Optimization of production and enzymatic efficiency for self-cyclizing nanodisc proteins.

Biochemical and biophysical studies of membrane proteins, and molecules that interact with cellular membranes, require an appropriate membrane model. Lipid nanodiscs formed by modified versions of apolipoprotein A1 (ApoA1), known as membrane scaffold proteins (MSPs), have emerged as attractive membrane models due to their high homogeneity and flat bilayer organization. The circular structure of MSPs has enabled the development of head-to-tail cyclized nanodiscs with improved monodispersity and thermal stability. Recently, a method called autocyclization was described to produce cyclic MSPs, in which a ligase is fused to the MSP to promote intramolecular ligation. Under dilute conditions, autocyclases follow a unimolecular pathway where polymerization is strongly suppressed. The efficiency of the autocyclase reaction depends on the intramolecular dynamics of the fusion protein, particularly the peptide "linker" connecting the ligase to the MSP. Previous research has shown that increasing linker dynamics enhances the unimolecular reaction rate but also reduces in vivo stability. Here, we sought to modify the autocyclase design to increase the unimolecular reaction rate while minimizing in vivo degradation by altering both the linker sequence and the ligase recognition site. Our results provide an improved autocyclase design that maintains the elevated reaction rate conferred by a disordered linker without the previously observed rapid in vivo degradation. These findings improve access to cyclized nanodiscs as membrane models, facilitating future studies of membrane proteins and membrane-active molecules.