selectively pull out the proteins lining the surface of supramolecules. A quantitative comparison of the abundances of various proteins in the sample pulled out by the beads from supramolecules to their original abundances could provide the information on the spatial arrangement of these proteins in the supramolecule. The usefulness of these synthetic beads was successfully verified by trials using a synthetic protein complex consisting of three layers of different proteins on glass coverslip. By using these beads, we determined the interior or superficial locations of 5 major and 19 minor constituent proteins in the postsynaptic density (PSD), a large protein complex and the landmark structure of asymmetric synapses in the mammalian CNS. The results indicate that α,β-tubulins, dynein heavy chain, microtubule-associated protein 2, spectrin, neurofilament H and M subunits, a hsp70 protein, α-internexin, dynamin and PSD-95 protein reside in the interior of the PSD. Dynein intermediate chain, AMPA receptors, kainate receptors, N-cadherin, β-catenin, N-ethylmaleimide-sensitive factor, a hsc70 protein and actin reside on the surface of the PSD. The results further suggest that the N-methyl-D-aspartate receptors and the α-subunits of calcium, calmodulin-dependent protein kinase II are likely to reside on the surface of the PSD with, however, unique local protein organizations. Based on our results and the known interactions between various PSD proteins from data mining, a model for the molecular organization of the PSD is proposed.