A Structural Investigation of the Postsynaptic Density Protein, PSD-95

Abstract

The mechanism known as Long-Term Potentiation (LTP), which is perhaps the most established mechanism associated with memory and learning, describes the Hebbian postulate that "neurons that fire together, wire together". LTP refers to the upregulation of synaptic strength in response to high activity and can be subdivided into distinct phases. Early-LTP involves the phosphorylation of neurotransmitter receptors while Late-LTP involves the increase in expression of receptors at the postsynaptic membrane. These are long-term, stable perturbations of synaptic activity, lasting from hours to weeks. However, more subtle mechanisms controlling shorter-term plastic changes in synaptic function lie in the spatial distribution of postsynaptic receptors. In glutamatergic synapses within the mammalian CNS the distribution of neurotransmitter receptors is controlled by a scaffold of proteins under the postsynaptic membrane known as the PostSynaptic Density (PSD). This complex has been observed to change shape on a rapid scale, however little is understood about its macromolecular structure.The PSD anchors glutamate receptors within the postsynaptic membrane along with metabotropic receptors and cytoplasmic enzymes that modulate synaptic function and PSD and dendritic spine morphology. Much of what is understood on the macro-molecular structure of the PSD is based on knowledge of known binding partners. A recent paper within the group presented the PSD-95-Kir2.1 sub-complex of the PSD using a multi-disciplinary structural approach, blending Transmission Electron Microscopy (TEM) and solution scattering models with high-resolution X-ray and NMR structures for individual folded domains. In this thesis the solution state structure of PSD-95 is examined more closely. Deletion constructs of PSD-95 have been obtained by expressing subcloned fragments of the gene from Homo sapiens in an E. coli expression system and purifying them through a combination of IMAC, ion exchange chromatography and gel filtration. The PDZ region is rigorously described using Small Angle X-ray Scattering and is orthogonally validated by comparing the models against hydrodynamic data for the constructs. The PDZ12 region was crystallized and subjected to extensive optimisation to improve the resulting diffraction, falling just short of a molecular replacement structure. Attempts were made to crystallize the PDZ3 domain with the K¬ir¬2.1 C-terminus. PSD-95 dynamics was also investigated using Deuterium Exchange Mass Spectrometry (DXMS) to test a hypothesis for an internal binding interaction but was found not to be present using the methods developed.

Date of Award	31 Dec 2012
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Stephen Prince (Supervisor)