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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Methods Mol Biol. Author manuscript; available in PMC 2013 January 21.
Published in final edited form as:
PMCID: PMC3549666
NIHMSID: NIHMS427185

Allosteric mechanisms of G protein coupled receptor signaling: a structural perspective

Abstract

G protein-Coupled Receptors (GPCRs) use a complex series of intramolecular conformational changes to couple agonist binding to the binding and activation of cognate heterotrimeric G protein (Gαβγ). The mechanisms underlying this long-range activation have been identified using a variety of biochemical and structural approaches and have primarily used visual signal transduction via the GPCR rhodopsin and cognate heterotrimeric G protein transducin (Gt) as a model system. In this chapter, we will review the methods that have revealed allosteric signaling through rhodopsin and transducin. These methods can be applied to a variety of GPCR-mediated signaling pathways.

Keywords: G protein coupled receptor, Heterotrimeric G proteins, Rhodopsin, Transducin, Receptor-mediated nucleotide exchange

1. Introduction

GPCRs regulate physical processes ranging from vision to olfaction to cardiac contractility to neurotransmission, yet these receptors likely act via a conserved signaling cycle (Figure 1). In this cycle, the activation states of GPCRs and cognate G proteins are encoded into protein conformations that influence the binding of partner signaling molecules. For example, agonist binding to a GPCR induces a conformational change that forms a binding site for heterotrimeric G proteins 25 Å away (Figure 2) (1, 2). The high affinity GPCR-G protein complex promotes exchange of GDP for GTP in the guanine nucleotide-binding pocket of the of the G protein α subunit (Gα) another 30 Å away from the proposed receptor-binding site (3, 4). The identity of the bound nucleotide then influences the formation of an effector-binding site, yet another 10 Å from bound GTP (Figure 3) (57). The activation steps in this complex series have been investigated by a number of complementary methods.

Figure 1
Summary of the G Protein Signaling Cycle
Figure 2
Differences in Conformation Changes Between Rhodopsin and Opsin
Figure 3
The binding regions of G proteins

1.1 Introduction to Allostery in Receptor Activation

Each GPCR has incredible fidelity to a small set of cognate ligands that act as agonists, antagonists, or inverse agonists to promote different activation states (8). Despite this diversity, GPCRs all share a common architecture comprising seven transmembrane helices (9, 10), and several conserved sequence motifs believed to be critical for regulation of receptor activation (1115). As a result, many of the general mechanisms of activation are assumed to be shared amongst all GPCRs.

The best-characterized GPCR, rhodopsin, is responsible for perception of light under low light conditions. Numerous properties of this photoreceptor have made it amenable to characterization by both in vivo and in vitro methods. Rhodopsin is naturally abundant (16), easily purified, stable in a variety of detergents, and has spectroscopic properties that provide a convenient method to monitor activation. In addition, rhodopsin covalently ligates an inverse agonist, 11-cis-retinal, that locks it into an inactive conformation, which decreases the conformational heterogeneity. Accordingly, rhodopsin was the first GPCR to be characterized by x-ray crystallography (17), and eight years elapsed before the structure of other GPCRs (1822) verified the conserved arrangement of transmembrane helices in the family.

Cryoelectron microscopy (10, 23), site-directed spin labeling, and electron paramagnetic resonance spectroscopy (SDSL-EPR) (24, 25) suggested that large conformational changes accompany rhodopsin activation, the most dramatic of which is the rigid body movement of helix VI away from helix III (Figure 3). This helix movement has long been hypothesized to be required for the formation of a G protein-binding site, and the recent crystal structures of opsin (2) and opsin in complex with a high affinity peptide mimicking the C-αt subunit (1) confirmed this hypothesis.

1.2 Introduction to Allostery in Heterotrimeric G Proteins

Heterotrimeric G proteins comprise Gα, Gβ, and Gγ (26). Upon activation by GPCRs, heterotrimeric G proteins disassociate into GTP-bound Gα, and Gβγ, both of which can interact with effector proteins to regulate downstream signaling (Figure 1) (27). In humans, there are currently 18 known G α subtypes belonging to 4 subfamilies, 5 Gβ isoforms, and 12 Gγ isoforms. Each has differing binding specificities for both effectors and GPCRs (28).

Gα and Gβ γ can act on a diverse variety of effectors, but like their cognate GPCRs, their mechanisms of activation and deactivation are likely conserved throughout the family. Numerous x-ray crystal structures have revealed how the binding of GDP, GDP-AlF4 or GTP γS to the G α subunit influences the conformations of three nearby surface loops known as switches I–III ( 4, 6, 29). These switch regions form a contiguous surface for effector binding (Figure 3b) (30).

While rhodopsin is by far the most extensively characterized GPCR, its cognate G protein Gt does not heterologously express to levels sufficient for biochemical characterization, which limits techniques that require mutagenesis. However, the large quantities of endogenously expressed transducin in bovine retina has allowed the native protein to be studied and crystallized. For techniques requiring mutagenesis, the close homolog of the Gαt subunit, Gαi (with 67% identity and 82% similarity to Gαt) is frequently used to understand general mechanisms of heterotrimeric G protein activation (3134). Gαi can be activated by rhodopsin, is easily expressed in bacteria, and can be reconstituted with Gβγ to form a fully functional chimeric system (46).

1.3 Introduction to Receptor-catalyzed Nucleotide Exchange

The rate-limiting step in GPCR signaling is receptor-catalyzed GDP release from the Gα subunit of the heterotrimer. A combination of site-directed mutagenesis (3639), peptide-mapping (3, 4043), and chemical crosslinking (4245) demonstrated that the α4–β6 loop, α3–β5 loop, N-terminus, and C-terminus of the Gα subunit as well as the C-termini of the G β G γ subunits are implicated in binding to rhodopsin. While these motifs are distantly arranged in the primary sequence, they form a contiguous surface on the folded protein (Figure 3a) (46, 47).

The α5 helix, located at the C-terminus of the Gα subunit, is perhaps the best-studied region of receptor interaction and independently binds within a pocket on activated receptor (1). Multiple studies have suggested that receptor induced rotation and translation of the α5 helix is a requirement for GDP release from Gα (26), and movement of the α5 helix dipole may be important for weakening the interactions between the bound nucleotide and Gα (43).

Much less is understood about how receptor interactions with the Gβγ subunits contribute to G protein activation. Until recently, two major hypotheses existed: the gearshift model (50) and the lever-arm model (51). Mutagenesis was used to support both hypotheses. The gearshift model proposes that the Gβ subunit rotates into the GTP-binding domain of the Gα subunit and pushes the helical domain away from the bound nucleotide. In comparison, the lever-arm model proposes that nucleotide exchange is catalyzed by induced tilt of the Gβγ subunits, which act as a lever to open the binding pocket through interactions with switch II. New insights into the mechanism of G protein activation were revealed by two recent crystal structures of activated β2 adrenergic receptor (β2AR). The first structure was that of the activated form of the receptor stabilized by llama antibodies termed nanobodies (52). The second structure was that of the activated receptor stabilized by its physiologically-relevant signaling partner, the Gαsβγ heterotrimer (Gs) (53). Both structures exhibited conformational changes in transmembrane helices 5 and 6. The β2AR-Gs structure additionally confirmed the stabilizing effects of G αβγ association on the agonist-bound state of the receptor and the presence of a translation of the a5 helix of Gα. Unanticipated observations revealed by the β2AR-Gs structure include ordering of intracellular loop 2 on β2AR, a lack of extensive contacts between the receptor and the Gβγ subunits of Gαsβγ, and a large displacement of the helical domain relative to the Ras-like GTPase domain of Gαs. These data, taken together, offer a possible mechanism for receptor-catalyzed GDP release from Gαβγ that is largely consistent with the models suggested by biochemical and structural data obtained thus far.

2. Materials

2.1. Dark-adapted Rod Outer Segment (ROS) membrane isolation

  1. 200 Bovine retinas (see Note 1).
  2. Isolation Buffer: 90 mM KCl, 30 mM NaCl, 2 mM MgCl2, 10 mM 3-(N-morpholino)propanesulfonic acid (MOPS), 0.1 mM ethylenediaminetetraacetic acid (EDTA), pH 8.0.
  3. 26% Sucrose Solution: Isolation Buffer containing 26% w/v sucrose, 1 mM dithiothreitol and 50 μM phenylmethylsulfonyl fluoride (PMSF) (see Notes 2, 3).
  4. 30% Sucrose Solution: Isolation Buffer containing 30% w/v sucrose, 1 mM DTT, and 50 μM PMSF
  5. Gradient Solution 1: 0.84 M sucrose, 10 mM MOPS, 60 mM KCl, 30 mM NaCl, and 2 mM MgCl2.
  6. Gradient Solution 2: 1.0 M sucrose, 10 mM MOPS, 60 mM KCl, 30 mM NaCl, and 2 mM MgCl2.
  7. Gradient Solution 3: 1.14 M sucrose, 10 mM MOPS, 60 mM KCl, 30 mM NaCl, and 2 mM MgCl2.
  8. 10 mg/mL aprotinin.
  9. 10 mg/mL leupeptin.
  10. 2.5 mg/mL pepstatin.

