Synthesis and validation of chemical probes for interrogating PDZ domains

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2011.Page 150 blank. Cataloged from PDF version of thesis.Includes bibliographical references.Macromolecular protein complexes at neuronal synapses are critical for establishing synaptic plasticity, which is the basis of information storage in the brain. These complexes consist of many PDZ domain-containing proteins. PDZ domains bind selectively to peptide sequences at the C-termini of partner proteins, and they play an essential role in the regulation of synaptic macromolecular complexes. While many PDZ domain-containing proteins are well characterized, much remains to be learned about their binding dynamics. The goal of the research presented herein is to gain a quantitative understanding of the complex PDZ-domain binding dynamics through the design, synthesis, and application of sensitive and selective biophysical probes. The peptidyl probes are based on three critical elements: solvatochromic fluorophores, molecular caging, and multivalency. This thesis presents the development of a systematic approach to screen for selective solvatochromic fluorophore-based probes for class I PDZ domains, and the biophysical characterization of probes for the PSD-95 and Shank3 PDZ domains. These probes have been utilized to examine the effects of Shank3 PDZ domain dimerization on ligand binding in vitro, and the quantitative results presented here implicate PDZ domain dimerization as a potential modular control mechanism for ligand-binding in the biological context. Further development of the probes via the application of a novel C-terminal caging strategy is also discussed. Validation in vitro shows the utility of a C-terminal 1-(2-nitrophenyl)ethyl cage for blocking the interaction between probe and cognate PDZ domain, and demonstrates the release of viable probe upon photoactivation of the molecular cage. This thesis also presents the in vivo application of PDZ domain probes, using the simple yet powerful eukaryotic C. elegans model system. Progress toward solvatochromic fluorophore-based probes for C. elegans Lin-10 PDZ domains is presented. Additionally, the design and synthesis of a bivalent peptidyl inhibitor, based on the C. elegans C-terminal sequence of STG-2 (a protein of the GLR-1 receptor complex), is discussed. This STG-2 sequence is a putative PDZ domain ligand, and its ortholog in higher eukaryotes functions in receptor-mediated multivalent interactions with partner PDZ domains. Electrophysiological data presented in this thesis suggest the effectiveness of the bivalent STG-2 peptide in the inhibition of the critical C. elegans GLR- 1 neural receptor complex.by Wendy S. Iskenderian-Epps.Ph.D

FRET-Capture: A Sensitive Method for the Detection of Dynamic Protein Interactions

The FRET-Capture approach exploits a bound solvatochromic fluorophore, 4-N,N-dimethylamino-1,8-naphthalimide, as a FRET donor in both inter- and intramolecular energy transfer. A unique feature of this method is the additional level of signal selectivity as the FRET signal is only turned on when the donor is specifically bound to the protein of interest, eliminating high background and false positive signals.National Institutes of Health (U.S.) (Grant R01 EB010246

SHANK3 mutations identified in autism lead to modification of dendritic spine morphology via an actin-dependent mechanism

Genetic mutations of SHANK3 have been reported in patients with intellectual disability, autism spectrum disorder (ASD) and schizophrenia. At the synapse, Shank3/ProSAP2 is a scaffolding protein that connects glutamate receptors to the actin cytoskeleton via a chain of intermediary elements. Although genetic studies have repeatedly confirmed the association of SHANK3 mutations with susceptibility to psychiatric disorders, very little is known about the neuronal consequences of these mutations. Here, we report the functional effects of two de novo mutations (STOP and Q321R) and two inherited variations (R12C and R300C) identified in patients with ASD. We show that Shank3 is located at the tip of actin filaments and enhances its polymerization. Shank3 also participates in growth cone motility in developing neurons. The truncating mutation (STOP) strongly affects the development and morphology of dendritic spines, reduces synaptic transmission in mature neurons and also inhibits the effect of Shank3 on growth cone motility. The de novo mutation in the ankyrin domain (Q321R) modifies the roles of Shank3 in spine induction and morphology, and actin accumulation in spines and affects growth cone motility. Finally, the two inherited mutations (R12C and R300C) have intermediate effects on spine density and synaptic transmission. Therefore, although inherited by healthy parents, the functional effects of these mutations strongly suggest that they could represent risk factors for ASD. Altogether, these data provide new insights into the synaptic alterations caused by SHANK3 mutations in humans and provide a robust cellular readout for the development of knowledge-based therapies