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Fluorophore Labeling, Nanodisc Reconstitution and Single-molecule Observation of a G Protein-coupled Receptor
G蛋白偶联受体的荧光标记、磷脂纳米盘重建和单分子观察   

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Abstract

Activation of G protein-coupled receptors (GPCRs) by agonist ligands is mediated by a transition from an inactive to active receptor conformation. We describe a novel single-molecule assay that monitors activation-linked conformational transitions in individual GPCR molecules in real-time. The receptor is site-specifically labeled with a Cy3 fluorescence probe at the end of trans-membrane helix 6 and reconstituted in phospholipid nanodiscs tethered to a microscope slide. Individual receptor molecules are then monitored over time by single-molecule total internal reflection fluorescence microscopy, revealing spontaneous transitions between inactive and active-like conformations. The assay provides information on the equilibrium distribution of inactive and active receptor conformations and the rate constants for conformational exchange. The experiments can be performed in the absence of ligands, revealing the spontaneous conformational transitions responsible for basal signaling activity, or in the presence of agonist or inverse agonist ligands, revealing how the ligands alter the dynamics of the receptor to either stimulate or repress signaling activity. The resulting mechanistic information is useful for the design of improved GPCR-targeting drugs. The single-molecule assay is described in the context of the β2 adrenergic receptor, but can be extended to a variety of GPCRs.

Keywords: G-protein coupled receptors(G蛋白偶联受体), β2 adrenergic receptor(β2肾上腺素能受体), Single-molecule fluorescence(单分子荧光), Phospholipid nanodiscs(磷脂纳米盘), Conformational dynamics(构象动力学)

Background

GPCRs mediate cellular communications, both locally and over long distances, especially in the endocrine system. For instance, the cellular response to hormones such as adrenaline is mediated through adrenergic receptors, of which the β2 adrenergic receptor (β2AR) is a prominent member. β2AR is expressed throughout the human body and is especially important in pulmonary, cardiac and immunological systems. Pharmacologically, agonists targeting β2AR are medically proven to alleviate acute asthma attacks, since activation of β2AR relaxes smooth muscle lining in the respiratory tracts. At the molecular level, β2AR binds extracellular ligands and transmits signals across the cell membrane to intracellular effectors, such as G proteins or β arrestin. A variety of β2AR ligands are known, and these are classified as agonists or inverse agonists, depending on whether they stimulate signaling activity or reduce the activity below the basal level, respectively (Baker, 2010). Crystallographic studies have revealed the three-dimensional structures of β2AR in both inactive (Cherezov et al., 2007) and active (Rasmussen et al., 2011) conformations. However, less is known about how the receptor converts from the inactive to active conformation during signaling and how these transitions are linked to ligand binding.

Observation of single receptor molecules can reveal spontaneous conformational transitions and the influence of ligands on conformational switching, providing a unique perspective on receptor activation (Lamichhane et al., 2015). Development of a single-molecule assay requires labeling the receptor with a fluorescent reporter group at an informative position and a method for reconstituting individual receptor molecules in a membrane-like environment. A Cy3 fluorophore attached to the cytoplasmic end of trans-membrane helix 6 is a suitable reporter of conformational transitions, since the fluorescence quantum yield is sensitive to the local protein environment, a phenomenon referred to as protein-induced fluorescence enhancement (Hwang et al., 2011; Stennett et al., 2015). Moreover, phospholipid nanodiscs provide an ideal system for reconstitution and observation of single receptor molecules (Bayburt and Sligar, 2010). Individual labeled receptors in nanodiscs can be monitored over extended time periods by total internal reflection fluorescence (TIRF) microscopy, directly revealing transitions between inactive and active-like receptor conformations. The concept of the assay is illustrated in Figure 1. Statistical analysis of a collection of receptor molecules provides the rate constants for conformational exchange and the equilibrium distribution of the two conformational states, information that is difficult to obtain otherwise. These analyses can be readily performed in the absence or presence of various β2AR ligands, providing detailed mechanistic information on the linkage of receptor activation to ligand binding. Such information should be useful in the design of improved GPCR-targeting drugs with finely tuned pharmacological efficacies and reduced side effects. Here we describe the protocols to label β2AR with a Cy3 fluorophore, to reconstitute the labeled receptor in nanodiscs, to attach receptor-nanodisc complexes to a microscope slide and the procedures used to record and analyze single-molecule TIRF microscopy data. Although we describe these methods in the context of β2AR, they are equally applicable to a variety of GPCRs.


Figure 1. Experimental system to monitor conformational transitions of β2AR at the single-molecule level (reproduced from Lamichhane et al., 2015). A. An individual receptor molecule (black) labeled with Cy3 (red sphere) incorporated in a phospholipid nanodisc is tethered to a quartz surface coated with polyethylene glycol (wavy lines) via biotin (orange circles) and streptavidin (dark blue rectangles). The labeled receptor is illuminated in the evanescent field of a totally internally reflected 532 nm laser beam (green). The cartoon of the receptor-nanodisc complex is adapted from Bayburt and Sligar (2010). B. Expanded view of a single receptor-nanodisc complex, showing the receptor exchanging between inactive (blue) and active (red) conformations, with corresponding changes in the local environment of the Cy3 probe attached to Cys265 (light or dark red spheres, respectively). The transparent cylinder represents an abstraction of the lipid bilayer.

Materials and Reagents

  1. PD-10 desalting column (GE Healthcare, catalog number: 17085101 )
  2. 2 ml Eppendorf tube
  3. Quartz microscope slides 1” x 3” x 1 mm thick, with a small diameter hole drilled at each end (http://finkenbeiner.com/quartzslides.php)
  4. Microscope cover slips 22 x 40-1 (Fisher Scientific, catalog number: 12-545-C )
  5. Double sided tape (Scotch, 3M)
  6. Pipette tip
  7. 100 kDa MWCO vivaspin 2 concentrators polyethersulfon (PES) membrane (Sartorius, catalog number: VS1041 )
  8. Cy3 maleimide, mono-reactive dye (GE Healthcare, catalog number: PA13131 )
  9. Timolol maleate salt (Sigma-Aldrich, catalog number: T6394 )
  10. cOmpleteTM, Mini, EDTA-free protease inhibitor cocktail (Roche Diagnostics)
  11. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S9888 )
  12. Talon metal affinity resin (Takara Bio, catalog number: 635502 )
  13. Imidazole (Sigma-Aldrich, catalog number: I5513 )
  14. DMSO (Thermo Fisher Scientific, InvitrogenTM, catalog number: D12345 )
  15. SDS-PAGE gels (Invitrogen NuPAGE Bis-Tris Pre-cast gels)
  16. Purified membrane scaffold protein 1 (MSP1), expressed in E. coli, as described (Ritchie et al., 2009)
  17. 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) (Avanti Lipids Polar, catalog number: 850457P )
  18. 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) (Avanti Lipids Polar, catalog number: 840034P )
  19. 16:0 biotinyl Cap PE (Avanti Lipids Polar, catalog number: 870277P )
  20. Bio-beads SM-2 resin (Bio-Rad Laboratories, catalog number: 1523920 )
  21. Ni-NTA resin (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88222 )
  22. Glycerol (Sigma-Aldrich, catalog number: G6279 )
  23. Grease (Borer Chemie, catalog number: glisseal N )
  24. Neutravidin (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 31000 )
  25. 4-(2-hydroyethyl)-1-piperazineethanesulfonic acid (HEPES) (Sigma-Aldrich, catalog number: H3375 )
  26. Magnesium chloride (MgCl2) (anhydrous) (Sigma-Aldrich, catalog number: M8266 )
  27. Potassium chloride (KCl) (Sigma-Aldrich, catalog number: P9541 )
  28. Ethylenediaminetetraacetic acid (EDTA) (0.5 M solution) (Sigma-Aldrich, catalog number: 03690 )
  29. N-dodecyl- β-D-maltopyranoside (DDM) (Anatrace, catalog number: D310 )
  30. Cholesteryl hemisuccinate (CHS) (Sigma-Aldrich, catalog number: C6512 )
  31. ATP (Sigma-Aldrich, catalog number: A26209 )
  32. Phosphate-buffered saline (PBS) (Fisher Scientific, catalog number: 70-011-044 )
  33. Trolox (Acros Organics, catalog number: 218940050 )
  34. Glucose (Sigma-Aldrich, catalog number: 158968 )
  35. Glucose oxidase (Sigma-Aldrich, catalog number: G2133 )
  36. Catalase (Sigma-Aldrich, catalog number: C3155 )
  37. Low salt wash buffer (see Recipe)
  38. High salt wash buffer (see Recipe)
  39. Solubilization buffer (see Recipe)
  40. Wash buffer 1 (see Recipe)
  41. Wash buffer 2 (see Recipe)
  42. Labeling buffer (see Recipe)
  43. SEC elution buffer (see Recipe)
  44. IMAC elution buffer (see Recipe)
  45. Final buffer (see Recipe)
  46. Imaging buffer (see Recipe)

Equipment

  1. 100 ml glass tissue homogenizer (Sigma-Aldrich, catalog number: T2567 )
  2. Beckman Ultra centrifuge
  3. Ti45 rotor (Beckman Coulter, model: Type 45 Ti , catalog number: 339160)
  4. Ti70 rotor (Beckman Coulter, model: Type 70 Ti , catalog number: 337922)
  5. Beckman X-12R centrifuge(Beckman Coulter, model: Allegra® X-12R ) or equivalent
  6. AKTAxpress FPLC system (GE Healthcare, model: AKTAxpress )
  7. Superdex 200 Increase 100/300 size exclusion column (GE Healthcare, catalog number: 17517501 )
  8. Chromatography column packed with 1-2 ml Ni-NTA agarose beads (referred to below as Ni column)
  9. Axiovert 200 microscope (Carl Zeiss, model: Axiovert 200 ) or equivalent
  10. Water-immersion C-Apochromat 63x/1.2 W objective (Carl Zeiss, model: C-Apochromat 63x/1.2 W Corr ) or equivalent
  11. Charge-coupled device (EMCCD) camera (Andor Technology, model: DU-897E iXon+ EMCCD ) or equivalent
  12. 532 nm (green) laser (CrystaLaser, catalog number: CL532-050-S ) or equivalent 

Software

  1. Data acquisition software (available from https://cplc.illinois.edu/software/)
  2. MATLAB scripts
  3. Igor software

Procedure

The protocols described here were developed for a beta 2 adrenergic receptor (β2AR) construct containing a thermo-stabilizing E122W mutation (Roth et al., 2008), C-terminal truncation at residue 348, deletion of residues 245 to 259 in the third intracellular loop (ICL3), a FLAG tag at the N-terminus and a 10x His tag at the C-terminus. The construct also contains C327S and C341S mutations, leaving just a single reactive cysteine residue (Cys265) for fluorophore labeling. The deletions, truncations and cysteine mutations do not disrupt binding of ligands to the receptor (Liu et al., 2012). The procedures to express the receptor in sf9 insect cells infected with high-titer recombinant virus are described elsewhere (Cherezov et al., 2008).

  1. Receptor purification and Cy3 labeling
    1. Lyse and wash frozen β2AR biomass (5 L, roughly 0.5 mg receptor/1 L biomass) using a 100 ml glass tissue homogenizer. Cell lysis is chiefly achieved through the use of hypertonic (low salt) buffer and the mechanical force of homogenization. Wash lysed biomass with the low salt wash buffer and the high salt wash buffer 3 times, respectively (500 ml per wash). Use Beckman Ultra centrifuge to pellet membrane fragment suspension (158,000 x g, Ti45 rotor, 35 min, 4 °C) in between washes. The washed membranes can be stored at -80 °C for up to two weeks or 4 °C overnight. Ideally, the washed membrane should be aliquoted, so that each aliquot of membranes contains roughly 0.5-1 mg of final purified receptor.
    2. Treat one aliquot of washed membrane with 1 mM timolol (dissolved in ddH2O), 1 crushed Roche protease inhibitor tablet for 30 min at 4 °C. This step can be done by adding a previously prepared 100 mM timolol stock (stored at 4 °C) and crushed Roche inhibitor directly to the suspended membrane. Then, thoroughly mix the treated membrane with the solubilization buffer at 1:1 ratio for 3 h at 4 °C.
    3. Remove membrane pellet from supernatant using Beckman Ultra centrifuge (rotor Ti70) at 265,000 x g, 35 min, 4 °C. Add stock 5 M NaCl, 4 M imidazole to the supernatant, so that the final concentration is 800 mM NaCl, 20 mM imidazole. Add 0.5 ml washed talon slurry to the supernatant and incubate at 4 °C overnight.
    4. Pellet talon resin using Beckman X-12R centrifuge (or equivalent) at 500 x g, 1 min, 4 °C and remove supernatant. Wash with 50 ml wash buffer 1, 10 ml wash buffer 2, 10 ml labeling buffer. Add 5 ml labeling buffer on top of the pelleted resin, and 20 μl of 5 mg/ml (in DMSO) Cy3 stock (approximately 25 μM final concentration, whereas the receptor concentration is approximately 1-5 μM). Incubate at 4 °C for 2 h in the dark.
    5. Load talon resin onto an empty column and wash with 50 ml wash buffer 2 using gravity flow. Elute purified, labeled receptor using 1 ml fractions of elution buffer. Run SDS-PAGE to visualize the elution pattern. Concentrate Cy3-labeled receptors using a 100 kDa MWCO vivaspin2 concentrator.

  2. Nanodisc preparation
    1. Mix labeled receptor, purified MSP1 and phospholipid mixture at a molar ratio 1:10:700 in a 2 ml Eppendorf tube. The phospholipid mixture contains POPC, POPS and biotinyl Cap PE in the ratio 67.5%, 27.5%, and 5%. Add Bio-Bead resin and incubate overnight at 4 °C.
    2. Remove Bio-Bead resin by pelleting. Purify nanodiscs by size exclusion chromatography using an AKTAxpress FPLC system, superdex 200 Increase 100/300 GL column and SEC elution buffer. Representative elution profiles of membrane protein-nanodisc complexes are presented elsewhere (Ritchie et al., 2009). Remove empty nanodiscs by immobilized metal affinity chromatography (IMAC) using an AKTAxpress FPLC system, Ni column and IMAC elution buffer. Exchange buffer to the final buffer using a PD-10 desalting column (GE Healthcare).
      Note: Ni-NTA and talon (Co2+) resins are both used to purify the receptor protein with poly-histidine tag, but differ in that 1) Ni2+ has greater affinity for histidine, conferring a larger binding capacity per ml of resin, whereas 2) Co2+ has greater selectivity toward histidine. As such talon resins are preferable to be used on crude lysate, as described in step A3 above, whereas Ni-NTA is preferable for the final purification step described here.

  3. Surface immobilization of receptor-nanodisc complexes
    1. Clean quartz slides and cover slips, passivate the surfaces with polyethylene glycol (5% biotinylated), as described previously (Lamichhane et al., 2010).
    2. Prepare a microfluidic channel about 4-5 mm wide on the quartz slide using double sided tape, fix cover slip on top of the double sided tape and close the two ends of channel using silicon grease at the time of experiment (Figure 2). This device is subsequently referred to as the sample chamber.


      Figure 2. Illustration of the microfluidic sample chamber. Buffers or sample solutions are introduced through the entrance port via a pipette tip, as shown. Any excess solution flows out of the exit port, as shown.

    3. Fill the sample chamber with 0.2 mg/ml neutravidin and incubate for five minutes followed by final buffer wash.
    4. Fill the sample chamber with 100 μl of receptor-nanodisc complexes (approximately 1 nM concentration), incubate for 10 min to allow complexes (containing 5% biotinylated lipids) to bind to neutravidin on the surface, then flush the chamber with imaging buffer.

  4. Single-molecule fluorescence data acquisition
    1. A microscope capable of total internal reflection fluorescence (TIRF) measurements, equipped with a 532 nm (green) laser for excitation (prism-based or objective-based illumination) and an intensified charge coupled device (CCD) camera capable of detecting the fluorescence emission from single fluorophores (Berezhna et al., 2012) is used to record a TIRF image of the surface at regular time intervals (e.g., every 100 msec or shorter, depending on the frame time of the CCD camera) for a total period of at least 100 sec. Many discrete fluorescent spots should be visible in the image, each representing individual receptor-nanodisc complexes (Figure 3A).
    2. Add a ligand, such as the agonist formoterol or inverse agonist ICI 118,551 (1 mM), to the sample chamber, allow ligand to interact with receptor (30 min to 1 h) and repeat the data acquisition. Alternatively, prepare a new sample chamber containing a pre-incubated mixture of receptor-nanodisc complexes and ligand.
    3. As a control to test for non-specific binding, add receptor-nanodisc complexes to a surface lacking neutravidin. Very few fluorescent spots should be visible on the surface (Figure 3B).


      Figure 3. TIRF images of immobilized receptor-nanodisc complexes and representative time trace for a single receptor-nanodisc complex (reproduced from Lamichhane et al., 2015). A. Typical TIRF image showing a 5 x 5 μm region (approximate dimensions) of immobilized Cy3-labeled β2AR reconstituted in nanodiscs. Each spot is due to the fluorescence emitted from an individual receptor-nanodisc complex. The very bright spots are due to a small fraction of receptor-nanodisc aggregates, which are readily identified and excluded from the data analysis. B. Corresponding TIRF image for a control surface lacking neutravidin. The number of fluorescent spots is greatly reduced relative to part A, indicating a negligible level of non-specific adsorption of receptor-nanodisc complexes. C. Representative fluorescence intensity versus time trace from a single nanodisc-bound receptor, showing repeated two-state intensity jumps prior to an irreversible photobleaching event. The red line is the best fit from Hidden Markov modeling. The single-step photobleaching transition confirms that a single receptor molecule is contained within the nanodisc.

