Quantifying interactions of a membrane protein embedded in a lipid nanodisc using fluorescence correlation spectroscopy.
Using fluorescence correlation spectroscopy, we measured a dissociation constant of 20 nM between EGFP-labeled LcrV from Yersinia pestis and its cognate membrane-bound protein YopB inserted into a lipid nanodisc. The combination of fluorescence correlation spectroscopy and nanodisc technologies provides a powerful approach to accurately measure binding constants of interactions between membrane bound and soluble proteins in solution. Straightforward sample preparation, acquisition, and analysis procedures make this combined technology attractive for accurately measuring binding kinetics for this important class of protein-protein interactions.
Lawrence Livermore National Laboratory, Livermore, California.
Sonny
S
Ly
Lawrence Livermore National Laboratory, Livermore, California.
Feliza
F
Bourguet
Lawrence Livermore National Laboratory, Livermore, California.
Nicholas O
NO
Fischer
Lawrence Livermore National Laboratory, Livermore, California.
Edmond Y
EY
Lau
University of California at Davis, Department of Radiation Oncology, Sacramento, California. Electronic address: coleman16@llnl.gov.
Matthew A
MA
Coleman
University of California at Davis, Department of Radiation Oncology, Sacramento, California. Electronic address: laurence2@llnl.gov.
Ted A
TA
Laurence
D000942:Antigens, Bacterial / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry*; D008567:Membranes, Artificial; D049329:Nanostructures / Q000737:chemistry*; D052899:Pore Forming Cytotoxic Proteins / Q000378:metabolism*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000379:methods*
D000942:Antigens, Bacterial; C043908:LcrV protein, Yersinia; D008051:Lipid Bilayers; D008567:Membranes, Artificial; D052899:Pore Forming Cytotoxic Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins