利用光敏阿米洛利光亲和标记牛心脏亚线粒体颗粒中呼吸链复合物I

Masatoshi Murai; Hideto Miyoshi

doi:10.21769/BioProtoc.3349

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Peer-reviewed

Photoaffinity Labeling of Respiratory Complex I in Bovine Heart Submitochondrial Particles by Photoreactive [¹²⁵I] amilorides

利用光敏阿米洛利光亲和标记牛心脏亚线粒体颗粒中呼吸链复合物I

MM Masatoshi Murai

HM Hideto Miyoshi email

发布: 2019年09月05日第9卷第17期 DOI: 10.21769/BioProtoc.3349 浏览次数: 4103

评审: Alexandros AlexandratosAlexander GalkinAnonymous reviewer(s)

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Abstract

The architecture of quinone/inhibitor-access channel in proton-translocating NADH-quinone oxidoreductase (respiratory complex I) was modeled by X-ray crystallography and cryo-EM, however, it remains debatable whether the channel model reflects the physiologically relevant state present throughout the catalytic cycle. Using photoreactive [¹²⁵I]amilorides, we demonstrated that amiloride-type inhibitors bind to the interfacial region of multiple subunits (49-kDa, ND1, PSST, and 39-kDa subunits), which is difficult to reconcile with the current channel model. This report describes the procedures for photoaffinity labeling of bovine submitochondrial particles by photoreactive [¹²⁵I]amilorides. The protocol could be widely applicable for the characterization of various biologically active compounds, whose target protein remains to be identified or characterized.

Keywords: Photoaffinity labeling (光亲和标记)

Respiratory complex I (呼吸链复合物I)

Amilorides (阿米洛利)

Enzyme inhibitor (酶抑制剂)

Mitochondria (线粒体)

Bioenergetics (生物能量学)

NADH-quinone oxidoreductase (NADH泛醌氧化还原酶)

Chemical biology (化学生物学)

Background

Proton-translocating NADH-quinone oxidoreductase (respiratory complex I) is a multi-subunit membrane protein complex, which catalyzes the initial step of mitochondrial/bacterial electron transport chains (Hirst, 2013). The recent progress in X-ray crystallography and cryo-EM enabled modeling the entire structure of complex I. The structural studies proposed the quinone/inhibitor-access channel model to explain how quinone/inhibitor bind to the enzyme (Sazanov, 2015; Wirth et al., 2016). However, since there is currently no structural data of complex I with bound-quinone or inhibitor, it remains debatable whether the channel model reflects the physiologically relevant state present throughout the catalytic cycle.

Photoaffinity labeling technique, which uses a synthetic ligand that possesses photoreactive group such as diazirine and phenyl-azido, is the most commonly used method of affinity-based protein modification (Hatanaka and Sadakane, 2002). It provides a powerful means of investigating interactions between biologically active compounds and the proteins of interest. Recently, to get insights into the structure of quinone/inhibitor-binding site in mitochondrial complex I, we carried out photoaffinity labeling using photoreactive [¹²⁵I]amilorides ([¹²⁵I]PRA3, [¹²⁵I]PRA4, [¹²⁵I]PRA5, and [¹²⁵I]PRA6) with bovine heart submitochondrial particles (SMPs) (Uno et al., 2019). The radioisotope (¹²⁵I) was used as a tracer, which enables the experiment with the lowest concentrations of ligands (1-10 nM). These concentrations are approximately two orders of magnitude lower than those of alkyne-tagged amilorides (PRA1 and PRA2, Murai et al., 2015), which can be conjugated with biotin or fluorophores via Cu⁺-catalyzed click chemistry (Wang et al., 2003) after cross-linking reaction. This advantage may minimize the possibility of non-specific labeling, which is a primary cause of false-positive results.

We herein describe the detailed procedures of the photoaffinity labeling of complex I in bovine SMPs by [¹²⁵I]PRA5 as an example (Figure 1), which includes 1) preparation of ¹²⁵I-tagged photoreactive ligand, 2) cross-linking of bovine SMPs by the ligand, and 3) identification of the labeled protein(s).

Figure 1. Schematic presentation of photoaffinity labeling of bovine heart SMPs by [¹²⁵I]amilorides

