Core D5: Phage display synthetic toxin pipeline

University Of Chicago, Chicago IL

Investigators

Linked publications & trials

Abstract

The Membrane Protein Structural Dynamics (MPSD) Consortium seeks to achieve mechanistic understanding of membrane protein operation by linking structure, dynamics and function. Membrane proteins change their conformation to operate. Our purpose is to study different conformational states associated with function and to map the pathway that links operating and resting conformations. Naturally-occurring peptide toxins have become an integral part of research on many membrane proteins. This core employs a new, high-throughput methodology that exploits the structural robustness of natural peptide toxin scaffolds and the power of phage display technology. The purpose is to produce novel synthetic toxins that bind to specific membrane receptors in site and/or state-dependent manner with high affinity and selectivity. This method extends the proven strategy of using high-affinity peptide ligands to study membrane proteins beyond a handful of natural toxins that have been isolated. Peptide toxins isolated from spiders, scorpions, snails and snakes have been potent analytic tools to advance understanding of channels, pumps, transporters, and hormone receptors in vitro and in vivo revealing the roles of these membrane proteins in physiology and their mechanisms of action [1]. In the wild, toxins act to immobilize prey;they are potent (pM-nM affinity) and broadly effective on a wide spectrum of membrane targets. Many toxins lock target receptors in unique functional states. Natural toxins are small (-10-80 amino acids) and are constructed on resilient structural scaffolds that tolerate wide residue diversity to yield products with markedly different properties. Laboratory synthesis of peptide toxins using bacteria or by de novo chemical methods has proven straightfoHA/ard in most cases. These strategies improve yield compared to isolation of natural products and, significantly, allow incorporation of useful modifications such as residue alterations to improve target specificity or affinity, to alter impact on receptor function, or to attach cargo for delivery to specific cellular and molecular locations [2, 3]. Natural toxins and their synthetic variants have been used to identify membrane receptor subtypes in different tissue and subcellular locales [4];distinguish roles in physiology and disease [5];delineate molecular mechanisms [6];immunopurify target receptors [7];to treat pain via blockade of ion channels [8];and, of unique relevance here, to define receptor structure as a function of conformational state using biophysical [9], optical [3] and computational methods [10]. Even though the predicted diversity of the natural peptide "toxome" extrapolated from biochemical and genetic studies is vast (>11 million), specific targets are not identified for most of the hundreds of toxins that have been isolated and studied. Those toxins that bind to known receptors are often of low affinity or cross-react with related targets. This state-of-affairs is easily understood: neither their purpose in the wild nor non-directed searches for target receptors favor isolation of specific, high-affinity toxins. Here, these problems are avoided by cloning toxins based on their functional attributes.