Analysis of Thylakoid Membrane Protein Complexes by Blue Native Gel Electrophoresis

Summary

A protocol for the elucidation of plant thylakoid protein complex organization and composition with blue native polyacrylamide gel electrophoresis (BN-PAGE) and 2D-SDS-PAGE is described. The protocol is optimized for Arabidopsis thaliana, but can be used for other plant species with minor modifications.

Abstract

Photosynthetic electron transfer chain (ETC) converts solar energy to chemical energy in the form of NADPH and ATP. Four large protein complexes embedded in the thylakoid membrane harvest solar energy to drive electrons from water to NADP⁺ via two photosystems, and use the created proton gradient for production of ATP. Photosystem PSII, PSI, cytochrome b₆f (Cyt b₆f) and ATPase are all multiprotein complexes with distinct orientation and dynamics in the thylakoid membrane. Valuable information about the composition and interactions of the protein complexes in the thylakoid membrane can be obtained by solubilizing the complexes from the membrane integrity by mild detergents followed by native gel electrophoretic separation of the complexes. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an analytical method used for the separation of protein complexes in their native and functional form. The method can be used for protein complex purification for more detailed structural analysis, but it also provides a tool to dissect the dynamic interactions between the protein complexes. The method was developed for the analysis of mitochondrial respiratory protein complexes, but has since been optimized and improved for the dissection of the thylakoid protein complexes. Here, we provide a detailed up-to-date protocol for analysis of labile photosynthetic protein complexes and their interactions in Arabidopsis thaliana.

Introduction

Large multisubunit protein complexes photosystem PSI and PSII, Cyt b₆f and ATPase coordinate the production of NADPH and ATP in photosynthetic light reactions. In higher plant chloroplasts, the complexes are located in the thylakoid membrane, which is a structurally heterogeneous membrane structure, comprising appressed grana and non-appressed stroma thylakoids. Blue native polyacrylamide gel electrophoresis (BN-PAGE) is an extensively used method in the analysis of large multisubunit protein complexes in their native and biologically active form. The method was established for the dissection of mitochondrial membrane protein complexes¹, but has later been customized for the separation of protein complexes from the thylakoid membrane network³. The method is suitable (i) for the purification of individual thylakoid protein complexes for structural analysis, (ii) for determining native interactions between protein complexes and (iii) for the analysis of overall organization of the protein complexes upon changing environmental cues.

Prior to the separation, protein complexes are isolated from the membrane with carefully chosen nonionic detergents, which are generally mild and preserve the native structure of the protein complexes. Detergents contain hydrophobic and hydrophilic sites and form stable micelles above a certain concentration, called a critical micellar concentration (CMC). Increasing the detergent concentration above the CMC results in disruption of the lipid-lipid interactions and in the solubilization of protein complexes. The choice of detergent depends on the stability of the protein complex of interest and on the solubilization capacity of the detergent. Routinely used detergents include α/β-dodecyl-maltoside and digitonin. Following the solubilization of protein complexes in their native state, insoluble material is removed by centrifugation. In higher plants, the thylakoid membrane is highly heterogenic in structure and some detergents (e.g., digitonin) selectively solubilize only a specific fraction of the membrane³. Therefore, to characterize the protein complex organization or the interactions between the protein complexes, it is crucial to always determine the solubilization capacity of the chosen detergent by determining the chlorophyll content and the chlorophyll a/b ratio of supernatant to assess the yield and the represented thylakoid (sub)domain, respectively, of the solubilized fraction. The chlorophyll a/b ratio in intact thylakoids of growth-light acclimated plants is typically around 3, whereas the chl a/b value of thylakoid fractions enriched either in grana or stroma thylakoids falls below (~2.5) or exceeds (~4.5) the value of the total thylakoids, respectively.

