Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

Chemical crosslinking provides information about protein?protein interactions in the context of intact protein complexes; therefore, it is particularly suited to the analysis of multiprotein complexes. Rather than a single distinct technique, chemical crosslinking represents a smorgasbord of techniques that differ significantly both in chemistry and in scope. This unit will attempt to guide the reader through the complexities of crosslinking to find the most suitable approach for a given biological question. Sample protocols for two crosslinking methods considered to be particularly useful for the analysis of large multiprotein complexes are provided: His6?mediated crosslinking and photoinducible label transfer crosslinking.

Keywords: Photoinducible crosslinking; label transfer; oxidative crosslinking; His6?mediated crosslinking; immunoblotting; MALDI?TOF?MS

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Strategic Planning
Basic Protocol 1: His6‐Mediated Crosslinking
Basic Protocol 2: Bait‐Localized Photoactivatable Crosslinking
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1: His6‐Mediated Crosslinking

Materials

Saccharomyces cerevisiae cultures: source of multiprotein complexes, either wild‐type cells or cells in which putative target polypeptides are genomically epitope‐tagged (see Klein et al., )
YEPD medium (see recipe )
Extract buffer (see recipe )
0.5‐mm glass beads, (e.g., Scientific Instruments), prechilled to −20°C
Siliconized glass wool, sterile (see recipe )
Ammonium sulfate, finely ground with mortar and pestle or with coffee grinder
1 M KOH
Dialysis buffer (see recipe )
Glutathione‐Sepharose immobilized proteins (see recipe ) in A50 buffer (see recipe )
- ∼80 µg His6‐GST‐TEVcs‐bait protein
- ∼20 µg His6‐GST‐TEVcs (control)
A50 buffer (see recipe )
Wash buffer: A50 buffer (see recipe ) without DTT
4× and 2× SDS sample buffer (see recipe )
12 mM nickel acetate
12 mM magnesium monoperphtalate (MMPP; Aldrich): prepare fresh just before use
TEV protease cleavage buffer (see recipe )
TEV protease (Invitrogen or USB)

Visible light spectrophotometer
50‐ml centrifuge tube
Refrigerated centrifuge
30‐ml round‐bottom Corex tube
10‐ml syringe
Ultracentrifuge (e.g., Beckman 70.1Ti) and corresponding ∼10‐ml ultracentrifuge tubes, precooled to 4°C
25‐ml beakers
Magnetic stir plate and stir bars
Dialysis tubing or dialysis cassettes (10,000 to 12,000 kDa MWCO; e.g. Slide‐A‐Lyzer cassettes from Pierce)
0.5‐ml microcentrifuge tubes
Nutator

Additional reagents and equipment for determining protein concentration ( appendix 3H ), performing SDS‐PAGE (unit 6.1 ), and detecting proteins by immunoblotting (unit 6.2 )

Basic Protocol 2: Bait‐Localized Photoactivatable Crosslinking

Materials

600 mM N‐[(2‐pyridyldithio)ethyl]‐4‐azidosalicylamide (PEAS) stock solution: prepare by dissolving the solid compound (Invitrogen) in DMSO; store up to 2 years at −70°C in an amber bottle
0.1 M sodium phosphate buffer, pH 7.4 ( appendix 2A )
Na¹²⁵ I in NaOH solution, pH 8.0‐12.0 (e.g., Amersham Biosciences, specific activity >0.6 TBq/mg iodide, 15 Ci/mg iodide)
50 mM tyrosine in water
7.5 µM purified yeast transcription factor TFIIB: pass the purified protein sample through a desalting column (Chen and Hahn, ) to remove the reducing agent in the purification buffer and elute in TFIIB conjugation buffer (see recipe )
TFIIB conjugation buffer
Dry Ice
Multiprotein complex: e.g., RNA polymerase II pre‐initiation complex (PIC) on beads formed using the immobilized template assay (Chen and Hahn, )
Acetate transcription buffer (see recipe )
Reducing agent: dithiothreitol or 2‐mercaptoethanol

IODO‐GEN precoated iodination tube (Pierce)
Desalting column: NAP‐5 Sephadex G‐25 column (Amersham Biosciences)
Scintillation counter for gamma counting
−70°C freezer
Magnet
UV irradiation device: ∼350‐nm wavelength

Additional reagents and equipment for performing SDS‐PAGE (unit 6.1 ) and autoradiography (unit 6.3 )

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Figures

Figure 17.10.1 Crosslinker groups that react with lysine or cysteine residues on proteins of interest and the resulting crosslinking adducts. R is the nonreactive moiety of the crosslinker.

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Figure 17.10.2 Photoactivatable crosslinking and label transfer. (A ) Aryl azide photoactivation and formation of crosslinking adducts. UV illumination of aryl azides triggers photolysis to form short‐lived nitrenes that either directly, or after ring expansion, attack chemical bonds in target proteins. The most frequently attacked groups are primary amines (thick arrows). R₁ , nonreactive moiety of the crosslinker; R₂ , target protein. (B ) Label‐transfer crosslinking. A labeled ~undefined) photoactivatable crosslinker with a cleavable disulfide (S‐S) bond is first covalently attached to a bait protein that is subsequently incubated with a target complex. UV illumination triggers crosslinking with directly interacting target subunits. Reducing agents cleave the disulfide bond, thereby transferring the label to the target polypeptide.

