Abstract
This protocol was developed to functionalize styrene maleic acid (SMA) by direct fluorescent labeling in an easy way, accessible to biochemistry laboratories. This novel method is based on the coupling of carboxylic acids to primary amines using a carbodiimide, a reaction commonly used for protein chemistry. The procedure uses the hydrolyzed styrene-maleic acid copolymer and occurs entirely in aqueous solution with mild conditions compatible with many biomolecules.
Keywords: SMALPs, Membrane solubilization, Nanodiscs, Fluorescent labeling, Nanodiscs modification
Background
Characterization of membrane proteins in-vitro can be very challenging (Grisshammer and Tate, 1995). In addition to difficulties with over-expression and membrane isolation, membrane proteins need to be extracted from their native environment. The necessary solubilization step commonly requires the use of detergent to replace the native lipid environment, this can often lead to the loss of structure and/or activity of the membrane protein (Duquesne et al., 2016). For this reason, several alternative options such as amphipols (Popot, 2010) have been developed to avoid some of the difficulties associated with detergents and maintain the solubility of membrane protein. A few years ago Styrene Maleic Acid (SMA) copolymer became more frequently used as an alternative to low molecular weight detergent strategies (Dorr et al., 2014; Jamshad et al., 2015; Prabudiansyah et al., 2015). SMA has been shown to be able to spontaneously solubilize biological membranes and give disc-shape particles with an average size of 10 nm. These nanodiscs (called SMALPs) contain a mixture of protein embedded in lipids from membrane and SMA copolymer maintaining the particle in solution (Knowles et al., 2009). SMALPs are compatible with most common biochemical approaches such as affinity purification or size exclusion chromatography. One important advantage of using SMA is the “almost native” environment they provide to membrane proteins, allowing preservation of key lipids often involved in maintaining membrane protein structure or function. Protein characterization sometimes requires labeled material, but fluorescent tags can also alter protein structure. Chemically modified SMA allows labeling of nanodiscs without protein modification and so could be of interest for various types of analysis. Previous studies have used labeled amphipols with different chemistries such as poly-histidine (Giusti et al., 2015); biotin (Charvolin et al., 2009); and DNA oligonucleotides tags (Le Bon et al., 2014) to immobilize membrane proteins. Fluorescent-labeled SMA can be employed in a similar way with lipid environment being preserved allowing for the study of membrane proteins in a native-like environment using fluorescence correlation spectroscopy or energy transfer measurements. This protocol aims to chemically modify SMA in solution based on the reaction coupling of carboxylic acids to primary amines using a carbodiimide, a reaction commonly used for protein cross-linking (Carraway and Koshland, 1972). This experiment was previously done with a 2:1 styrene-maleic anhydride form as the starting point (Lindhoud et al., 2016). Our protocol maintains mild conditions compatible with many biomolecules throughout the procedure. This makes the chemistry easily accessible to biological laboratories, and will allow a wide range of molecules to be attached to the SMA.
Materials and Reagents
Equipment
Procedure
Data analysis
The degree of SMA labeling is calculated based on the decomposition of the absorption spectrum of the washed labeled SMA-SH reaction (Figure 5). The quantity of SMA is calculated from the absorption at 260 nm and fluorophore from absorption at its absorption maximum (500 for Atto488 and 550 nm for Atto532, respectively). An absorption spectrum of the labeled SMA-SH solution is recorded from 700 to 200 nm (with 1 nm increment at medium speed) in a quartz cuvette on a UV spectrometer (Shimadzu). It can be necessary to dilute labeled SMA-SH solution prior to measure absorbance to avoid intensity saturation. Spectra are extracted in .csv format. The degree of labeling is then calculated using the following formula: The extinction coefficient for SMA was ϵSMA of 6989M-1•cm-1 (Grethen et al., 2017; Oluwole et al., 2017). The extinction carboxylate coefficients of Atto dyes were ϵAtto of 9 x 104 M-1•cm-1 and 1.5 x 105 M-1•cm-1 for Atto488 and Atto532 respectively, based on the Atto-TEC literature. Dye absorbance is measured at it maximum, 500 nm for Atto488 in our example. Figure 5. Absorption spectra of SMA-SH labeled with Atto-488. Absorption at 260 nm, corresponding to SMA-SH and 500 nm for Atto488 are used to calculate the degree of labeling as mentioned in the formula.
Recipes
Acknowledgments
The authors thank V. Prima, JP Duneau and Y. Rhamani for valuable technical assistance and very helpful discussions. This work was supported by CNRS and Espoir contre la Mucoviscidose. This protocol has been used in Schmidt and Sturgis, 2018.
Competing interests
The authors declare no conflicts of interest.
References
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