2.2 Rhodopsin-Gt separation: ROS membrane urea wash

  1. Dark-adapted ROS membranes isolated from retinas.
  2. EDTA Buffer: 10 mM Tris(hydroxymethyl)aminomethane (Tris-Cl) (see Note 4), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM DTT, pH 7.5 (see Note 2).
  3. Urea Buffer: 10 mM Tris-Cl, 1 mM EDTA, 1 mM DTT, 6 M urea, pH 7.5.
  4. Buffer A: 10 mM MOPS pH 7.5, 2 mM MgCl2, 200 mM NaCl, 1 mM DTT, 0.1 mM PMSF.

2.3 Rhodopsin extraction from native membranes

  1. Urea-washed, dark-adapted ROS membranes.
  2. Concanavalin A (ConA) Binding Buffer: 20 mM Tris-Cl pH 7.4, 250 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 1 mM MnCl2.
  3. Rhodopsin Solubilization Buffer: 80 mM SOL-grade n-Dodecyl-β-D-maltoside (DDM) (Anatrace) (see Note 5) dissolved in ConA Binding Buffer (see Note 6).

2.4 Purification of detergent extracted rhodopsin by ConA affinity chromatography

  1. ConA Sepharose column (GE Healthcare).
  2. ConA Binding Buffer: 20 mM Tris-Cl pH 7.4, 250 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 1 mM MnCl2 (see note 4).
  3. Wash Buffer 1: ConA Binding Buffer containing 0.5 mM ANAGRADE DDM (Anatrace) (see notes 5 and 6).
  4. Wash Buffer 2: 20 mM Tris-Cl pH 7.4, 100 mM NaCl, 0.5 mM ANAGRADE DDM (Anatrace), pH 7.4.
  5. Elution Buffer 1: 20 mM Tris-Cl pH 7.4, 100 mM NaCl, 0.5 mM ANAGRADE DDM (Anatrace)400 mM α-D methylglucoside (Acros Organics), pH 7.4.
  6. Elution Buffer 2: 20 mM Tris-Cl pH 7.4, 100 mM NaCl, 0.5 mM ANAGRADE DDM (Anatrace), 500 mM sucrose, pH 7.4.
  7. Extra Meta II Exchange Buffer: 50 mM HEPES pH 8.0, 100 mM NaCl, 1 mM MgCl2, 0.5 mM DDM.

2.5 Determining the concentration of rhodopsin

  1. Rhodopsin in native membranes or purified into detergent micelles.
  2. 20 mM hexadecyl-cetyltrimethylammonium chloride (HTAC) buffer (Sigma Aldrich).

2.6 Metarhodopsin II (meta II) assay

  1. 10 μM purified rhodopsin.
  2. 10 μM purified transducin.
  3. Meta II Buffer: 50 mM HEPES pH 8.0, 100 mM NaCl, 1 mM MgCl2, 1 mM DTT (see Note 2).

2.7 Crystallization of rhodopsin

  1. Pre-greased 24-well Linbro plates (Hampton Research).
  2. Siliconized Glass or Plastic cover slips (Hampton Research) (see Note 7).
  3. Refrigerant-based propelled compressed air cleaner, such as Dust-off.

2.8 Light-adapted ROS membrane isolation

  1. 200 Bovine retinas (see Note 1).
  2. Isolation Buffer: 90 mM KCl, 30 mM NaCl, 2 mM MgCl2, 10 mM MOPS, 0.1 mM EDTA, pH 8.0.
  3. 26% Sucrose Solution: Isolation Buffer containing 26% w/v sucrose, 1 mM dithiothreitol (DTT), and 50 μM phenylmethylsulfonyl fluoride (PMSF) (see Notes 2 and 3).
  4. 30% Sucrose Solution: Isolation Buffer containing 30% w/v sucrose, 1 mM DTT, and 50 μM PMSF.
  5. Gradient Solution 1: 0.84 M sucrose, 10 mM MOPS, 60 mM KCl, 30 mM NaCl, and 2 mM MgCl2.
  6. Gradient Solution 2: 1.0 M sucrose, 10 mM MOPS, 60 mM KCl, 30 mM NaCl, and 2 mM MgCl2.
  7. Gradient Solution 3: 1.14 M sucrose, 10 mM MOPS, 60 mM KCl, 30 mM NaCl, and 2 mM MgCl2.
  8. 10 mg/mL aprotinin.
  9. 10 mg/mL leupeptin.
  10. 2.5 mg/mL pepstatin.

2.9 Isolation and purification of Gt

  1. Isotonic Buffer: 5 mM Tris-Cl, 130 mM KCl, 0.6 mM MgCl2, 1 mM EDTA, 5mM βME or 1 mM DTT, 0.1 mM PMSF, pH 8.0 (see Notes 24).
  2. Hypotonic Buffer: 5 mM Tris-Cl, 0.6 mM MgCl2, 1 mM EDTA, 5mM βME or 1 mM DTT, 0.1 mM PMSF, pH 8.0.
  3. Hypotonic buffer + GTP: Hypotonic Buffer containing 0.1 mM guanosine-5′-triphosphate (GTP) (see Note 8).

2.10 Expression of Gαi1

  1. 2X YT (Yeast-Tryptone) Media: 1.6% w/v Bacto-tryptone, 10% w/v Bacto-yeast extract, 0.5% w/v NaCl, autoclaved.
  2. 100 mg/mL ampicillin, filter sterilized (see Note 9).
  3. Isopropyl-β-D-thiogalactopyranoside (IPTG), 1.0 M in distilled water, filter sterilized.

2.11 Purification of Gαi1

  1. Lysis Plus Buffer: 50mM NaH2PO4, 300 mM NaCl, 5 mM β-mercaptoethanol (βME), 5mM imidazole pH 8.0. Add 5 mM βME or 1 mM DTT, 20 μM GDP, 0.1 mM PMSF, 10 μg/mL aprotinin, 10 μg/mL leupeptin, and 2.5 10 μg/mL pepstatin (see Notes 2 and 3).
  2. Loading Buffer: 50 mM NaH2PO4, 300 mM NaCl, 5 mM βME or 1 mM DTT, pH 8.0.
  3. Wash Buffer: 50 mM NaH2PO4, 300 mM NaCl, 5 mM βME or 1 mM DTT, 10 mM imidazole, pH 8.0.
  4. Elution Buffer: 50 mM NaH2PO4, 300 mM NaCl, 5 mM βME or 1 mM DTT, 100 mM imidazole, pH 8.0.
  5. Dialysis Buffer: 50 mM Tris-Cl pH 8.0, 50 mM NaCl, 1 mM MgCl2, 10% (v/v) glycerol (see Note 4). Add 0.1 mM PMSF, 5 mM βME or 1 mM DTT, and 20 μM GDP right before buffer is used.
  6. Buffer A: 50 mM Tris-Cl pH 8.0, 50 mM NaCl, 2 mM MgCl2, 5mM βME or 1 mM DTT.
  7. Buffer B: 50 mM Tris-Cl pH 8.0, 1 M NaCl, 2 mM MgCl2, 5mM βME or 1 mM DTT.
  8. Storage Buffer: 50 mM Tris-Cl pH 8.0, 50 mM NaCl, 2 mM MgCl2, 5 mM βME or 1 mM DTT, 10 μM GDP
  9. Collection Buffer: 50 mM Tris-Cl pH 8.0, 50 mM NaCl, 2 mM MgCl2, 5 mM βME or 1 mM DTT 100 μM GDP
  10. i Buffer: 50 mM Tris-Cl pH 8.0, 200 mM NaCl, 2 mM MgCl2, 1 mM EDTA pH 8.0, 1 mM DTT, 20 μM GDP.
  11. TALON or Nickel NTA Resin (Clontech).
  12. SOURCE 15Q Anion Exchange resin (GE Healthcare).
  13. Superdex 200 GL 10/300 Gel Filtration Column (GE Healthcare).

