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May 2020

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FRET-based Microscopy Assay to Measure Activity of Membrane Amino Acid Transporters with Single-transporter Resolution
用荧光共振能量转移的显微技术测定单转运体膜氨基酸转运体的活性   

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Abstract

Secondary active transporters reside in cell membranes transporting polar solutes like amino acids against steep concentration gradients, using electrochemical gradients of ions as energy sources. Commonly, ensemble-based measurements of radiolabeled substrate uptakes or transport currents inform on kinetic parameters of transporters. Here we describe a fluorescence-based functional assay for glutamate and aspartate transporters that provides single-transporter, single-transport cycle resolution using an archaeal elevator-type sodium and aspartate symporter GltPh as a model system. We prepare proteo-liposomes containing reconstituted purified GltPh transporters and an encapsulated periplasmic glutamate/aspartate-binding protein, PEB1a, labeled with donor and acceptor fluorophores. We then surface-immobilize the proteo-liposomes and measure transport-dependent Fluorescence Resonance Energy Transfer (FRET) efficiency changes over time using single-molecule Total Internal Reflection Fluorescence (TIRF) microscopy. The assay provides a 10-100 fold increase in temporal resolution compared to radioligand uptake assays. It also allows kinetic characterization of different transport cycle steps and discerns kinetic heterogeneities within the transporter population.

Keywords: Single-molecule FRET (单分子荧光共振能量转移), Glutamate transporter (谷氨酸转运蛋白), Transport assay (转运试验), TIRF microscopy (全内反射荧光显微镜), Amino-acid sensor (氨基酸的传感器)

Background

Membrane resident secondary active transporters or solute carriers (SLC) mediate cellular uptake of amino acids, hormones, neurotransmitters, vitamins, and drugs, among other solutes. They couple concentrative substrate uptake to energetically favorable dissipation of electrochemical gradients of ions maintained primarily through the work of Na+/K+ ATPases (Lingrel and Kuntzweiler, 1994). The structural mechanisms of many secondary active transporters have come to light over the last decades (Grewer and Rauen, 2005; Shi, 2013; Vandenberg and Ryan, 2013; Drew and Boudker, 2016).


Measuring radioligand uptake into cells, membrane vesicles, or proteoliposomes is a traditional means to assay transport (Nimigean, 2006; Geertsma et al., 2008; Volpe, 2016). The cells and membrane vesicles provide the most native environment. By contrast, proteoliposomes allow precise control over compositions of membranes and luminal solutions. Broadly, purified transporters are reconstituted into liposomes with internal and external buffers providing the necessary ionic gradients. Gradient-dependent accumulation of the radiolabeled substrates in proteoliposomes is then measured. Specifically, a radiolabeled substrate is added to the external buffer, aliquots are taken at different time points and filtered. Finally, radioactivity trapped in the filters within the vesicles is measured. Initial rates of transport are measured as functions of substrate concentrations, lipid compositions, or temperature. These ensemble-based measurements provide mean uptake rates averaged over the population of transporters and over time. A shortcoming of the method relies on estimating what fraction of the transporters is reconstituted in an active form. Thus, accurate turnover times may be challenging to determine. Furthermore, the manual pipetting of aliquots limits the temporal resolution to 5 s or more, as reported in studies with various channels and transporters, including primary and secondary solute transporters and potassium channels (Knol et al., 1996; Heginbotham et al., 1998; Jung et al., 1998; van der Heide and Poolman, 2000; Enkvetchakul et al., 2004; Borths et al., 2005; Yamashita et al., 2005; Boudker et al., 2007).


We describe a single-molecule Fluorescence Resonance Energy Transfer (smFRET)-based uptake assay of liposome-reconstituted purified transporters (Figure 1). The current embodiment of this assay provides a temporal resolution of approximately 50 ms, a 100-fold increase compared to traditional radioligand uptake assays. The number and orientation of transporters in vesicles can be controlled. The assay detects the time it takes for each transporter to transport the first substrate molecule (during this time, the protein binds, translocates and releases the substrate into the vesicle lumen) and allows estimation of the mean turnover time of each transporter. A single measurement follows the activity of ca. 100-500 individual transporters simultaneously. The need for radioactive materials, which might be difficult and expensive to procure and are harmful to researchers and the environment, is eliminated.


