Abstract
Peroxiredoxin 1 (Prdx1) is a ubiquitous peroxide scavenging enzyme with secondary signalling and chaperone functions. Like many other peroxiredoxins, Prdx1 exists in a state of dynamic equilibrium between an obligate homo-dimeric state and a decameric toroidal state consisting of five dimeric subunits. This equilibrium is known to be influenced by a number of factors in vitro and is thought to play a role in regulating functions and protein-protein interactions of Prdx1 in vivo. Investigations of these dynamics in vivo have been hampered by a lack of experimental techniques. Here we have begun developing oligomeric selective Prdx1 nanobodies to enable investigation of the relationship between oligomeric state and function. An initial selection of nanobody candidates from the panning of a yeast synthetic library have been tested experimentally and have not yielded high affinity Prdx1 binding. A number of potential issues with the original nanobody development owing to the dynamic structural qualities of Prdx1 have been identified and addressed. Modified techniques and quality control protocols have enabled the development of a 2nd generation of nanobody candidates. These nanobody candidates have been tested for affinity and selectivity, with several binding with at least moderate affinity and some potential for selectivity. Ideally this work will culminate with the development and characterisation of several nanobodies with high affinity and selectivity for either the dimeric or decameric state of Prdx1. This would enable their use as diverse experimental probes for investigations of the relationship between quaternary structure and function in
Prdx1.