Data analysis

  1. Use data acquisition software (available from https://cplc.illinois.edu/software/) to acquire the emission intensity of each identified spot on the surface at each time point.
  2. Filter the data to reject spots that: (a) show very high emission intensity, indicative of receptor-nanodisc aggregates, or (b) show photoblinking transitions (emission intensity transiently samples a zero-intensity state). For subsequent analysis, select time traces that show a single-step photobleaching transition, indicative of a single receptor molecule (Figure 3B).
  3. For each selected time trace, establish the background intensity level after the single photobleaching transition. Correct the intensity at all preceding time points accordingly. Steps 2 and 3 (Data analysis) can be automated using suitable MATLAB scripts.
  4. The majority of selected time traces should show reversible transitions between two distinct intensity levels (Figure 3C). Using at least 100 traces, construct binned intensity histograms representing the intensity states sampled in the entire collection (using Igor software). The histograms typically reveal two distinct peaks. Fit the histograms with two Gaussian functions to identify the centers and relative areas of each peak (using Igor or Origin software).
  5. Repeat this analysis for the data obtained in the presence of agonist or inverse agonist ligands. The intensity peak representing the active state of the receptor will be enhanced in the presence of agonist, while the peak representing the inactive state will be enhanced in the presence of inverse agonist, thereby establishing the identities of the two peaks. The peak areas report the equilibrium distribution of inactive and active conformational states of the receptor.
  6. Fit each selected intensity trace using Hidden Markov analysis (McKinney et al., 2006) to determine the dwell times spent in each state prior to transition to the other (example of fit shown in Figure 3C). Construct histograms of the dwell times for each state from the entire collection of complexes. Fit the histograms with single-exponential decay functions to determine rate constants for the activation or deactivation transitions (using Igor software). If the single-exponential fits are poor, based on the reduced chi-square value, refit the histograms with bi-exponential functions (Lamichhane et al., 2015).

Recipes

  1. Low salt wash buffer
    10 mM HEPES, pH = 7.5, 20 °C
    10 mM MgCl2
    20 mM KCl
    2 mM EDTA
  2. High salt wash buffer
    10 mM HEPES, pH = 7.5, 20 °C
    10 mM MgCl2
    20 mM KCl
    1 M NaCl
  3. Solubilization buffer
    100 mM HEPES, pH = 7.5, 20 °C
    300 mM NaCl
    1% (w/v) DDM, 0.2% CHS (w/v)
  4. Wash buffer 1
    50 mM HEPES, pH = 7.5, 20 °C
    800 mM NaCl
    10 mM MgCl2
    20 mM imidazole
    0.05% (w/v) DDM, 0.01% (w/v) CHS
    8 mM ATP
  5. Wash buffer 2
    50 mM HEPES, pH = 7.5, 20 °C
    150 mM NaCl
    20 mM imidazole
    0.05% (w/v) DDM, 0.01% (w/v) CHS
  6. Labeling buffer
    50 mM HEPES, pH = 7.5, 20 °C
    150 mM NaCl
    0.05% (w/v) DDM, 0.01% (w/v) CHS
    GPCR ligand
  7. SEC elution buffer
    50 mM PBS, pH = 7.4, 20 °C
  8. IMAC elution buffer
    50 mM HEPES, pH = 7.5, 20 °C
    150 mM NaCl
    200 mM imidazole
    0.05% (w/v) DDM, 0.01% (w/v) CHS
  9. Final buffer
    50 mM HEPES, pH = 7.5, 20 °C
    150 mM NaCl
  10. Imaging buffer (final buffer plus oxygen scavenging system)
    50 mM HEPES, pH = 7.5, 20 °C
    150 mM NaCl
    2 mM trolox
    2-5% (w/v) glucose
    50 µg/ml glucose oxidase
    10 µg/ml catalase

Acknowledgments

This work was supported by the Roadmap Initiative grant P50 GM073197 from the National Institutes of Health. The protocol described here is adapted from the work of Lamichhane et al. (2015).

References

  1. Baker, J. G. (2010). The selectivity of beta-adrenoceptor agonists at human β1-, β2- and β3-adrenoceptors. Br J Pharmacol 160(5): 1048-1061.
  2. Bayburt, T. H. and Sligar, S. G. (2010). Membrane protein assembly into Nanodiscs. FEBS Lett 584(9): 1721-1727.
  3. Berezhna, S. Y., Gill, J. P., Lamichhane, R. and Millar, D. P. (2012). Single-molecule Forster resonance energy transfer reveals an innate fidelity checkpoint in DNA polymerase I. J Am Chem Soc 134(27): 11261-11268.
  4. Cherezov, V., Liu, J., Griffith, M., Hanson, M. A. and Stevens, R. C. (2008). LCP-FRAP assay for pre-screening membrane proteins for in meso crystallization. Cryst Growth Des 8(12): 4307-4315.
  5. Cherezov, V., Rosenbaum, D. M., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., Choi, H. J., Kuhn, P., Weis, W. I., Kobilka, B. K. and Stevens, R. C. (2007). High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 318(5854): 1258-1265.
  6. Hwang, H., Kim, H. and Myong, S. (2011). Protein induced fluorescence enhancement as a single molecule assay with short distance sensitivity. Proc Natl Acad Sci U S A 108(18): 7414-7418.
  7. Lamichhane, R., Liu, J. J., Pljevaljcic, G., White, K. L., van der Schans, E., Katritch, V., Stevens, R. C., Wuthrich, K. and Millar, D. P. (2015). Single-molecule view of basal activity and activation mechanisms of the G protein-coupled receptor β2AR. Proc Natl Acad Sci U S A 112(46): 14254-14259.
  8. Lamichhane, R., Solem, A., Black, W. and Rueda, D. (2010). Single-molecule FRET of protein-nucleic acid and protein-protein complexes: surface passivation and immobilization. Methods 52(2): 192-200.
  9. Liu, J. J., Horst, R., Katritch, V., Stevens, R. C. and Wuthrich, K. (2012). Biased signaling pathways in β2-adrenergic receptor characterized by 19F-NMR. Science 335(6072): 1106-1110.
  10. McKinney, S. A., Joo, C. and Ha, T. (2006). Analysis of single-molecule FRET trajectories using hidden Markov modeling. Biophys J 91(5): 1941-1951.
  11. Rasmussen, S. G., Choi, H. J., Fung, J. J., Pardon, E., Casarosa, P., Chae, P. S., Devree, B. T., Rosenbaum, D. M., Thian, F. S., Kobilka, T. S., Schnapp, A., Konetzki, I., Sunahara, R. K., Gellman, S. H., Pautsch, A., Steyaert, J., Weis, W. I. and Kobilka, B. K. (2011). Structure of a nanobody-stabilized active state of the β2 adrenoceptor. Nature 469(7329): 175-180.
  12. Ritchie, T. K., Grinkova, Y. V., Bayburt, T. H., Denisov, I. G., Zolnerciks, J. K., Atkins, W. M. and Sligar, S. G. (2009). Chapter 11 - Reconstitution of membrane proteins in phospholipid bilayer nanodiscs. Methods Enzymol 464: 211-231.
  13. Roth, C. B., Hanson, M. A. and Stevens, R. C. (2008). Stabilization of the human β2-adrenergic receptor TM4-TM3-TM5 helix interface by mutagenesis of Glu122(3.41), a critical residue in GPCR structure. J Mol Biol 376(5): 1305-1319.
  14. Stennett, E. M., Ciuba, M. A., Lin, S. and Levitus, M. (2015). Demystifying PIFE: The photophysics behind the protein-induced fluorescence enhancement phenomenon in Cy3. J Phys Chem Lett 6(10): 1819-1823.

简介

通过激动剂配体激活G蛋白偶联受体(GPCR)是通过从无活性受体构象向活性受体构象的转变来介导的。我们描述了一种新颖的单分子测定法,可以实时监测单个GPCR分子中的激活连锁构象转换。受体在跨膜螺旋6末端用Cy3荧光探针进行位点特异性标记,并在连接到显微镜载玻片的磷脂纳米圆盘中重构。然后通过单分子全内反射荧光显微镜随时间监测单个受体分子,显示无活性和活性样构象之间的自发转变。该测定提供关于无活性和活性受体构象的平衡分布以及构象交换的速率常数的信息。实验可以在不存在配体的情况下进行,显示负责基础信号传导活动的自发构象过渡,或者存在激动剂或反向激动剂配体,揭示配体如何改变受体的动力学刺激或抑制信号传导活性。所得到的机械信息对于改进的GPCR靶向药物的设计是有用的。单分子测定法在β2肾上腺素能受体的背景下进行了描述,但可扩展到多种GPCRs。
【背景】GPCR介导本地和远距离的细胞通讯,特别是内分泌系统。例如,细胞对激素如肾上腺素的反应是通过肾上腺素能受体介导的,其中β2肾上腺素能受体(β2AR)是突出的成员。 β2AR在整个人体中表达,在肺,心脏和免疫系统中尤其重要。药理学上,靶向β2AR的激动剂在医学上被证明可缓解急性哮喘发作,因为β2AR的活化可以缓解呼吸道平滑肌的内衬。在分子水平上,β2AR结合细胞外配体,并将信号跨越细胞膜传递给胞内效应物,如G蛋白或β抑制蛋白。各种β2AR配体是已知的,并且它们被分类为激动剂或反向激动剂,这取决于它们是分别刺激信号活动还是降低活性低于基础水平(Baker,2010)。晶体学研究揭示了β2AR在无活性(Cherezov等,2007)和活性(Rasmussen等,2011)构象中的三维结构。然而,关于受体在信号传导过程中如何从无活性转变为活性构象,以及这些转变如何与配体结合相关的知之甚少。
观察单受体分子可以显示自发构象转变和配体对构象切换的影响,为受体激活提供独特的视角(Lamichhane等,2015)。单分子测定的开发需要在信息位置用荧光报告基团标记受体,以及在膜样环境中重建各个受体分子的方法。连接到跨膜螺旋6的细胞质末端的Cy3荧光团是构象转换的合适报告物,因为荧光量子产率对局部蛋白质环境敏感,这被称为蛋白质诱导的荧光增强现象(Hwang et al。 ,2011; Stennett等人,2015)。此外,磷脂纳米圆盘提供了重建和观察单一受体分子的理想系统(Bayburt和Sligar,2010)。通过全内反射荧光(TIRF)显微镜可以在延长的时间内监测纳米圆盘中的单个标记受体,直接显示无活性和活性样受体构象之间的转换。测定的概念如图1所示。受体分子集合的统计分析提供了构象交换的速率常数和两种构象状态的平衡分布,否则难以获得的信息。这些分析可以在不存在或存在各种β2AR配体的情况下容易地进行,提供关于受体活化与配体结合的连接的详细的机理信息。这些信息应该在设计改进的GPCR靶向药物时具有微调的药理作用和减少的副作用。这里我们描述用Cy3荧光团标记β2AR的方案,重建纳米圆盘中的标记受体,将受体 - 纳米晶体复合物附着到显微镜载玻片上,以及用于记录和分析单分子TIRF显微镜数据的程序。虽然我们在β2AR的上下文中描述这些方法,但它们同样适用于各种GPCR。

关键字:G蛋白偶联受体, β2肾上腺素能受体, 单分子荧光, 磷脂纳米盘, 构象动力学

材料和试剂

  1. PD-10脱盐柱(GE Healthcare,目录号:17085101)
  2. 2 ml Eppendorf管
  3. 石英显微镜滑动1"x 3"x 1 mm厚,每端都钻有一个小直径的孔( http://finkenbeiner.com/quartzslides.php
  4. 显微镜盖板22 x 40-1(Fisher Scientific,目录号:12-545-C)
  5. 双面胶带(苏格兰,3M)
  6. 移液器尖端
  7. 100 kDa MWCO vivaspin 2浓缩器聚醚砜(PES)膜(Sartorius,目录号:VS1041)
  8. Cy3马来酰亚胺,单活性染料(GE Healthcare,目录号:PA13131)
  9. 噻吗洛尔马来酸盐(Sigma-Aldrich,目录号:T6394)
  10. cOmplete TM ,Mini,EDTA-free蛋白酶抑制剂混合物(Roche Diagnostics)
  11. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S9888)
  12. Talon金属亲和树脂(Takara Bio,目录号:635502)
  13. 咪唑(Sigma-Aldrich,目录号:I5513)
  14. DMSO(Thermo Fisher Scientific,Invitrogen TM,目录号:D12345)
  15. SDS-PAGE凝胶(Invitrogen NuPAGE Bis-Tris预制凝胶)
  16. 纯化的膜支架蛋白1(MSP1),表达于E。 (Ritchie等人,2009)
  17. 1-棕榈酰-2-油酰-sn-甘油基-3-磷酸胆碱(POPC)(Avanti Lipids Polar,目录号:850457P)
  18. 1-棕榈酰-2-油酰-sn-甘油基-3-磷酸-L-丝氨酸(POPS)(Avanti Lipids Polar,目录号:840034P)
  19. 16:0生物素基帽PE(Avanti Lipids Polar,目录号:870277P)
  20. 生物珠SM-2树脂(Bio-Rad Laboratories,目录号:1523920)
  21. Ni-NTA树脂(Thermo Fisher Scientific,Thermo Scientific TM,目录号:88222)
  22. 甘油(Sigma-Aldrich,目录号:G6279)
  23. 油脂(Borer Chemie,目录号:glisseal N)
  24. 中性粒细胞(Thermo Fisher Scientific,Thermo Scientific TM ,目录号:31000)
  25. 4-(2-羟乙基)-1-哌嗪乙磺酸(HEPES)(Sigma-Aldrich,目录号:H3375)
  26. 氯化镁(MgCl 2)(无水)(Sigma-Aldrich,目录号:M8266)
  27. 氯化钾(KCl)(Sigma-Aldrich,目录号:P9541)
  28. 乙二胺四乙酸(EDTA)(0.5M溶液)(Sigma-Aldrich,目录号:03690)
  29. N-十二烷基-β-D-麦芽糖苷(DDM)(Anatrace,目录号:D310)
  30. 胆固醇半琥珀酸盐(CHS)(Sigma-Aldrich,目录号:C6512)
  31. ATP(Sigma-Aldrich,目录号:A26209)
  32. 磷酸盐缓冲盐水(PBS)(Fisher Scientific,目录号:70-011-044)
  33. Trolox(Acros Organics,目录号:218940050)
  34. 葡萄糖(Sigma-Aldrich,目录号:158968)
  35. 葡萄糖氧化酶(Sigma-Aldrich,目录号:G2133)
  36. 过氧化氢酶(Sigma-Aldrich,目录号:C3155)
  37. 低盐洗涤缓冲液(见配方)
  38. 高盐洗涤缓冲液(见配方)
  39. 溶解缓冲液(见配方)
  40. 洗涤缓冲液1(参见食谱)
  41. 洗涤缓冲液2(参见食谱)
  42. 标签缓冲液(见配方)
  43. SEC洗脱缓冲液(见配方)
  44. IMAC洗脱缓冲液(见配方)
  45. 最终缓冲(见配方)
  46. 成像缓冲区(请参阅配方)

设备

  1. 100毫升玻璃组织匀浆器(Sigma-Aldrich,目录号:T2567)
  2. 贝克曼超离心机
  3. Ti45转子(Beckman Coulter,型号:45 Ti,目录号:339160)
  4. Ti70转子(Beckman Coulter,型号:70 Ti,目录号:337922)
  5. Beckman X-12R离心机(Beckman Coulter,型号:Allegra X-12R)或等效物
  6. AKTAxpress FPLC系统(GE Healthcare,型号:AKTAxpress)
  7. Superdex 200增加100/300尺寸排除列(GE Healthcare,目录号:17517501)
  8. 色谱柱填充1-2ml Ni-NTA琼脂糖珠(以下称为Ni柱)
  9. Axiovert 200显微镜(Carl Zeiss,型号:Axiovert 200)或等效物
  10. 水浸式C-Apochromat 63x/1.2W物镜(Carl Zeiss,型号:C-Apochromat 63x/1.2W Corr)或等效物
  11. 电荷耦合器件(EMCCD)摄像头(Andor Technology,型号:DU-897E iXon + EMCCD)或等效物
  12. 532nm(绿色)激光(CrystaLaser,目录号:CL532-050-S)或等效物

软件

  1. 数据采集软件(可从 https://cplc.illinois.edu/software/
  2. MATLAB脚本
  3. Igor软件

程序

本文描述的方案是针对含有热稳定性E122W突变的β2肾上腺素能受体(β2亚型AR)构建体开发的(Roth等,2008),C-残基348处的末端截短,第三细胞内环(ICL3)中残基245至259的缺失,N-末端的FLAG标签和C末端的10x His标签。该构建体还含有C327S和C341S突变,仅留下单个反应性半胱氨酸残基(Cys265)用于荧光标记。缺失,截短和半胱氨酸突变不会破坏配体与受体的结合(Liu et al。,2012)。在高滴度重组病毒感染的sf9昆虫细胞中表达受体的程序在其他地方描述(Cherezov等人,2008)。