To provide negative charge to the protein complexes, Coomassie brilliant blue (CBB) dye is added to the solubilized sample. Due to the charge shift, protein complexes migrate towards the anode and are separated on an acrylamide (AA) gradient according to their molecular mass and shape. Effective and high-resolution separation is achieved by using a linear acrylamide concentration gradient. During the electrophoresis, the protein complexes migrate towards the anode until they reach their size-dependent pore-size limit. The pore-size of polyacrylamide gel depends on (i) the total acrylamide/bis-acrylamide concentration (T) and (ii) on the cross-linker bis-acrylamide monomer concentration (C) relative to the total monomers⁴. After the separation with BN-PAGE, the protein complexes can be further subdivided into their individual protein subunits by second-dimension (2D)-SDS-PAGE. Here, we describe a detailed protocol for the analysis of thylakoid membrane protein complexes by BN-PAGE/2D-SDS-PAGE.

Protocol

1. Preparing BN Gel1,2,3 Set up the gel caster with 8 cm x 10 cm plates (rectangular glass and notched alumina plate) according to manufacturer's instructions using 0.75 mm spacers. Place a gradient mixer on a stir plate and connect it with the peristaltic pump by a tubing. Attach a syringe needle to the other end of the tubing and place the needle between the glass and…

Representative Results

A representative 2D-BN/SDS-PAGE system in Figure 1 demonstrates the separation of digitonin and β-DM-solubilized thylakoid protein complexes and their detailed protein subunit composition. The protein complex pattern of digitonin solubilized thylakoids (horizontal gel on the top on the top of Figure 1A) contains the PSII-LHCII-PSI megacomplex, two large PSII-LHCII supercomplexes (sc), PSI-LHCII supercomplex, PSI monomer (m),…

Discussion

The photosynthetic energy conversion machinery is composed of large multisubunit protein complexes, which are embedded in the thylakoid membrane. This protocol describes a basic method for analysis of the plant thylakoid protein complexes from Arabidopsis thaliana with BN-PAGE combined with 2D-SDS-PAGE. The protocol is also suitable for the analysis of thylakoid protein complexes from tobacco and spinach thylakoids, but might require small adjustments.

For the solubilization of membra…

Materials

6-aminocaproic acid (ACA)	Sigma-Aldrich	A2504
BisTris	Sigma-Aldrich	B4429
Sucrose	Sigma-Aldrich	S0389
Acrylamide (AA)	Sigma-Aldrich	A9099	Caution: Neurotoxic!
n-dodecyl-β-D-maltoside	Sigma-Aldrich	D4641
Tricine	Sigma-Aldrich	T0377
Tris	Sigma-Aldrich	T1503
SDS	VWR	442444H
Urea	VWR	28877.292
Glycerol	J.T. Baker	7044
Sodium Fluoride (NaF)	J.T. Baker	3688
EDTA disodium salt	J.T. Baker	1073
Digitonin	Calbiochem	300410	Caution:Toxic!
Pefabloc SC	Roche	11585916001
Serva Coomassie Blue G	Serva	35050
β-mercaptoethanol	Bio-Rad	1610710
APS (Ammonium persulfate)	Bio-Rad	161-0700
TEMED (Tetramethylethylenediamine)	Bio-Rad	1610801
(N,N'-Methylene)-Bis-Acrylamide	Omnipur	2610
Glycine	Fisher	G0800
Prestained Protein Marker, Broad Range (7-175 kDa)	New England Biolabs	P7708
Falcon, Conical Centrifuge Tubes 15 ml	Corning	352093
Dual gel caster with 10 x 8 cm plates	Hoefer	SE215
Gradient maker SG5	Hoefer
0.75 mm T-spacers	Hoefer	SE2119T-2-.75
Sample gel comb, 0.75 mm	Hoefer	SE211A-10-.75
Mighty Small SE250 vertical electrophoresis system	Hoefer	SE250
IPC-pump	Ismatec
Power supply, PowerPac HV	Bio-Rad	164-5097
Centrifuge	Eppendorf	5424R
Rocker-Shaker	Biosan	BS-010130-AAI
PROTEAN II xi Cell	Bio-Rad	1651813