Single-molecule fluorescence spectroscopy using phospholipid bilayer nanodiscs.
Nanodiscs are a new class of model membranes that are being used to solubilize and study a range of integral membrane proteins and membrane-associated proteins. Unlike other model membranes, the Nanodisc bilayer is bounded by a scaffold protein coat that confers enhanced stability and a narrow particle size distribution. The bilayer diameter can be precisely controlled by changing the diameter of the protein coat. All these properties make Nanodiscs excellent model membranes for single-molecule fluorescence applications. In this chapter, we describe our work using Nanodiscs to apply total internal reflection fluorescence microscopy (TIRFM), fluorescence correlation spectroscopy (FCS), and Förster resonance energy transfer (FRET) to study the integral membrane protein cytochrome P450 3A4 and the peripheral membrane-binding proteins islet amyloid polypeptide (IAPP) and alpha-synuclein, respectively. The monodisperse size distribution of Nanodiscs enhances control over the oligomeric state of the membrane protein of interest, and facilitates accurate solution-based measurements as well. Nanodiscs also comprise an excellent system to stably immobilize integral membrane proteins in a bilayer without covalent modification, enabling a range of surface-based experiments where accurate localization of the protein of interest is required.
Nath
Abhinav
A
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut, USA.
Trexler
Adam J
AJ
Koo
Peter
P
Miranker
Andrew D
AD
Atkins
William M
WM
Rhoades
Elizabeth
E
D000682:Amyloid / Q000737:chemistry / Q000378:metabolism; D051544:Cytochrome P-450 CYP3A / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D058228:Islet Amyloid Polypeptide; D008051:Lipid Bilayers / Q000737:chemistry*; D008075:Lipoproteins, HDL / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008968:Molecular Conformation; D049329:Nanostructures / Q000737:chemistry*; D010316:Particle Size; D010743:Phospholipids / Q000737:chemistry*; D011485:Protein Binding; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods; D013499:Surface Properties; D051844:alpha-Synuclein / Q000737:chemistry / Q000378:metabolism
D000682:Amyloid; D058228:Islet Amyloid Polypeptide; D008051:Lipid Bilayers; D008075:Lipoproteins, HDL; D008565:Membrane Proteins; D010743:Phospholipids; D051844:alpha-Synuclein; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human