Lsm proteins as ring tectons
thesisposted on 2022-03-28, 12:31 authored by Dominic Logel
Bionanotechnology is beginning to exploit naturally occurring proteins as building blocks for creating nanoscale materials and complex devices. One example of interest is the Lsm family of proteins, which spontaneously assemble in vivo either as homomeric (archaea or prokarya), or heteromeric (eukarya) oligomers comprising six or seven Lsm protomers which play a role in RNA metabolism. At Macquarie, several Lsm proteins have beeen reengineered to form novel artificial RNA-binding rings. In this thesis I explore biophysical characteristics of new versions of yeast-derived tectons: utilisng polyproteins Lsm[1+4] and Lsm[5+6]. For the first time, the Lsm[5+6]₄ tecton has been cleanly produced, and its stability evaluated, whilst pure Lsm[1+4] proved to be difficult to obtain. This thesis also describes the first steps towards a crystal structure of a ring assembly formed by R65PLsmα. This mutant has been proposed as a variant of the heptameric Lsmα₇, possibly comprising a hexameric organisation. I successfully generated many crystal forms, which led on to provide diffraction of good quality. Results from structure solving allowed definition of the correct number of components, which was not able to be determined from biophysical solution methods.