Quantifying interactions of a membrane protein embedded in a lipid nanodisc using fluorescence correlation spectroscopy.

ABSTRACT Using ﬂuorescence correlation spectroscopy, we measured a dissociation constant of 20 nM between EGFP-labeledLcrVfromYersiniapestisanditscognatemembrane-boundproteinYopBinsertedintoalipidnanodisc.Thecombinationof ﬂuorescence correlation spectroscopy and nanodisc technologies provides a powerful approach to accurately measurebinding constants of interactions between membrane bound and soluble proteins in solution. Straightforward sample prepara-tion,acquisition,andanalysisproceduresmakethiscombinedtechnologyattractiveforaccuratelymeasuringbindingkineticsforthis important class of protein-protein interactions. Received for publication 17 September 2013 and in ﬁnal form 12 December 2013. *Correspondence: laurence2@llnl.gov or coleman16@llnl.gov Interactions involving membrane proteins are integral to amultitude of cellular processes, including signal transduc-tion, energy production and conversion, cell adhesion, andforeign molecule identiﬁcation. More than half of all phar-maceutical drugs target membrane proteins, further illus-trating their importance in human health (1). Due to this,there is a continuing high demand for methods that canscreen, validate, and quantify interactions involving mem-brane proteins. Unfortunately, the quantitative techniquesavailable to characterize protein-protein interactions aremost often directed toward soluble proteins, and are oftendifﬁcult to apply to membrane proteins (2). Recently,advances in lipid nanodisc technologies, often referred toasreconstitutedhigh-densitylipoproteins,haveenabledbio-physical and biochemical studies of solubilized membraneproteins. The nativelike lipid environment of nanodiscsmaintains protein functionality, opening a realm of possibil-ities in analyzing membrane protein function and dynamicsin solution (3).Nanodiscs are discoidal cell membrane mimetics that are8–20 nm in diameter, consisting of a lipid bilayer stabilizedby two peripheral apolipoprotein A-I proteins (4). Theseparticles provide an excellent alternative to traditionallipid-based platforms (e.g., liposomes) for membrane pro-tein solubilization and interrogation. The diameter of nano-disc can be engineered to accommodate different-sizedmembrane proteins by varying lipid composition. As such,nanodiscs represent an important platform for expression,isolation, and study of functional membrane proteins andthe multiprotein complexes they form. Several groupshave successfully reconstituted a variety of membrane pro-teins, including bacteriorhodopsin (5), G-protein coupledreceptors (6), and cytochrome P450 (7) to name a few.To produce solubilized, discrete membrane proteins, weutilizedacell-freeexpressionapproachtoembedmembraneproteins directly into nanodiscs formed in situ. Thisapproach allows for enhanced puriﬁcation and rapid label-ing of proteins of interest where traditional approaches areunsuccessful (8). Fluorescence correlation spectroscopy(FCS) analysis coupled with nanodisc technology hasbeen successfully used to monitor small ligand binding in-teractions with membrane proteins (6,8) and to measurelipid-protein interactions at the single molecule level (9).Here, weshow that FCS can be used to measureinteractionsbetween soluble proteins and cognate membrane proteinsinserted into nanodiscs produced using cell-free expressionmethodologies.FCS uses correlation analysis of ﬂuorescence arisingfrom randomly diffusing molecules to measure diffusionconstants, and hencehydrodynamic radii, of species in solu-tion.DiffusiontimesmeasuredbyFCSareinverselypropor-tional todiffusion constants. Ashiftto slower diffusion timeindicates an increase in the hydrodynamic radius due tobinding. Due to this size sensitivity, FCS has been proposedrepeatedly as a method to quantify molecular interactions insolution(10).However,animportantdifﬁcultyinFCSariseswhen characterizing interactions between two proteins ofsimilar size. The diffusion time is inversely proportionalto the cube root of the molecular mass t