2.12 Intrinsic tryptophan fluorescence assay

  1. NaF, 500 mM in distilled water.
  2. AlCl3, 10 mM in distilled water.
  3. Storage Buffer: 50 mM Tris-Cl pH 8.0, 50 mM NaCl, 2 mM MgCl2, 5 mM βME or 1 mM DTT, 10 μM GDP (see Notes 2 and 4).

2.13 Determining the concentration of Gt or Gαi1

  1. Quick Start Bradford Protein Assay Kit containing 1X dye reagent and seven BSA standards ranging in concentration from 0.125 – 2.0 mg/mL (BIORAD).
  2. 1:10 and 1:50 dilutions of purified Gt or Gαi1 sample.
  3. 1 mL quartz cuvettes.

2.14 Crystallization of Gt or Gαi1

  1. Pre-greased 24-well Linbro plates (Hampton Research).
  2. Siliconized Glass or Plastic cover slips (Hampton Research) (see Note 7).
  3. Refrigerant-based propelled compressed air cleaner, such as Dust-off.

2.15 Chemical Crosslinking of Rhodopsin-Gt

  1. Dark-adapted ROS membranes.
  2. Buffer A: 10 mM MOPS pH 7.4, 200 mM NaCl, 1 mM MgCl2, 1 mM DTT, 1 mM EDTA, 0.1 mM PMSF (see Notes 2 and 3).
  3. 3,3′-dithio-bis succini- midylpropionate (DTSSP), cleavable crosslinking agent (Pierce).
  4. 1M Tris-Cl pH 7.5 (see Note 4).
  5. 100 μM GTPγS.
  6. 50 mM DTT.

2.16 In vitro Rho-Gt complex formation

  1. Purified Gt.
  2. Purified rhodopsin.
  3. Rho-Gt Buffer: 50 mM HEPES pH 8.0, 100 mM NaCl, 1 mM MgCl2, 0.5 mM DDM, 40 μM all-trans retinal (see Notes 10 and 11).
  4. Superdex 200 GL 10/300 Gel Filtration Column (GE Healthcare).

2.17 Receptor-catalyzed GTPγS exchange assay

  1. 200 nM purified Gt
  2. 100 nM purified rhodopsin.
  3. Gt Activation Buffer: 10 mM MOPS, 130 mM NaCl, 1mM MgCl2, pH 7.2.
  4. 10 μM GTPγS.

3. Methods

Large quantities of pure protein are essential for studying biophysical properties of GPCRs and heterotrimeric G proteins. Therefore, the identification of mechanisms of GPCR signaling has been facilitated by reproducible methods for obtaining and purifying rhodopsin, transducin, and Gαi. Spectroscopic monitoring is commonly used to follow the activation state in these proteins, while x-ray crystal structures have provided snapshots of stable signaling intermediates.

3.1 Dark-adapted Rod Outer Segment (ROS) membrane isolation

  1. All steps are to be performed under dim red light and on ice or at 4°C.
  2. Thaw bovine retinas from 200 bovine eyes on ice and decant into a 600 mL beaker. Wash the containers with 30% Sucrose Solution, pool the wash with the thawed retinas, and bring the final volume to 200 mL with 30% Sucrose Solution.
  3. Stir the retina slurry for 1 hour at 4°C to separate the outer segments from the rod cell inner segments and basolateral membrane.
  4. Evenly distribute approximately 30 mL of the retina slurry into eight Oak Ridge centrifuge tubes and balance with 30% Sucrose Solution. Cap and invert the tubes 2–3 times to evenly mix the solution. Centrifuge for 20 minutes at 4°C in a Sorvall SS-34 rotor at 4,000 rpm (or 2,000 rcf(max) in a similar rotor).
  5. Carefully pour the supernatant into a beaker placed on ice and set aside (see Notes 12 and 17). Resuspend each pellet with a small volume of 30% Sucrose Solution. Evenly distribute the resuspended pellet into four clean Oak Ridge centrifuge tubes. Rinse the old centrifuge tubes with a small volume of 30% Sucrose Solution. Evenly distribute this wash solution into each of the new centrifuge tubes and balance with 30% Sucrose Solution. Cap and invert the tubes 2–3 times to evenly mix the solution. Centrifuge at 4°C for 4 minutes in a Sorvall SS-34 rotor at 6,000 rpm (or 4,300 rcf(max) in a similar rotor).
  6. Carefully pool the supernatant with the supernatant from the previous round of centrifugation. The pellet may now be discarded. Evenly distribute the pooled supernatant into four, clean Oak Ridge centrifuge tubes and balance with Isolation Buffer. Cap and invert the tubes 2–3 times to evenly mix the solution. Centrifuge for 20 minutes at 4°C in a Sorvall SS-34 rotor at 19,000 rpm (or 43,000 rcf(max) in a similar rotor).
  7. Discard the supernatant and resuspend the pellets with 1 mL of 26% Sucrose Solution for every two pellets. Pool the resuspended pellet slurry in a clean beaker placed on ice. Wash the centrifuge tubes with 1 mL of 26% Sucrose Solution for every two tubes. Pool the wash solution with the resuspended pellet slurry.
  8. Prepare a sucrose gradient by layering each of the Sucrose Gradient Solutions (13) in six 36 mL Sorvall AH629 swinging bucket centrifuge tubes (or similar). To prepare this gradient, first use a large needle or pipette to carefully layer 7 mL of Sucrose Solution 1 on the bottom of each centrifuge tube. Next, using the same pipette, layer 7 mL of Solution 2 by inserting the pipette tip through Solution 1 and touching the bottom of the centrifuge tube below Solution 1. Dispensing Solution 2 will cause Solution 1 to rise. Finally, pipette 7 mL of Solution 3 below Solution 2 using the same technique. Dispensing Solution 3 will cause Solutions 1 and 2 to rise. The final order of the sucrose solutions is Sucrose Solution 3 on the bottom, Sucrose Solution 2 in the middle, and Sucrose Solution 1 on top (see Note 13).
  9. Carefully layer the pellet slurry on top of each of each tube, balance with 26% Sucrose Solution, and centrifuge at 4°C for 30 minutes in a Sorvall AH-629 swinging bucket rotor at 25,000 rpm (or a similar rotor at 112,000 rcf(max)).
  10. Once the spin is complete, there will be a gradient of different colored layers in each of the centrifuge tubes. The bottom layer will be somewhat translucent, the next layer will be opaque, and the top layer will, again, be translucent. The orange layer in the center is the ROS membrane layer. Carefully discard the upper layer using a pipette and collect the opaque layer in the middle (see Note 14).
  11. Pool the ROS membrane layers, evenly distribute into clean Oak Ridge centrifuge tubes and balance with Isolation Buffer. Cap and invert the tubes 2–3 times to evenly mix the solution. Centrifuge for 20 minutes at 4 °C in a Sorvall SS-34 rotor at 19,000 rpm (or 43,000 rcf(max) in a similar rotor) to pellet the ROS membranes. During this centrifugation step, prepare 15 mL of Isolation Buffer containing 5 μg/mL pepstatin, 10 μg/mL aprotinin, and 10 μg/mL leupeptin.
  12. Discard the supernatant and resuspend each pellet with 1 mL of freshly prepared Isolation buffer containing 2.5 μg/mL pepstatin, 10 μg/mL aprotinin, and 10 μg/mL leupeptin Thoroughly homogenize the resuspended ROS membranes by pipetting up and down. Pool the ROS membranes in a 15 mL conical tube placed on ice.
  13. Wash the empty centrifuge tubes with enough of the remaining freshly prepared Isolation Buffer so that the final volume of ROS membranes does not exceed 15 mL. Dark-adapted ROS membranes can be stored in tubes wrapped in aluminum foil at −80°C for long-term storage.

3.2 Rhodopsin-transducin separation: dark-adapted ROS membrane urea wash

  1. All steps are to be performed under dim red light and on ice or at 4°C.
  2. Thaw dark-adapted ROS membranes on ice and transfer to a 40 mL glass homogenizer. Fill the homogenizer with EDTA Buffer and homogenize ROS membranes (see Note 15). Pour the homogenate into ultracentrifuge tubes and balance with EDTA buffer (see Note 16). Cap and invert the tubes 2–3 times to evenly mix the solution. Centrifuge for 30 minutes in a Ti-70 rotor at 30,000 rpm or 92,000 rcf (max) in a similar rotor.
  3. Discard the supernatant. Resuspend each pellet with 1 mL EDTA Buffer, and transfer to a glass homogenizer. Rinse the tubes with a small volume of EDTA Buffer, pool with the resuspended pellets in the homogenizer, fill the homogenizer with EDTA Buffer, and homogenize (see Note 17). Evenly distribute the homogenate to new ultracentrifuge tubes and balance with EDTA Buffer. Cap and invert the tubes 2–3 times to evenly mix the solution. Centrifuge for 30 minutes in a Ti-70 rotor at 30,000 rpm (or 92,000 rcf (max) in a similar rotor).
  4. Discard the supernatant and resuspend each pellet with 1 mL of Urea Buffer. Transfer the pellet resuspension to a glass homogenizer. Rinse the tubes with a small volume of Urea Buffer, pool with the resuspended pellets, fill the homogenizer with Urea Buffer, and homogenize. Evenly distribute the homogenate to clean ultracentrifuge tubes and balance with Urea Buffer. Cap and invert the tubes 2–3 times to evenly mix the solution. To pellet the membranes, centrifuge for 30 minutes in a Ti-70 rotor at 45,000 rpm (or 208,000 rcf (max) in a similar rotor).
  5. Discard the supernatant and resuspend the pellets with Buffer A (see Note 17). Transfer the pellet resuspension to a glass homogenizer. Rinse the tubes with a small volume of Buffer A, pool with the resuspended pellets, fill the homogenizer with Buffer A, and homogenize. Evenly distribute the homogenate to clean ultracentrifuge tubes and balance with Buffer A. Cap and invert each tube 2–3 times to evenly mix the solution. Centrifuge for 30 minutes in a Ti-70 rotor at 30,000 rpm (or 92,000 rcf (max) in a similar rotor).
  6. Discard the supernatant and resuspend each pellet with 1 mL of Buffer A by pipetting up and down with a pipette in the centrifuge tubes. Prepare 100 μL aliquots of the urea-washed, dark-adapted ROS membranes in 650 μL tubes, wrap in foil, and store at −80°C.

3.3 Rhodopsin extraction from native membranes

  1. All steps are to be performed under dim red light and on ice or at 4°C.
  2. Thaw 3–4 aliquots of urea washed, dark-adapted ROS membranes on ice and pool in a 1.5 mL tube. Use 400 μL of ConA Binding Buffer wash each tube and pool with the thawed membranes (see Note 18).
  3. Centrifuge the membranes for 25 minutes at 4°C at top speed (20,000 RCF(max)) in a benchtop Eppendorf Centrifuge.
  4. Discard the supernatant and resuspend the pellet with 400 μL Rhodopsin Solubilization Buffer (see Note 19) by pipetting up and down until the resuspension is no longer cloudy. Incubate the resuspended pellet on ice for 45 minutes. Mix the sample by pipetting every 15 minutes during the incubation.
  5. After 45 minutes, centrifuge the sample for 25 minutes at 4°C at top speed (20,000 RCF(max)) in a benchtop Eppendorf Centrifuge.
  6. Collect the supernatant (~500 μL) with a pipette and discard the pellet.

3.4 Purification of detergent extracted rhodopsin by ConA affinity chromatography

  1. Assemble a light-protected “ConA Purification Hutch” in a 4°C environment to protect the purification apparatus from light. Place a clamp stand for the ConA Sepharose column, a peristaltic pump, and a tube rack inside of the hutch (see Figure 4).
    Figure 4
    ConA Purification Hutch
  2. Attach a 30 mL syringe to the top of a 1 mL ConA column for holding buffers (see Note 20). Load 10 mL of Wash Buffer 1 to wash off the ethanol-containing storage buffer in which the column is packed. Equilibrate the column in 30 mL of Wash Buffer 1 in a continuous loop using a peristaltic pump flowing at 0.3 mL/min for at least 48 hours (see Note 21).
  3. Dilute ~500 μL of detergent-extracted rhodopsin (see Subheading 4) to a total volume of 10 mL with ConA Binding Buffer (see Note 22). When the volume of Wash Buffer 1 in the syringe is ≤ mL, load the rhodopsin fraction. Flow the rhodopsin load fraction in a continuous loop over the column using a peristaltic pump flowing at 0.3 mL/min for 4 hours (see Note 23).
  4. Collect the flowthrough and all subsequent fractions for later analysis of the purification by SDS-PAGE (see Figure 5 for expected results). When the volume of loaded protein in the syringe is ≤ mL, load 10 mL of Wash Buffer 1 and collect the flowthrough as Wash 1.
    Figure 5
    ConA Purification Fractions Analyzed by SDS-PAGE
  5. When the volume of loaded Wash Buffer 1 in the syringe is ≤ mL, load 5 mL of Wash Buffer 2 and collect the flowthrough as Wash 2.
  6. When the volume of loaded Wash Buffer 2 in the syringe is ≤ mL, load 50 mL of Elution Buffer 1 and collect the flowthrough as Elution 1. This fraction contains ConA purified rhodopsin.
  7. When the volume of loaded Elution Buffer 1 in the syringe is ≤ mL, load 5 mL of Elution Buffer 2 and collect the flowthrough as Elution 2. This fraction should also contain ConA purified rhodopsin and can be pooled with Elution 1.
  8. Concentrate the pooled Elution fractions in a 30 kDa molecular weight cutoff Amicon concentrator wrapped in aluminum foil. Centrifuge the concentrator in a benchtop centrifuge at 4°C at 1,600 rcf(max) in 20 minute intervals until the total volume is ~2 mL (see Note 24).
  9. Using the same concentrator, buffer exchange the concentrated, ConA purified rhodopsin by diluting to 50 mL in Extra Meta II Exchange Buffer and serial centrifugation at 4°C and 1,600 rcf(max) in 20 minute intervals. Concentrate the sample to a volume of ~200–500 μL and measure the concentration using the rhodopsin concentration assay (see Subheading 5).

3.5 Determining the concentration of rhodopsin

  1. The concentration of solubilized rhodopsin is determined by measuring its absorbance at 500 nm and 650 nm both before and after photobleaching in the presence of 20 mM fluorescence enhancing detergent, HTAC.
  2. Record an absorption spectrum from 350–650 nm for 96 μL of 20 mM HTAC buffer added to an appropriate cuvette. Use this measurement to establish a baseline.
  3. Add 4 μL of a dark-adapted rhodopsin sample to 96 μL of HTAC buffer in the cuvette and mix thoroughly by pipetting up and down. Record a second absorption spectrum from 350–650 nm for the dark-adapted sample.
  4. Photobleach the rhodopsin-containing sample in the cuvette by pulsing with a burst of light from a camera flash, such as a Vivitar 283. Thoroughly mix the sample by pipetting up and down in the cuvette, being careful to avoid generating air bubbles, and pulse the sample with light a second time (see Notes 25 and 27). Mix the sample again and record a third spectrum from 350–650 nm for the light-adapted sample.
  5. Calculate the difference in the absorbance between 650 nm and 500 nm for both the dark-adapted and light-adapted readings using Equation 1:
    equation M1
  6. Calculate the difference in this value between dark-adapted and light-adapted rhodopsin using Equation 2:
    equation M2
  7. Use this value to then calculate the concentration with ‚Beer’s Law (Equation 3):
    equation M3
    where A corresponds to the absorbance value, ε corresponds to the molar extinction coefficient for rhodopsin, b corresponds to the path length (in cm), and C corresponds to concentration (in units of molarity (mol/L)). The molar extinction coefficient is 42,000 M−1cm−1 for rhodopsin in ROS membranes and 40,600 M−1cm−1 for detergent-extracted rhodopsin. Use the latter value to determine the concentration of purified rhodopsin (see Note 26).
  8. See Figure 6 for expected results.
    Figure 6
    Solubilized Rhodopsin Absorption Spectrum

3.6 Metarhodopsin II (meta II) assay

The metarhodopsin II assay can be used to determined the stability of light-activated rhodopsin, meta II, in the conditions in which it has been purified. Chimeric, heterotrimeric G protein (Gαiβγ) can be substituted for Gt to characterize the contribution of mutations on protein complex stability.

  1. Prepare a 10 μM sample of purified, dark-adapted rhodopsin (see Subheadings 4 and 5) in Meta II Buffer and mix with 10 μM purified Gt (see Subheadings 9 and 13) in Meta II Buffer.
  2. Incubate the sample on ice for 15 minutes in the dark or under dim red light. Pipette the incubated sample into an appropriate cuvette, place in a spectrophotometer, and collect an absorption spectrum from 350–650 nm for the dark-adapted sample.
  3. Light-activate the sample by pulsing with two bursts of light from a camera flash. Wait 1 minute to allow the sample to be completely activated, mix thoroughly by pipetting, and collect a second absorption spectrum from 350–650 nm for the light-adapted sample (see Note 27).
  4. To determine the signal contributed by meta II, calculate the difference in the absorbance between 380 nm and 440 nm for both the dark-adapted and light-adapted readings using Equation 4.
    equation M4
  5. Then determine the meta II signal by calculating the difference in ΔA380–440 between dark-adapted and light-adapted rhodopsin using Equation 5.
    equation M5
  6. See Figure 7 for expected results.
    Figure 7
    Metarhodopsin II Absorption Spectrum

3.7 Crystallization of Rhodopsin

While multiple methods of crystallization exist (sitting drop, hanging drop, batch, etc), the hanging drop vapor diffusion method is the most commonly used (Figure 8). In this method, a closed system is designed in which a droplet of purified protein mixed with reservoir solution, usually in a 50:50 ratio, is allowed to equilibrate with a solution contained in a reservoir below. As water evaporates from the droplet hanging above the reservoir solution, the solubility of the purified protein decreases, the concentration of precipitant increases, and nucleation of crystals occurs. The system is then maintained at equilibrium until crystallization is complete.

Figure 8
Hanging Drop Vapor Diffusion
  1. Filter all reagents to be used in the crystallization screen and equilibrate at the crystallization temperature (see Notes 28 and 29).
  2. Mix 1 mL of reservoir solution in the bottom of each well of a pre-greased 24-well Linbro tray. Crystallization conditions for rhodopsin are given in Table 1. Place the tray on a rotating table or pipette reservoir solution up and down to mix the reagents.
    Table 1
    Crystallization of Rhodopsin
  3. Remove dust from siliconized cover slips using an air duster. Pipette 1–2 μL of purified protein onto the cover slip. Pipette an equal volume of reservoir solution on top of the protein drop (see Note 30). It is not necessary to mix this drop. Carefully invert the cover slip and place over the well from which the reservoir solution was taken. Gently push down on the cover slip to make sure the well is tightly sealed. There should be no gaps between the cover slip and silicone grease.
  4. Allow the crystallization reactions to equilibrate without disruption in a vibration-free location at the appropriate temperature for at least 24 hours before monitoring (see Notes 3133).
  5. Adjust crystallization conditions based on the results of preliminary screens (see Notes 34 and 35).

3.8 Light-adapted ROS membrane isolation

  1. All steps should be performed under ambient light and on ice or at 4°C.
  2. Thaw bovine retina preparations from 200 eyeballs on ice and decant into a 600 mL beaker. Wash the containers with 30% Sucrose Solution, pool the wash with the thawed retinas, and bring the final volume to 200 mL with 30% Sucrose Solution.
  3. After diluting the retinal slurry to 200 mL with 30% Sucrose Solution, photobleach the retinal slurry without stirring for 30 minutes in order to allow for the formation of the high affinity Rhodopsin-Gt complex. After 30 minutes, continue to photobleach the retinal slurry for an additional 1 hour with stirring to separate the outer segments from the rod cell inner segments and basolateral membrane.
  4. Repeat steps 4–13 as given in Subheading 1. It is not necessary to protect the purified membranes from the light.

3.9 Isolation and purification of transducin

  1. All steps should be performed at 4°C or on ice.
  2. Thaw light-adapted ROS membranes (see Subheading 8) and pour into a 40 mL glass homogenizer. Rinse the frozen ROS membrane container with Isotonic Buffer. Pool the wash solution with the thawed ROS membranes, fill the homogenizer with Isotonic Buffer, and homogenize.
  3. Evenly distribute homogenate into ultracentrifuge tubes and balance with Isotonic Buffer (see Note 16). Cap and invert the tubes 2–3 times to evenly mix the solution. Centrifuge for 20 minutes in a Ti-70 rotor at 30,000 rpm (or 92,000 rcf(max) in a similar rotor).
  4. Discard the supernatant and resuspend the pellets with a small volume of Isotonic Buffer. Transfer the resuspended pellets to the glass homogenizer. Rinse each centrifuge tube with Isotonic Buffer. Pool the wash solution with resuspended pellets, fill the homogenizer with Isotonic Buffer, and homogenize.
  5. Repeat steps 3 and 4 twice, followed by step 3 a third time for a total of four washes with Isotonic Buffer.
  6. After the fourth Isotonic Buffer wash, discard the supernatant and resuspend the pellets with a small volume of Hypotonic buffer. Transfer the resuspended pellets to a glass homogenizer. Rinse each centrifuge tube with Hypotonic Buffer, pool the wash solution with resuspended pellets, fill the homogenizer with Hypotonic Buffer, and homogenize.
  7. Evenly distribute homogenate into ultracentrifuge tubes and balance with Hypotonic Buffer. Cap and invert the tubes 2–3 times to evenly mix the solution. Centrifuge for 20 minutes in a Ti-70 rotor at 30,000 rpm (or 92,000 rcf(max) in a similar rotor).
  8. Repeat steps 6 and 7 for a total of two Hypotonic Buffer washes.
  9. Prepare 400 mL of fresh Hypotonic Buffer containing 0.1 mM GTP (see Note 8). After the second Hypotonic Buffer wash, resuspend each pellet with 25 mL of Hypotonic Buffer containing 0.1 mM GTP. Transfer pellet resuspension to a glass homogenizer. Rinse each tube with a small-volume of the Hypotonic Buffer containing 0.1 mM GTP, and homogenize.
  10. Evenly distribute homogenate into ultracentrifuge tubes and balance with Hypotonic Buffer containing 0.1 mM GTP. Cap and invert the tubes 2–3 times to evenly mix the solution. Centrifuge for 45 minutes in a Ti-70 rotor at 30,000 rpm (or 92,000 rcf(max) in a similar rotor).
  11. Decant the supernatant into a clean container and store on ice. This supernatant contains Gt. Repeat steps 9 and 10 for a total of two washes with Hypotonic Buffer containing 0.1 mM GTP.
  12. Pool the supernatant with the supernatant from the previous round of washing with Hypotonic Buffer containing 0.1 mM GTP. The pellet can now be discarded.
  13. Concentrate Gt-containing supernatant to 1 mL. Particulate contaminants and any remaining membranes may be removed by spinning at top speed in a benchtop Eppendorf centrifuge at 4°C for 30 minutes (optional).
  14. If the purified Gt is to be stored for use at a later time, add glycerol to a final concentration of 10% and freeze the purified protein at −20°C for short-term storage, or −80°C for long-term storage (see Note 36).
  15. See Figure 9 for expected results of an SDS-PAGE analysis of Gt purification fractions.
    Figure 9
    Purification of Gt

3.10 Expression ofGαi1

  1. i subunits are usually expressed with hexahistidine-tags and from expression vectors with ampicillin-resistance. If your mutant is expressed using an alternative antibiotic resistance marker, use the appropriate antibiotic.
  2. Transform BL21 cells with plasmid DNA encoding Gα and plate onto a luria-broth (LB) agar plate containing 100 μg/mL ampicillin. Incubate the plate overnight at 37°C to allow colonies to grow (see Note 37).
  3. The day before you begin cell culture, prepare eight 2L baffled flasks containing 500 mL 2X YT media and one 500 mL baffled flask containing 150 mL of 2X YT media. Autoclave and cool the media to room temperature.
  4. In the morning, prepare a starter culture by pipetting 2 mL 2X YT medium out of the 150 mL flask into a 14 mL cell culture tube and supplementing with 100 μg/mL ampicillin Inoculate this starter culture with a single colony from the overnight LB agar plate, by transferring on a sterile pipette tip or toothpick (see Note 38). Incubate this culture for 8 hours at 37°C while shaking.
  5. After 8 hours, supplement the 500 mL baffled flask containing 150 mL 2X YT media with 100 μg/mL ampicillin and inoculate with your 2 mL starter culture. Incubate this culture overnight, at room temperature (22–25°C), while shaking.
  6. On the following morning, supplement each 2 L baffled flask containing 500 mL 2X YT media with 100 μg/mL Ampicillin and inoculate with 10 mL of the 150 mL 2X YT overnight culture. Incubate the cultures at room temperature while shaking until optical density at 600 nm (OD600) reaches 0.3–0.4 (see Note 39).
  7. Once the OD600 reaches the appropriate level, induce protein expression by adding 30 μM IPTG to each flask. Incubate the cultures at room temperature while shaking for 16–18 hours (see Note 40).
  8. After 18 hours, stop the cell growth and harvest cells for purification (see Note 41).

3.11 Purification of Gαi1

  1. Distribute overnight cultures (see Subheading 10) into 1L centrifuge bottles, balance each bottle, and pellet the cells by centrifuging for 15 minutes at 4°C at 5,000 rpm in Sorvall F9S rotor (or 4,500 rcf(max)).
  2. Discard the supernatant and resuspend the cell pellets with 25 mL of Lysis Plus Buffer on ice. Pool the cell resuspension in a sonication beaker on ice and bring the total volume to 100 mL with Lysis Plus Buffer.
  3. Lyse the cell resuspension by sonicating at 50% amplitude in 20 second intervals for a total of 4 minutes. Rest the cells for 40 seconds between each sonication cycle. Supplement the cell lysate with 0.1 mM PMSF after each minute of sonication. Add 0.1 mM GDP to the lysate, transfer to ultracentrifuge tubes (see Note 16), and balance the tubes with Lysis Plus Buffer. Cap and invert the tubes 2–3 times to mix the solution. Centrifuge at 4°C for 1 hour in a Ti-70 rotor at 50,000 rpm (or 257,000 rcf(max) in a similar rotor). During this time, begin to equilibrate the resin for affinity chromatography (step 5).
  4. Collect the supernatant and discard the pellet. The supernatant is your cleared lysate. Supplement the lysate with 0.1 mM PMSF and store on ice if the resin for affinity chromatography is not fully prepared.
  5. Equilibrate 5 mL of Ni-NTA resin or Co2+-TALON resin (10 mL of a 50% slurry) per 4 L of cell culture in Lysis Plus Buffer. To do this, add 5 mL slurry to each of two 50 mL conical tubes, fill each tube to 50 mL with Lysis Plus Buffer, and mix by gently inverting (see Note 42). Pellet the resin by centrifugation at 4°C and 700 rcf(max) for 2 minutes, discard supernatant, and resuspend each resin pellet with 25 mL (10 × column volume) of Lysis Buffer. Repeat centrifugation and resuspension steps 3–5 times (see Note 43).
  6. Once equilibration of the resin is nearly complete, add DNase and RNase to the cleared lysate and incubate at room temperature for ~ 5–10 min to digest genomic DNA and decrease the viscosity of your lysate. Filter the lysate through a 0.45 μm filter.
  7. Pellet the resin by centrifugation, discard the supernatant, and mix each 2.5 mL aliquot of equilibrated resin with 50 mL lysate in conical tubes for 1 hour at 4°C while rotating.
  8. Pour each lysate-resin mixture into a separate 20 mL gravity flow column and collect the lysate flowthrough (see Note 44).
  9. Wash the tubes containing the filtered lysate-resin mixture with 10 mL of Loading Buffer to recover any remaining protein or resin and add to the gravity flow columns.
  10. Add 25 mL (10 × column volume) of Loading Buffer to each column and collect the flowthrough as Wash fraction 1 in a new collection tube.
  11. Add 25 mL of Wash Buffer (10 × column volume) to each column and collect the flowthrough as a Wash fraction 2 in a new collection tube.
  12. Add 5 mL of Elution Buffer (10 × column volume) to each column and collect the flowthrough as an Elution Fraction 1 in a new collection tube.
  13. Pool the elution fractions, transfer to 10 kDa molecular weight cutoff dialysis tubing, and dialyze the sample in 1.8 L of Dialysis Buffer overnight at 4°C with stirring.
  14. On the following morning, collected the dialyzed protein sample, and concentrate to 5 mL in a 10 kDa molecular weight cut off Amicon concentrator. Pass the concentrated, dialyzed protein sample through a 0.22 μm Whatman filter.
  15. Connect a column packed with 5 mL of SOURCE 15Q resin (GE Healthcare) to an HPLC or FPLC system. Place one buffer inlet into Buffer A, and the other inlet into Buffer B. Wash the attached column with 15 mL (3 × column volume) of Buffer B.
  16. Following Buffer B, wash the column with 30 mL (6 × column volume) of Buffer A. Load the filtered protein sample onto a 5 mL sample loop and flow Buffer A over the column at 1.0 mL/min until the entire volume of protein sample has been loaded. While the sample is loading, add 300 μL of Collection Buffer to pre -chilled test tubes (see Note 45 ).
  17. Flow a linear gradient of Buffer B at 1.0 mL/min so that the final concentration of the buffer flowing over the column at the end of 30 minutes is 20% Buffer B and 80% Buffer A. Once the gradient reaches 20% Buffer B, flow Buffer B in a linear gradient at 1.0 mL/min so that the final concentration at the end of 10 minutes is 100% Buffer B and 0% Buffer A (see Note 46). Collect 500 μL fractions in the pre-chilled test tubes containing 300 μL of Collection Buffer until the gradient is complete.
  18. Use the intrinsic tryptophan fluorescence assay to test the activity of each fraction (see Subheading 12). Pool fractions with a minimum of 40% increase in intrinsic tryptophan activation by AlF4-, and analyze purity by SDS-PAGE (see Note 47).
  19. Subject pooled fractions to Bradford Assay (see Subheading 13) to determine the concentration of the pooled protein. If necessary, concentrate the pooled fractions to 2 mL using a 10 kDa molecular weight cutoff Amicon concentrator. Purified protein can be stored at −80°C as 0.5 mg protein aliquots in 10% glycerol or immediately purified by gel filtration chromatography.
  20. For gel filtration chromatography, equilibrate a Superdex 200 10/300 GL gel filtration column in Gαi Buffer (see Notes 48 and 49).
  21. Filter the protein sample using a 0.22 μm SpinX filter and load onto your column (see Note 50).
  22. Collect 500 μL fractions corresponding to purified Gα, which is expected to elute 15.5 mL after sample injection onto a 24 mL Supderdex 200 10/300 GL gel filtration column (see Note 51). Analyze the fractions for purity by SDS-PAGE (see Note 52) and test for activity using the intrinsic tryptophan fluorescence assay (see Subheading 12). Pure, active protein fractions can then be pooled, concentrated, and frozen at −80°C for long-term storage.
  23. See Figure 10 for expected results of an SDS-PAGE analysis of Gα purification fractions.
    Figure 10
    Purification of Gαi1

3.12 Intrinsic Tryptophan Fluorescence Assay

  1. Set excitation wavelength to 280 nm and emission wavelength to 340 nm on a spectrofluorometer.
  2. Add 900 μL of Storage Buffer to a quartz cuvette and zero the spectrofluorometer.
  3. Add 20 μL of purified protein (~200 nM) to 900 μL of Storage Buffer in the cuvette, mix thoroughly, and record basal fluorescence.
  4. Add AlF4 by mixing 20 μL of 500 mM NaF (10 mM final concentration) and 8 μL of 10 mM AlCl3 (80 μM final concentration) simultaneously in the cuvette containing protein in Storage Buffer (see Note 53). Mix thoroughly by pipetting and record fluorescence emission at 340 nm.
  5. Calculate the percentage increase in fluorescence between the two readings using the following formula:
    equation M6
  6. See Figure 11 for expected results.
    Figure 11
    Intrinsic Tryptophan Fluorescence of Heterologously Expressed G3 subunits

3.13 Determining the concentration of Gt or Gαi1

  1. Warm 1X Quick Start Bradford dye to room temperature (see Notes 54 and 55).
  2. Add 20 μL of water or buffer for a blank standard, each BSA standard, and 1:10 or 1:50 dilutions of purified, soluble Gt or Gαi1 into 1 mL cuvettes (see Notes 56 and 57).
  3. Add 1mL of room temperature 1X Quick Start dye to each cuvette.. Mix each sample by pipetting and incubate at room temperature for at least 5 minutes, but no more than 1 hour (see Note 58).
  4. Measure the absorbance of each sample at 595 nm. Use the blank sample prepared with water or buffer to zero the instrument before recording the absorbance for the standards and purified Gαi1 or Gt.
  5. Use a graphing program to plot the absorbance values and to fit the data to a line with the following equation:
    equation M7
    where A595 corresponds to the absorbance at a wavelength of 595 nm for each standard, C corresponds to the concentration of each known standard, b is the y-intercept of the line fit to the data collected, and m is the slope of the line.
  6. After determining the equation of the line that best fits the absorbance data collected for the known standards, use the recorded A595 collected for the protein sample of unknown concentration to determine its concentration (C).