Materials and Reagents

1.5 ml Screw-capped microtube (Watson, catalog number: 139-115-112C)
1.5 ml microtube (Safe-Lock Tube, Eppendorf, catalog number: 0030120086)
Pipette tips (Gilson)
Membrane filter unit (Amicon-Ultra, 100 kDa, Merck Millipore, catalog number: UFC510096)
Reverse-phase HPLC column (COSMOSIL 5C18-MSII, 4.6 mm x 200 mm, Nacalai Tesque)
TLC plate (Merck Millipore, catalog number: 1.05554.0001)
Plastic wrap
Amiloride-tin-precursor (A21a, 1.0 mM in ethanol, Figure 1A, The synthetic procedures are described in Uno et al., 2019)
[¹²⁵I]NaI (carrier-free, 2,000 Ci/mmol, Perkin-Elmer, catalog number: NEZ033A)
Chloramine T (Wako Pure Chemicals, catalog number: 032-02182)
Sodium hydrogen sulfite (NaHSO₃, Wako Pure Chemicals, catalog number: 198-01371)
Bovine submitochondrial particles (SMPs)
Notes:
SMPs are inside-out vesicles of inner mitochondrial membrane.
Mitochondria were isolated from bovine heart as described elsewhere (Smith, 1967).
SMPs were prepared on the basis of the previously described protocol (Matsuno-Yagi and Hatefi, 1985) using a sonication medium containing 0.25 M sucrose, 1.0 mM potassium succinate, 1.5 mM ATP, 10 mM MgCl₂, 10 mM MnCl₂, and 10 mM Tris/HCl (pH 7.4), followed by ultracentrifugation. They are stored in a buffer containing 250 mM sucrose and 10 mM Tris-HCl (pH 7.4) at -80 °C until used.
NativePAGE^TM Bis-Tris protein gels (4-16% precast gel, Thermo Fisher, catalog number: BN1004BOX)
NativePAGE^TM Runnning buffer and Cathode buffer additive (Thermo Fisher, catalog numbers: BN2001 and BN2002, respectively)
Serva Blue G (CBB G-250, Serva, catalog number: 35050.01)
n-Dodecyl-D-β-maltoside (DDM, Dojin, catalog number: D316)
Glycerol (Wako Pure Chemicals, catalog number: 075-00616)
Ethanol (Wako Pure Chemicals, catalog number: 057-00456)
Methanol (Wako Pure Chemicals, catalog number: 137-01823)
Acetic acid (Wako Pure Chemicals, catalog number: 017-00251)
Sucrose (Wako Pure Chemicals, catalog number: 192-00017)
MgCl₂ (hexahydrate, Wako Pure Chemicals, catalog number: 135-00165)
NaCl (Wako Pure Chemicals, catalog number: 195-01665)
Ponceau S (Nacalai Tesque, catalog number: 28322-72)
Tricine (Dojin, catalog number: GB05)
SDS (Wako Pure Chemicals, catalog number: 191-07145)
Mercaptethanol (Wako Pure Chemicals, catalog number: 137-06862)
Tris (Wako Pure Chemicals, catalog number: 011-20095)
Acrylamide (Wako Pure Chemicals, catalog number: 011-08015)
Bis-acrylamide (Wako Pure Chemicals, catalog number: 134-15081)
Urea (Wako Pure Chemicals, catalog number: 219-00175)
Silver Stain Kit (Wako Pure Chemicals, catalog number: 291-50301, compatible with MS)
Trifluoroacetic acid (TFA)
NADH (Oriental Yeast, catalog number: 44320000)
Labeling buffer (see Recipes)
4x BN-PAGE sample buffer (see Recipes)
Elution buffer (see Recipes)
4x Schägger sample buffer (see Recipes)
10x cathode buffer (see Recipes)
10x anode buffer (see Recipes)
AB-3 mix (see Recipes)
3x gel buffer (see Recipes)
1.0 M KPi buffer, pH 7.4 (see Recipes)
Gel composition for dSDS-PAGE (see Recipes)

Equipment

Pippetmann (Gilson)
-80 °C freezer (PHC, model: MDF-DU300H)
HPLC system (Shimazdu, model: LC-10AS)
Micro-centrifuge (CHIBITAN, Merck Millipore, model: XX42CF0RT)
γ-counter (COBRA^TM II, Packard)
Vacuum centrifugal evaporator (EYELA, model: CVE-2200)
Bio-imaging analyzer (FLA-5100 or Typhoon FLA-9000 , Fuji Film or GE healthcare, respectively)
Imaging plate (BAS IP MS 2040E, GE Healthcare)
Vortex mixer (AS ONE, model: Trio TM-1N)
Heat block (AS ONE, model: MyBL-10)
Long wavelength UV lamp (UVP, Black-lay model B-100AP, equipped with a 100 W bulb, 365 nm)
Electro-Eluter (Bio-Rad, model: 422)
Gel dryer (Bio-Rad, Model583, equipped with HydroTech vacuum pump)
Gel electrophoresis apparatus and glass plates (ATTO, AE-6500, 8 x 9 cm for mini-size gel format)

Software

Image analysis software such as Multi Gauge or Image Quant (Fuji Film or GE Healthcare, respectively)

Procedure

English

中文翻译

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Murai, M. and Miyoshi, H. (2019). Photoaffinity Labeling of Respiratory Complex I in Bovine Heart Submitochondrial Particles by Photoreactive [¹²⁵I] amilorides. Bio-protocol 9(17): e3349. DOI: 10.21769/BioProtoc.3349.
Uno, S., Kimura, H., Murai, M. and Miyoshi, H. (2019). Exploring the quinone/inhibitor-binding pocket in mitochondrial respiratory complex I by chemical biology approaches. J Biol Chem 294(2): 679-696.