We recently used this assay to study archaeal sodium and aspartate symporter, GltPh (Ciftci et al., 2020). We engineered a glutamate/aspartate FRET sensor based on a periplasmic glutamate/aspartate binding protein PEB1a by introducing two cysteine mutations for labeling with donor and acceptor fluorophores (ccPEB1a). The FRET efficiency of the labeled ccPEB1a increases as it binds an amino acid. We further mutated ccPEB1a to tune its aspartate affinity to allow tracking single- or up to 5-30 rounds of transport. GltPh containing a single cysteine mutation on the transporter's extracellular side was biotinylated and reconstituted into liposomes at 1:1,000 protein to lipid ratio (PLR) to enrich the population of proteoliposomes containing one transporter (Figure 1A). ccPEB1a was encapsulated by freeze-thaw cycles, and proteoliposomes were extruded through 0.1 µm filters, immobilized in smFRET imaging chambers, equipped with a rapid perfusion system, and imaged using TIRF microscopy (Figure 1B). The first half-cycle times for each transporter are determined manually from the onset of the FRET efficiency increase in a single-molecule trajectory following the substrate injection into the imaging chamber. The consequent continuous increase in FRET efficiency is fitted to a time-dependent aspartate binding equation (Ciftci et al., 2020) to obtain mean transporter turnover times (Figure 1C).


The assay was inspired by and, in part, based on a single-molecule assay developed for a neutral amino acid transporter MhsT utilizing periplasmic leucine, isoleucine, valine-binding protein LIV-BP (Fitzgerald et al., 2019). We expect analogous single-molecule transport assays to be developed for various other transporters, taking advantage of the natural diversity of periplasmic binding proteins (Berntsson et al., 2010) or other types of sensor proteins (Deuschle et al., 2005; Hou et al., 2011; Chen et al., 2013; Liu et al., 2015).



Figure 1. Single-molecule transport assay. A. Sample preparation flowchart. Biotinylated transporters are reconstituted into liposomes, the FRET sensor encapsulated, and proteoliposomes are immobilized in imaging chambers. B. Proteoliposomes are first imaged in a Resting buffer (K+), then a Non-activating buffer (K+/L-Asp) to test leakage, followed by an Activating buffer (Na+/L-Asp, blue circles) to initiate transport. Orange and purple arrows indicate the first half-cycle and consecutive turnovers, respectively. C. A representative single-molecule FRET efficiency trajectory.

Materials and Reagents

  1. 50 ml Syringe (BD, catalog number: 309654)

  2. Amicon Ultra-15 Centrifugal filters 100K (EMD Millipore, catalog number: UFC910096)

  3. Amicon Ultra-4 Centrifugal filters 100K (EMD Millipore, catalog number: UFC810096)

  4. Amicon Ultra-15 Centrifugal filters 10K (EMD Millipore, catalog number: UFC901024)

  5. Amicon Ultra-4 Centrifugal filters 10K (EMD Millipore, catalog number: UFC801024)

  6. Whatman Nucleopore Track-Etched membrane, 0.4 µm, 19 mm (Sigma-Aldrich, catalog number: WHA800282)

  7. Whatman Nucleopore Track-Etched membrane, 0.1 µm, 19 mm (Sigma-Aldrich, catalog number: WHA800309)

  8. Tube revolver/rotator for 1.5 ml reaction tubes

  9. General Long-Term Storage Cryogenic Tubes (NalgeneTM, Thermo Fisher Scientific, catalog number: 5000-0020)

  10. 50 ml tubes (Fisher Scientific, catalog number: 1443222)

  11. Glycerol (Fisher Scientific, catalog number: G334)

  12. Liquid N2

  13. Chloroform (Fisher Scientific, catalog number: 507517453)

  14. Triton X-100 (Sigma-Aldrich, catalog number: T9284)

  15. LB broth, miller (Fisher Bioreagents, catalog number: BP1426-2)

  16. Ampicillin, sodium salt (GoldBio, catalog number: A-301-100)

  17. Ni-NTA superflow (QIAGEN, catalog number: 30430)

  18. Thiol reactive dyes (LD555, LD655) (Lumidyne Technologies, catalog numbers: LD555-MAL, LD655-MAL)

  19. Dimethyl sulfoxide (DMSO) (Fisher Bioreagents, catalog number: BP231-100)

  20. Aspartate-glutamate binding protein, PEB1a (UniProt ID: M9QLL2. Mutations for site-specific labeling: C18S/N73C/K149C; mutations to tune affinity: A64F, D102L, Y198F, R89L)

  21. Escherichia coli (E. coli) Polar Lipid Extract (Avanti Polar Lipids, catalog number: 100600C-100mg)

  22. