Design of Fluorescent Membrane Scaffold Proteins for Nanodiscs

Abstract

Nanodiscs are nanoscale lipid bilayer membrane mimetics surrounded by two membrane scaffold proteins (MSP). They are widely used as soluble cassettes for membrane proteins and lipids in diverse applications in structural, functional, and biophysical studies. The original MSP was derived directly from human apolipoprotein A-1, and novel constructs have been adapted from this original design, including fluorescent nanodiscs of varying designs. However, chemical derivatization with fluorophores can be expensive, and prior designs of split fluorescent proteins fused to MSP were limited in the color pallet available. Here, we developed MSPs with a wide range of different fluorescent C-terminal protein tags, including a versatile HaloTag fusion. These fluorescent MSP were purified following typical MSP purification procedures with similar yield. Then, we demonstrate that fluorescent MSPs form nanodiscs with similar structure and stoichiometry to conventional MSP nanodiscs. They are also suitable for assembly of nanodiscs with embedded integral membrane protein. Finally, we apply these constructs to monitor peripheral membrane protein binding to lipids via fluorescence resonance energy transfer. These fluorescent MSP constructs enable a range of different applications and provide a versatile template for future design of nanodiscs with unique functions. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/716332v3_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1bf2d2borg.highwire.dtl.DTLVardef@2ea07eorg.highwire.dtl.DTLVardef@12aeccforg.highwire.dtl.DTLVardef@121430d_HPS_FORMAT_FIGEXP M_FIG C_FIG