3.14 Crystallization of Gα proteins

Table 2 summarizes the conditions used to determine the structures of a subset of 33 proteins known to interact with rhodopsin. These can be used to guide your own vapor diffusion experiment using Subheading 7.

Table 2
Crystallization of G protein α-subunits

3.15 Chemical Crosslinking of Rhodopsin to Gt

  1. Determine the concentration of rhodopsin in dark-adapted ROS membranes that are not urea-washed and resuspend to 10 μM in Buffer A (see Note 59).
  2. Light-activate the ROS-Gt sample by incubating on ice for 5 minutes under ambient light.
  3. Add a 10-fold molar excess of DTSSP to the light activated ROS sample and incubate for 30 minutes on ice.
  4. Add 1 M Tris-Cl pH 7.5 to a final concentration of 50 mM and incubate for 15 minutes at room temperature to terminate the crosslinking reaction.
  5. Centrifuge the crosslinked sample at 100,000 rcf(max) for 15 minutes at 4°C, discard the supernatant, and resuspend the pellet with Buffer A. Repeat membrane pellet wash three times. This removes Gt that is not associated with rhodopsin.
  6. After the third wash, resuspend the membrane pellet with Buffer A containing 100 μM GTPγS. Incubate the resuspended membranes for 5 minutes at 4°C.
  7. After 5 minutes, centrifuge the sample at 100,000 rcf(max) for 15 minutes at 4°C. Separate the supernatant from the pellet and analyze both by SDS-PAGE (see Figure 12 for expected results).
    Figure 12
    Chemical Crosslinking of Rhodopsin-Gt

3.16 In vitro Rho-Gt complex formation

  1. Mix stoichiometric amounts of dark-adapted ConA-purified rhodopsin with purified Gt and allow the sample to mix for 1 hour at 4°C in the dark.
  2. Light-activate rhodopsin and induce complex formation by pulsing the sample with bursts of light from a camera flash. Mix the sample by pipetting up and down and expose to a second burst of light from a camera flash. Incubate the light-activated sample for 20 minutes on ice under ambient light.
  3. Filter the sample through a 0.22 μm filter and load the activated complex on a Superdex 200 10/300 GL gel filtration column equilibrated with at least 10 column volumes of Rho-Gt Buffer (see Note 60).
  4. Monitor the absorbance of eluted species at 280 nm (total protein), 380 nm (meta II), and 440 nm (meta I) to quantify the completeness of rhodopsin activation and to separate heterogeneous species (see Figure 13a for expected results).
    Figure 13
    In vitro Formation of the Rhodopsin-Gt Complex
  5. Collect 500 μL fractions corresponding to purified Rhodopsin-Gt complex. The 2:1 complex is expected to elute at 11.5 mL and the 1:1 complex is expected to elute at 12.6 mL on a Superdex 200 10/300 GL column equilibrated in Rho-Gt Buffer (see Note 61). Analyze the fractions by SDS-PAGE (see Figure 13b for expected results) to verify the purity and presence of all components of the complex (see Notes 52 and 62). Analyze the activity of the complex using the meta II assay (see Subheading 6 or the GTPγS exchange assay (see Subheading 17).

3.17 Receptor-catalyzed GTP3S exchange assay

The rate of receptor-catalyzed nucleotide exchange can be measured by monitoring the increase in intrinsic tryptophan fluorescence in heterotrimeric G proteins in the presence of light activated rhodopsin. This assay can be applied to Gt or recombinant Gαi reconstituted with Gβγ (see Note 63 ).

  1. Mix 200 nM Gt or 200 nM Gαi mixed with 200 nM Gβγ in a cuvette containing Activation Buffer (see Note 64).
  2. Set the excitation wavelength to 280 nm and emission wavelength to 340 nm.
  3. Add 10 μM GTPγS and 100 nM purified rhodopsin, mix thoroughly by pipetting, and collect the emission spectrum at 340 nm for 40 minutes at 21°C.
  4. Normalize the data to the baseline (0%) and the fluorescence maximum (100%).
  5. Fit the normalized data to an exponential association curve to determine the receptor-catalyzed nucleotide exchange rate (see Figure 14 for expected results).
    Figure 14
    Example Optimization of a Crystallization Condition
Figure 15
Receptor-Catalyzed GTPγS Exchange

Footnotes

1Bovine eyes can be obtained from your local slaughterhouse.

2All solutions that include volatile reagents (DTT or βME) or protease inhibitors with short half-lives (PMSF) should be prepared fresh each time the solution is made.

3PMSF rapidly degrades in aqueous solutions where its half-life is approximately 110 minutes (56). Prepare stock solutions of this protease inhibitor in organic solvents such as acetone or isopropanol.

4The pH of all buffers containing Tris-Cl listed in protocols throughout this chapter should be adjusted with HCl.

5Many detergents are available in two grades. SOL-grade detergents are less pure and less expensive. These can be used for solubilization of membrane proteins but may be too heterogeneous to support crystallization. ANAGRADE detergents are of higher purity and should be used for techniques requiring homogenous solutions, like x-ray crystallography.

6All buffers used for chromatographic techniques should be filtered to remove large particles and dust. Buffers containing detergents should be filtered in sufficiently large bottles (use a 1 L bottle for filtering 500 mL of buffer) so that the bubbles generated during filtering will not be suctioned into the vacuum pump. If you are using Millipore Centricon® filters, add DDM after filtering. DDM will concentrate in Millipore Centricon® filters, which can affect the final concentration of detergent in your buffer.

7It is important to use high quality, siliconized or plastic cover slips versus non-siliconized glass cover slips. While more expensive, the hydrophobic surface of siliconized cover slips promotes protein droplets more conducive to crystallization.

8GTP can be hydrolyzed in aqueous solutions. Prepare this buffer right before use and add GTP from a fresh stock.

9Ampicillin is usually prepared as a 1000X (100 mg/mL) stock solution that can withstand long-term storage at −20°C. The stock solution can be made in either a water solution or a 70% ethanol solution. The latter will not freeze at −20°C, thus decreasing the wait time for thawing the reagent during experiments.

10The rhodopsin-Gt complex is light sensitive. Protect the gel filtration column from ambient light by performing gel filtration experiment under dim red light or by wrapping column in aluminum foil.

11The half-life of a rhodopsin-Gt complex in the presence of detergent micelles is approximately 30 minutes. Supplementing the gel filtration buffer or eluted protein fractions with all-trans retinal can improve the half-life of the complex (57).

12If the volume of the retinal slurry exceeds the volume held by the maximum number of centrifuge tubes that will fit in your rotor, leave the pellet intact, layer remaining slurry on top of the pellet, and repeat the centrifugation step until all of the retinal slurry is centrifuged as described. Pool the fresh supernatant with supernatant from the previous round of centrifugation.

13Sucrose Solution 1 is the least dense, while Sucrose Solution 3 is the densest. To ensure that you maintain a distinct interface, be slow to dispense each sucrose solution into each of the centrifuge tubes and slow to remove the pipette tip from the centrifuge tube once the solution has been dispensed.

14The sucrose layer below the ROS membrane layer and the pellet at the bottom of the centrifuge tube contain contaminants. To avoid these contaminants, remove the top layer by pipetting slowly and without disrupting the gradient.

15Homogenize all resuspended fractions by inserting and removing the plunger into the glass homogenizer at least 10 times. This will ensure adequate separation of the ROS membranes from other retinal components.

16Extra caution is required when balancing ultracentrifuge tubes. The weight of each tube should be within 0.1 g of each other.

17The pellet in this step of the purification contains the ROS membranes. It is very soft and may decant with the supernatant. Instead of decanting, pipette the supernatant off of the pellet to control the amount of pellet loss.

18Detergent in this step of the membrane wash will extract rhodopsin from the membrane pellet and result in a loss of protein. It is important to use a solution without any detergent when washing this membrane pellet.

19The volume of Solubilization Buffer should be proportional to the initial volume of urea-washed ROS membranes used in your ConA purification so that the final concentration of DDM after receptor solubilization is less than 40 mM.

20Be careful to avoid getting air on the column, which can damage the resin and prevent the elution buffer from accessing the air pockets (thus decreasing yield). Attach the syringe to the column using a “wet-connect” technique by adding buffer to an adaptor connected to the top of the column and filling the syringe tube with a small volume of buffer once it is connected to the column before the column plug on the bottom end is removed.

