Biophysical Characterization of Membrane Proteins Embedded in Nanodiscs Using Fluorescence Correlation Spectroscopy

Abstract

Proteins embedded in biological membranes perform essential functions in all organisms, serving as receptors, transporters, channels, cell adhesion molecules, and other supporting cellular roles. These membrane proteins comprise ~30% of all human proteins and are the targets of ~60% of FDA-approved drugs, yet their extensive characterization using established biochemical and biophysical methods has continued to be elusive due to challenges associated with the purification of these insoluble proteins. In response, the development of nanodisc techniques, such as nanolipoprotein particles (NLPs) and styrene maleic acid polymers (SMALPs), allowed membrane proteins to be expressed and isolated in solution as part of lipid bilayer rafts with defined, consistent nanometer sizes and compositions, thus enabling solution-based measurements. Fluorescence correlation spectroscopy (FCS) is a relatively simple yet powerful optical microscopy-based technique that yields quantitative biophysical information, such as diffusion kinetics and concentrations, about individual or interacting species in solution. Here, we first summarize current nanodisc techniques and FCS fundamentals. We then provide a focused review of studies that employed FCS in combination with nanodisc technology to investigate a handful of membrane proteins, including bacteriorhodopsin, bacterial division protein ZipA, bacterial membrane insertases SecYEG and YidC, Yersinia pestis type III secretion protein YopB, yeast cell wall stress sensor Wsc1, epidermal growth factor receptor (EGFR), ABC transporters, and several G protein-coupled receptors (GPCRs).

Keywords: cell-free expression; fluorescent correlation spectroscopy; membrane proteins; nanodiscs.

Figure 1

(A) Transmembrane protein embedded in a nanodisc lipid bilayer supported by (left) scaffold lipoproteins (NLPs) and (right) styrene maleic acid copolymers (SMALPs). (B) One-pot cell-free synthesis of membrane protein NLP complexes using a co-translational approach: transmembrane proteins and apolipoproteins are translated from cDNA simultaneously in the presence of liposomes using the translational machinery of an E. coli cell-free lysate, leading to the self-assembly of nanodisc loaded with the membrane protein of interest [41,42,43].

Figure 2

Fluorescence correlation spectroscopy (FCS) applied to membrane proteins embedded in nanodiscs. (A) FCS instrument schematic for a single-color system based on a confocal microscope. A tightly focused laser excitation combined with a confocal pinhole define a small, femtoliter-scale open detection volume. The emitted fluorescence signal is typically split and projected onto two detectors to eliminate detector dead time and afterpulsing effects. OBJ: objective; DM: dichroic mirror; L: lens; PH: pinhole; BS: beamsplitter; BP: bandpass filter; APD: avalanche photodiode. (B) As a molecule of the fluorescently labeled species, such as the membrane protein embedded in a nanodisc shown, diffuses through the open detection volume, the detected fluorescence signal fluctuates. Size of nanodisc in comparison to laser focus not drawn to scale. (C) The g⁽²⁾ correlation is calculated from the fluorescence signal (green data points). A simple diffusion model (Equation (1)) (black line) is fitted to the data, giving the diffusion time and concentration of the diffusing species. Data shown are of the human β-2 adrenergic receptor conjugated with the green fluorescent protein and embedded inside an Apo-A1 encircled DMPC nanodisc. Fitted diffusion time τ_D = 0.92 ± 0.02 ms.

Figure 3

Binding of nanodisc-embedded membrane proteins to smaller, fluorescently labeled ligands are easily detectable by FCS due to a large change in diffusion time. (A) Cartoon of the Y. pestis membrane protein YopB (a multimer) inserted into a 10 nm nanodisc and its cognate protein LcrV (PDB: 1R6F) labeled with EGFP. (B) FCS correlation curves of LcrV-EGFP in the presence of different concentrations of YopB-nanodisc (data selected from Ly et al. [89]). Addition of YopB-nanodisc shifts the correlation curve of LcrV-EGFP to the right. Intermediate concentrations of YopB-nanodisc result in a diffusion curve with two species present, one of free LcrV-EGFP and one bound to YopB-nanodisc. Curves are fitted to Equation (2) to obtain the diffusion times and fractions of the two species. (C) Fraction of LcrV-EGFP bound to YopB-nanodisc at different YopB-nanodisc concentrations. A fit to the Hill equation gives dissociation constant K_D = 20 ± 2 nM and Hill coefficient n = 1.4 [89].

Figure 4

FCS correlation curves and diffusion times of nanodisc-embedded membrane proteins among a large range of particle sizes. (A) A representative correlation curve, from data collected for 1 min, for each species given in (B). Blue shaded area represents the range in which curves for membrane proteins embedded in nanodiscs are expected to reside. Green shades represent data collected with 488 nm laser excitation, and red shades represent data collected with 561 nm laser excitation. Data were collected on a homemade FCS system with a calibrated detection volume of ~3 µm³. (B) Diffusion times of different species plotted against particle size (diameter). Error bars on diffusion times are standard deviations based on multiple measurements. Error bars on size are estimated from diameters of the species. Blue shaded area represents the range in which nanodisc-embedded membrane proteins are expected to reside. Atto488 (Sigma-Aldrich, St. Louis, MS, USA, product #41051); Alexa555: Alexa Fluor 555 NHS ester (Thermo Fisher Scientific, Carlsbad, CA, USA, product #A20009); FluoSphere505 and FluoSphere580: FluoSpheres fluorescent carboxylate-modified microspheres 0.04 µm (Thermo Fisher Scientific, product #F10720); TetraSpeck: TetraSpeck fluorescent microspheres 0.1 µm (Thermo Fisher Scientific, product #T7279).