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Figure 17.10.3 Oxidative crosslinking and formation of crosslinking adducts. Complexed transition metal ions (Me²⁺ ), e.g., Ni²⁺ chelated by hexahistidine (His6) and Ru²⁺ chelated by tris(2,2′‐bipyridyl), can be oxidized to highly electrophilic Me³⁺ species that subtract electrons from oxidizable amino acid side chains (e.g., Tyr, Trp, Cys) within the tagged protein. Tyrosine (Tyr ) is the amino acid side chain shown in the figure and the most efficient electron donor for His6‐mediated crosslinking. Subsequent proton release generates highly reactive radicals that can form adducts with susceptible amino acid residues in side chain or backbone components of the target protein.

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Figure 17.10.4 Structures of the lysine‐targeting homobifunctional crosslinker disuccinimidyl suberate (DSS) and the heterobifunctional crosslinker N ‐succinimidyl 3‐(2‐pyridylthio)propionate (SPDP). SPDP consists of a lysine‐targeting NHS group that is separated by a cleavable disulfide bond from a cysteine‐targeting pyridyldisulfide group.

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Figure 17.10.5 Cartoon comparing different methods for detection and analysis of crosslinking adducts. A protein complex consisting of subunits (a, b, c, and d; panel A contains only two subunits, a and b) is subjected to crosslinking. Reactions are separated by SDS‐PAGE and analyzed by Coomassie staining (A ), immunoblotting (B ) and (C ), autoradiography (D ), and MALDI‐TOF MS (E ). Samples have either been treated with diffusible crosslinkers (panels A, B, and E), with localized crosslinkers (panel C), or with‐label transfer crosslinkers (panel D). Please note that bifunctional crosslinkers (depicted by thin bars) can either covalently connect two amino acid side chains within or between subunits or modify a single amino acid chain. See text for further details. Abbreviation: *, radioactive label; −, without crosslinking; +, with crosslinking.

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Figure 17.10.6 Cartoon depicting steps in isolating interacting proteins using a bait protein. (A ) Baits that form subunits of stable protein complexes can be allowed to assemble in vivo prior to affinity purification of the bait‐containing protein complex. (B ) Baits that transiently interact with protein complexes can be first purified as glutathione Sepharose bead–immobilized recombinant bait proteins and allowed to interact in vitro with cell extracts. The bait (a) containing complexes are enriched or isolated by affinity purification, and the interacting protein (b) is crosslinked and analyzed (with or without removal of the His6‐GST‐TEVcs‐tag) by SDS‐PAGE. Abbreviations: H6, His6 tag; GST, glutathione‐ S ‐transferase tag; MMPP, magnesium monoperphtalate. Crosslinking is indicated by the large, bold X, and the cleavage site for TEV protease is indicated by a thick bar.

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Figure 17.10.7 Crosslinking of two acidic activation domains (ADs) with Taf12, TAF12 is a subunit of both coactivators SAGA (the example used for the gels shown in this figure) and TFIID (the example illustrating ), and the complex depicted at the top of the figure can represent either. Immobilized His6‐GST‐TEVcs‐AD fusion proteins of two different sizes (AD1 and AD2) were incubated with yeast whole cell extract. Native complexes bound to the bait proteins were crosslinked, and the crosslinking reaction mix was separated by SDS‐PAGE. (A ) Top: immunoblot with anti‐Taf12 antibodies. A, bottom: the same blot stripped and reprobed with an antibody against a different SAGA subunit, Ada2. The asterisk ~undefined) indicates residual TAF12 signal after stripping. (B ) Immunoblot of supernatants of the crosslinking reaction and a mock reaction. (C ) Part of the crosslinking reaction was treated with TEV protease prior to denaturation and immunoblotting with anti‐HA (heme agglutinin epitope tag) antibody. The cleavage site for TEV protease is indicated by a thick bar. Modified from Klein et al., ; see Klein et al., for details.

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Figure 17.10.8 Flow chart demonstrating a strategy for the use of protein‐protein photocrosslinking with radiolabeled bait protein to identify the protein binding target.

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Figure 17.10.9 Autoradiograms of radiolabeled crosslinker conjugation and photocrosslinking products from TFIIB complexes. (A ) Protein complexes formed using two TFIIB mutants (R37C and V35C) with surface cysteine residues conjugated to ¹²⁵ I‐PEAS and analyzed by SDS‐PAGE electrophoresis in the presence (+) and absence (−) of DTT. A complete loss of radiolabeling of the modified protein sample in the presence of 50 mM DTT indicates cleavage of the disulfide linkage between the crosslinker and the inserted surface cysteine residue of the protein. Each lane contains 10 µl of protein sample from the conjugation reaction. (B ) Protein complexes formed using two TFIIB mutants (R37C and V35C) with surface cysteine residues conjugated to ¹²⁵ I‐PEAS as well as a control protein (WT, wild type) without a surface cysteine in the formation of the polymerase II transcription pre‐initiation complex (PIC). Photocrosslinking samples treated with UV irradiation and DTT are indicated by +. Arrows point to the crosslinked polypeptides. Lane M contains ¹⁴ C molecular weight markers.

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Literature Cited

Literature Cited
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Identification and Analysis of Multiprotein Complexes Through Chemical Crosslinking

Abstract

Table of Contents

Materials

Basic Protocol 1: His6‐Mediated Crosslinking

Basic Protocol 2: Bait‐Localized Photoactivatable Crosslinking

Figures

Videos

Literature Cited