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.
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.
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.
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…
The authors have nothing to disclose.
This research was financially supported by the Academy of Finland (project numbers 307335 and 303757) and Solar Energy into Biomass (SE2B) Marie Skłodowska-Curie grant agreement (675006). The protocol is based on reference3.
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 |