  1. 受体纯化和Cy3标记
    1. 使用100ml玻璃组织匀浆器洗涤冷冻的β2亚型AR生物量(5L,约0.5mg受体/1L生物质)。细胞裂解主要通过使用高渗(低盐)缓冲液和均质化的机械力来实现。用低盐洗涤缓冲液和高盐洗涤缓冲液分别洗涤溶解的生物质3次(每次洗涤500ml)。使用Beckman Ultra离心机将膜片段悬浮液(158,000 x g,Ti45转子,35分钟,4℃)沉淀在洗涤液中。洗涤的膜可以在-80℃下储存长达两周或4℃过夜。理想情况下,洗涤的膜应该被等分,使得每个等分试样的膜含有大约0.5-1mg的最终纯化的受体。
    2. 用1mM噻吗洛尔(溶于ddH 2 O),1份破碎的Roche蛋白酶抑制剂片剂在4℃下处理一份洗涤的膜。该步骤可以通过加入预先制备的100mM噻吗洛尔原液(4℃储存)并将Roche抑制剂直接压碎至悬浮膜来完成。然后,将处理过的膜与增溶缓冲液以4:1的比例以1:1的比例充分混合3小时
    3. 使用Beckman Ultra离心机(转子Ti70),以265,000 x g,35分钟,4℃从上清液中除去膜沉淀。向上清液中加入5M NaCl,4M咪唑,使最终浓度为800mM NaCl,20mM咪唑。加入0.5毫升洗涤的塔隆浆液至上清液,并在4℃下孵育过夜。
    4. 使用Beckman X-12R离心机(或等同物),500×g,1分钟,4℃的粒状爪骨树脂,并除去上清液。用50ml洗涤缓冲液1,10ml洗涤缓冲液2,10ml标记缓冲液洗涤。在沉淀树脂的顶部加入5ml标记缓冲液,加入20μl5mg/ml(在DMSO中)Cy3原液(终浓度约为25μM,而受体浓度约为1-5μM)。在黑暗中4℃孵育2小时。
    5. 将塔隆树脂加载到空的柱子上,用50ml洗涤缓冲液2用重力流洗涤。用1ml洗脱缓冲液洗脱纯化的标记受体。运行SDS-PAGE以显示洗脱模式。使用100 kDa的MWCO vivaspin2浓缩器浓缩Cy3标记的受体
  2. Nanodisc准备
    1. 将混合标记的受体,纯化的MSP1和磷脂混合物以1:10:700的摩尔比混合在2ml Eppendorf管中。磷脂混合物含有POPC,POPS和生物素类Cap PE,比例分别为67.5%,27.5%,5%。加入生物珠树脂,并在4℃下孵育过夜。
    2. 通过造粒去除生物珠树脂。通过使用AKTAxpress FPLC系统的尺寸排阻色谱法纯化纳米圆盘,superdex 200增加100/300 GL柱和SEC洗脱缓冲液。膜蛋白 - 纳米复合物的代表性洗脱曲线在其他地方(Ritchie等人,2009)提出。通过使用AKTAxpress FPLC系统,Ni柱和IMAC洗脱缓冲液的固定化金属亲和层析(IMAC)去除空的纳米圆盘。使用PD-10脱盐柱(GE Healthcare)向最终缓冲液交换缓冲液。
      注意:Ni-NTA和talon(Co 2 + )树脂都用于用聚组氨酸标签纯化受体蛋白,但不同之处在于1)Ni 2 + 对组氨酸具有更大的亲和力,赋予每ml树脂更大的结合能力,而2) Co 2 + 对组氨酸具有更大的选择性。因为如上述步骤A3中所述,优选将这种爪子树脂用于粗裂解物,而Ni-NTA优选用于本文所述的最终纯化步骤。

  3. 受体 - 纳米复合体的表面固定
    1. 如前所述(Lamichhane等人,2010),清洁石英滑块和盖玻片,用聚乙二醇(5%生物素化)钝化表面。
    2. 使用双面胶带在石英玻片上制备约4-5毫米宽的微流体通道,将盖子滑动到双面胶带的顶部,并在实验时使用硅油封闭通道的两端(图2)。该装置随后称为样品室。


      图2.微流体样品室的图示。如图所示,缓冲液或样品溶液通过进样口通过移液管吸头引入。任何多余的溶液流出出口,如图所示
    3. 向样品室中加入0.2mg/ml中性抗生物素蛋白,孵育5分钟,最后缓冲液洗涤
    4. 向样品室填充100μl受体 - 纳米圆盘复合物(约1 nM浓度),孵育10 min,使复合物(含有5%生物素化脂质)与表面上的中性抗生物素蛋白结合,然后用成像缓冲液冲洗腔室。

  4. 单分子荧光数据采集
    1. 具有全内反射荧光(TIRF)测量的显微镜,其具有用于激发的532nm(绿色)激光(基于棱镜或基于物体的照明))和能够检测荧光发射的强化电荷耦合器件(CCD)照相机来自单荧光团(Berezhna等人,2012)用于以规则的时间间隔(例如,每100毫秒或更短)记录表面的TIRF图像,依赖于在CCD摄像机的帧时间上)总共至少100秒。许多离散的荧光斑点在图像中应该是可见的,每一个都代表着单独的受体 - 纳米圆盘复合体(图3A)
    2. 向样品室中加入配体如激动剂福莫特罗或反向激动剂ICI 118,551(1 mM),使配体与受体(30分钟至1小时)相互作用并重复数据采集。或者,准备一个含有受体 - 纳米复合物和配体的预孵育混合物的新样品室
    3. 作为测试非特异性结合的对照,将受体 - 纳米复合物添加到缺乏中性抗体的表面。在表面上应该看得很少的荧光斑点(图3B)。


      图3.固定的受体 - 纳米圆盘复合物的TIRF图像和单个受体 - 纳米圆盘复合物的代表性时间曲线(从Lamichhane等人,2015年转载)。显示在纳米圆盘中重构的固定化的Cy3标记的β2亚型的5×5μm区域(近似尺寸)的典型TIRF图像。每个点都是由于单个受体 - 纳米圆盘复合体发出的荧光。非常亮点是由于受体 - 纳米尺度聚集体的一小部分,容易被识别并排除在数据分析之外。 B.对照表面缺乏嗜中性粒细胞的相应TIRF图像。相对于部分A,荧光斑点的数量大大降低,表明受体 - 纳米复合物的非特异性吸附水平可以忽略不计。 C.代表荧光强度对来自单个纳米盘结合受体的时间曲线,显示在不可逆的漂白事件之前的重复的两态强度跳跃。红线是隐马尔可夫模型中最适合的。单步光漂白转移证实纳米圆盘中含有单一受体分子

数据分析

  1. 使用数据采集软件(可从 https://cplc.illinois.edu/software/获取) ),以获取每个时间点上每个识别点的发射强度。
  2. 过滤数据以排除:(a)显示非常高的发射强度,指示受体 - 纳米体聚集体,或(b)显示光致连接跃迁(发射强度瞬时采样零强度状态)。对于后续分析,选择显示单步光漂白转换的时间曲线,指示单个受体分子(图3B)。
  3. 对于每个选定的时间轨迹,在单次漂白过渡后建立背景强度水平。相应地纠正所有以前时间点的强度。步骤2和3(数据分析)可以使用合适的MATLAB脚本进行自动化。
  4. 大多数选定的时间轨迹应显示两个不同强度水平之间的可逆转换(图3C)。使用至少100条迹线,构建表示在整个集合中采样的强度状态的二进制强度直方图(使用Igor软件)。直方图通常显示两个不同的峰。使用两个高斯函数拟合直方图,以确定每个峰值的中心和相对面积(使用Igor或Origin软件)。
  5. 对激动剂或反向激动剂配体存在下获得的数据重复此分析。在激动剂的存在下,代表受体活性状态的强度峰将被增强,而在反向激动剂的存在下,代表无活性状态的峰将被增强,从而建立两个峰的身份。峰值区域报告受体的无活性和活性构象状态的平衡分布
  6. 使用隐马尔可夫分析(McKinney等人,2006)来适应每个所选择的强度轨迹,以确定在转换到另一状态之前在每个状态中花费的停留时间(图3C所示的拟合示例)。从整个复合体集合构建每个状态的停留时间的直方图。使用单指数衰减函数拟合直方图以确定激活或停用转换的速率常数(使用Igor软件)。如果单指数拟合差,则基于减少的卡方值,使用双指数函数重新设计直方图(Lamichhane等人,2015)。

