Abstract
FtsH is a membrane-bound protease that plays a crucial role in proteolytic regulation of many cellular functions. It is universally conserved in bacteria and responsible for the degradation of misfolded or misassembled proteins. A recent study has determined the structure of bacterial FtsH in detergent micelles. To properly study the function of FtsH in a native-like environment, we reconstituted the FtsH complex into lipid nanodiscs. We found that FtsH in membrane scaffold protein (MSP) nanodiscs maintains its native hexameric conformation and is functionally active. We further investigated the effect of the lipid bilayer composition (acyl chain length, saturation, head group charge and size) on FtsH proteolytic activity. We found that the lipid acyl chain length influences AaFtsH activity in nanodiscs, with the greatest activity in a bilayer of di-C18:1 PC. We conclude that MSP nanodiscs are suitable model membranes for further in vitro studies of the FtsH protease complex.
Highlights
The vast majority of proteins must fold into precise threedimensional conformations in order to gain functional activity
Purified Aquifex aeolicus FtsH (AaFtsH) was reconstituted into nanodiscs using the membrane scaffold protein MSP2N2 and POPC lipids
Reconstitution of AaFtsH in nanodiscs was confirmed by SDSPAGE, size exclusion chromatography (SEC)-MALS, and negative stain electron microscopy (EM) analysis
Summary
The vast majority of proteins must fold into precise threedimensional conformations in order to gain functional activity. How ever, in the cellular environment, proteins are prone to misfolding, causing the formation of aggregates and other toxic species [1]. To avoid these problems, cells are equipped with protein quality control ma chines, consisting of molecular chaperones and proteases [2,3]. Pro teases are responsible for the removal of misfolded and non-functional proteins from the cell. Proteolysis in bacteria is mediated by energy-dependent AAA+ (ATPases associated with various cellular ac tivities) proteases, that use ATP hydrolysis to unfold, translocate, and degrade protein substrates [4,5]. Various types of AAA+ proteases have been characterized in bacteria, including ClpXP, ClpAP, HslUV, Lon, and FtsH [4]
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