21It is important to equilibrate the ConA resin for at least 2 days in a buffer containing 0.5 mM (or more) DDM before it is used for the first time (SOL-GRADE detergent is acceptable for this). Most chromatography columns contain hydrophobic binding sites that will strip the detergent micelles from protein if they are not filled. This results in aggregation of membrane proteins and large decreases in yield.

22The detergent concentration in the solubilized rhodopsin sample needs to be sufficiently decreased by dilution with ConA Binding Buffer. While the effects of high detergent concentrations on ConA affinity chromatography are unknown, it is possible that the high concentration of DDM may interfere with rhodopsin-ConA interactions.

23The rhodopsin-containing load fraction needs to be looped over the conA column during the loading step because the kinetics of binding are slow.

24ConA-purified rhodopsin samples contain the sugars α-D methylglucoside and sucrose, which can stick to concentrator filters. During concentration, wash the sides of the filter with the concentrated protein sample before refilling with unconcentrated, purified protein to minimize the amount of protein stuck to the filter.

25While the sample is being light-activated with bursts of bright light, remember to close your eyes to preserve night-vision.

26The concentration measured in this experiment is for 4 μL of protein diluted to 100 μL (1:25 dilution). Be sure to account for such dilution factors when calculating the concentration. Multiply the concentration obtained using equation 3 by 25 to obtain the actual rhodopsin concentration in your undiluted sample.

27This photoactivation technique activates only 10% of the total rhodopsin population in your sample.

28Filter all buffers and protein samples before using in the vapor diffusion experiment to remove dust particles.

29The temperature of the reagents should match that of the crystallization system. Drastic changes in temperature often interfere with crystallization.

30Be careful to avoid air bubbles during pipetting. Watching your pipette as solutions are dispensed and stopping before air is dispensed can prevent bubble formation in your protein drop. If you do introduce an air bubble into the crystallization reaction, these can be removed by touching the droplet gently with a clean, dry pipette tip.

31Crystallization can be temperature sensitive. If you do not have a dedicated crystallization incubator, place the crystallization trays into a room with low temperature variation and away from windows, as sunlight can cause dramatic local temperature changes.

32Physical vibrations, such as those from air compressors, can impair crystallization. Store crystallization trays in a room that lacks signification vibrations. Avoid disrupting trays for a least one day after having set them up.

33Because rhodopsin undergoes a conformational change upon activation, dark-adapted rhodopsin crystals must be examined under dim red light, either by using a red filter fitted to a light microscope or with an infrared camera.

34The crystallization condition for obtaining the highest quality crystals can be optimized by slightly varying the components in the condition. This includes testing alternative precipitants, alternative salts, buffer pH, temperature, concentrations of each chemical, protein concentrations, hanging drop size, and the method of crystallization used. For example, in the hanging drop vapor diffusion example given in Figure 14a, the buffer identity and concentration are held constant, but salt and precipitant concentrations are varied. Condition optimization is often performed in 24-well crystallization trays with each well containing a unique crystallization condition. Figure 14b is an example of a optimization screen.

35While the majority of protein crystals exhibit the best diffraction when they are large, the best crystals of rhodopsin were very small and needle-shaped. The diffraction from rhodopsin crystals is inconsistent, with only a small percentage of optically equivalent crystals exhibiting reasonable diffraction.

36If Gt is to be separated into GDP-bound Gαt and Gβγ, concentrate the Gt-containing supernatant to ~7–8 mL. Add MgSO4 to a final concentration of 40 mM and purify the protein sample on a Blue Sepharose gel filtration column (GE Healthcare). Collect the fractions corresponding to Gα and Gβγ separately, and analyze their purity by SDS-PAGE.

37It is preferable to use cells that have been transformed with fresh plasmid DNA (less than 3 months old) immediately before cell expression. Both glycerol stocks of transformed cells and older DNA (even stored at −20 °C) commonly result in lower protein yields for unknown reasons.

38Wrap parafilm around the bacterial plate, to keep moisture and air out, and store at 4°C. Colonies from this plate can be used for protein growths for up to 1 week. After 1 week, yields may be compromised.

39Most cell cultures of Gα proteins expressed in BL-21 E. coli will reach an OD600 of 0.3–0.4 within 2–3 hours post-inoculation when grown at room temperature, however, this is dependent on the Gα mutant and expression system used. Monitor the absorbance carefully, as late induction can affect the yield.

40Induction for longer than 18 hours decreases the yield.

41Cell pellets can be stored at −80°C. Freezing and thawing the pellets has been shown to actually improve the protein yield by contributing to cell lysis.

42When resuspending the resin, gently invert the bottle until all of the resin is in solution. Shaking can cause the resin to break apart into small pieces, called fines, which can clog fritted filters and interfere with affinity chromatography. To remove fines, re-mix the resin in the desired buffer and allow it to settle for ~5 minutes. This is enough time to allow the beads to settle, but the small particles remain in solution. Decant the resin supernatant, add fresh buffer, and re-mix by inverting. Repeat the process until the resin settles within 5 minutes and leaves behind a clear supernatant.

43Commercially available resin is usually stored in ethanol, which will precipitate the protein if it is not completely exchanged prior to use. Perform 3–5 successive washes of the resin slurry using 10 column volumes of Loading Buffer per wash or by continuous flow of Loading Buffer over resin in a gravity flow column to remove the ethanol.

44While G proteins are stable at room temperature, it is beneficial to perform all steps of purification at 4°C.

45If anion exchange is performed at room temperature, be sure to collect protein fractions on ice.

46A slow, low salt buffer gradient followed by a fast, high salt buffer gradient is needed to separate Gα proteins from contaminants by anion exchange chromatography.

47Purified Gα is best resolved on 10% polyacrylamide gels.

48Filter all buffers and protein samples prior to gel filtration chromatography as large particles and aggregates can irreversibly damage both the column and the instrument.. Small volumes of buffer or protein can be spin-filtered on a Spin-X column, while large volumes can be vacuum filtered.

49Gα subunits more stable in Tris-Cl buffers than HEPES buffers. The inclusion of a guanine nucleotide is critical for the stability of the Gα subunit, and GDP is most frequently used.

50For achieving the best resolution, use a sample volume of 500 μL or less for gel filtration purification of proteins on a 24 mL Superdex 200 10/300 GL gel filtration column.

51i purified on a column equilibrated in 50 mM Tris-Cl pH 7.5 buffer containing 200 mM NaCl will elute at 15.5 mL. This is not necessarily the case for Gαi purified in other buffers. If using other buffers, the gel filtration column should be calibrated in that buffer using standards.

52The Gt heterotrimer and Gαi are both best resolved on a 10% polyacrylamide gel. Of the three Gt subunits, Gγ is the smallest (approximately 8 kDa) and is not observed on 10% acrylamide gels.

53AlF4 is highly unstable in solution for extended periods of time, and must be formed by mixing AlCl3 and NaF immediately before intrinsic tryptophan fluorescence is measured.

54In the case of limiting quantities of protein, this assay can also be performed in a 96-well plate using 5 μL of protein and 250 μL of 1X Quick Start Bradford dye.

55Mix the dye thoroughly by inverting the bottle multiple times before use so that all samples develop evenly.

56Use a clean pipette tip for each sample to avoid cross-contamination, as this will affect the fit of a regression line to your data.

57In order to determine the appropriate dilution of your protein of unknown concentration, try loading different dilutions of the purified sample on a gel and analyze by SDS-PAGE. Compare the intensity of each dilution to that of a protein sample of known concentration. You will want to pick a dilution that falls into the range of the protein standard concentrations.

58The Bradford dye is light sensitive. Keep the samples protected from ambient light during the incubation step.

59If substituting buffers, do not use Tris-Cl or any buffer that contains a primary amine, as these will interfere with the cross-linking reaction.

60For the reasons discussed in Note 21, it is important to equilibrate the gel filtration column in buffer containing detergent for a minimum of 48 hours prior to the first use. To save money on detergent, a fixed volume of buffer may be looped over the column.

61The given elution volumes are for rhodopsin-Gt complexes purified on a Superdex 200 10/300 GL gel filtration column equilibrated in Rho-Gt Buffer. This is not necessarily the volume at which the complex will elute if purified in different buffers or different detergents.

62Many membrane proteins will aggregate upon boiling in SDS-PAGE buffer. Room temperature incubation is sufficient.

63To reconstitute Gt using recombinant Gα and GPγ, first mix purified proteins in equimolar amounts and incubate on ice for 10 minutes prior to addition to the sample cuvette containing buffer.

64The Activation Buffer should not contain GDP. The presence of free GDP in solution will inhibit GTPγS binding.

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