食谱

  1. 低盐洗涤缓冲液
    10mM HEPES,pH = 7.5,20℃ 10mM MgCl 2
    20 mM KCl
    2 mM EDTA
  2. 高盐洗涤缓冲液
    10mM HEPES,pH = 7.5,20℃ 10mM MgCl 2
    20 mM KCl
    1 M NaCl
  3. 溶解缓冲液
    100mM HEPES,pH = 7.5,20℃ 300 mM NaCl
    1%(w/v)DDM,0.2%CHS(w/v)
  4. 洗涤缓冲液1
    50mM HEPES,pH = 7.5,20℃ 800 mM NaCl
    10mM MgCl 2
    20mM咪唑
    0.05%(w/v)DDM,0.01%(w/v)CHS
    8 mM ATP
  5. 洗涤缓冲液2
    50mM HEPES,pH = 7.5,20℃ 150 mM NaCl
    20mM咪唑
    0.05%(w/v)DDM,0.01%(w/v)CHS
  6. 标签缓冲区
    50mM HEPES,pH = 7.5,20℃ 150 mM NaCl
    0.05%(w/v)DDM,0.01%(w/v)CHS
    GPCR配体
  7. SEC洗脱缓冲液
    50mM PBS,pH = 7.4,20℃
  8. IMAC洗脱缓冲液
    50mM HEPES,pH = 7.5,20℃ 150 mM NaCl
    200毫克咪唑
    0.05%(w/v)DDM,0.01%(w/v)CHS
  9. 最终缓冲区
    50mM HEPES,pH = 7.5,20℃ 150 mM NaCl
  10. 成像缓冲液(最终缓冲液加除氧清除系统)
    50mM HEPES,pH = 7.5,20℃ 150 mM NaCl
    2 mM trolox
    2-5%(w/v)葡萄糖
    50μg/ml葡萄糖氧化酶
    10μg/ml过氧化氢酶

致谢

这项工作得到了美国国立卫生研究院路线图倡议拨款P50 GM073197的支持。这里描述的协议改编自Lamichhane等人的工作。 (2015年)。

参考

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  2. Bayburt,TH和Sligar,SG(2010)。 Membrane蛋白质组装到纳米圆盘中。 FEBS Lett 584(9):1721-1727。
  3. Berezhna,SY,Gill,JP,Lamichhane,R.和Millar,DP(2012)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/22650319"target ="_ blank">单分子福斯特共振能量转移揭示了DNA聚合酶I中的先天保真检查点。 J. Am Chem Soc 134(27):11261-11268。 br />
  4. Cherezov,V.,Liu,J.,Griffith,M.,Hanson,MA和Stevens,RC(2008)。< a class ="ke-insertfile"href ="http://www.ncbi.nlm。 nih.gov/pubmed/19234616"target ="_ blank"> LCP-FRAP测定用于筛选内膜结晶中的膜蛋白。 Cryst Growth Des 8(12):4307- 4315.
  5. Cherezov,V.,Rosenbaum,DM,Hanson,MA,Rasmussen,SG,Thian,FS,Kobilka,TS,Choi,HJ,Kuhn,P.,Weis,WI,Kobilka,BK and Stevens,RC(2007) ; 工程化人类β的高分辨率晶体结构2 - 肾上腺素能G蛋白偶联受体。 科学 318(5854):1258-1265。
  6. Hwang,H.,Kim,H。和Myong,S。(2011)。蛋白质诱导的荧光增强作为具有短距离灵敏度的单分子测定。 Proc Natl Acad Sci USA 108(18):7414-7418。
  7. Lamichhane,R.,Liu,JJ,Pljevaljcic,G.,White,KL,van der Schans,E.,Katritch,V.,Stevens,RC,Wuthrich,K。和Millar,DP(2015)。 class ="ke-insertfile"href ="http://www.ncbi.nlm.nih.gov/pubmed/26578769"target ="_ blank"> G蛋白偶联的基础活性和活化机制的单分子观点受体β2亚型.Rep Natl Acad Sci USA 112(46):14254-14259。
  8. Lamichhane,R.,Solem,A.,Black,W.和Rueda,D。(2010)。蛋白质核酸和蛋白质 - 蛋白质复合物的单分子FRET:表面钝化和固定。方法 52(2):192-
  9. Liu,JJ,Horst,R.,Katritch,V.,Stevens,RC和Wuthrich,K。(2012)。以19F-NMR为特征的β2亚肾上腺素能受体中的偏倚信号通路科学 335( 6072):1106-1110。
  10. McKinney,SA,Joo,C. and Ha,T。(2006)。  使用隐马尔科夫模型分析单分子FRET轨迹。 Biophys J 91(5):1941-1951。
  11. Rasmussen,SG,Choi,HJ,Fung,JJ,Pardon,E.,Casarosa,P.,Chae,PS,Devree,BT,Rosenbaum,DM,Thian,FS,Kobilka,TS,Schnapp,A.,Konetzki,I 。,Sunahara,RK,Gellman,SH,Pautsch,A.,Steyaert,J.,Weis,WI和Kobilka,BK(2011)。< a class ="ke-insertfile"href ="http: .ncbi.nlm.nih.gov/pubmed/21228869"target ="_ blank">β2肾上腺素受体的纳米体稳定的活性状态的结构。大自然 469(7329):175-180。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Lamichhane, R., Liu, J. J., Pauszek III, R. F. and Millar, D. P. (2017). Fluorophore Labeling, Nanodisc Reconstitution and Single-molecule Observation of a G Protein-coupled Receptor. Bio-protocol 7(12): e2332. DOI: 10.21769/BioProtoc.2332.
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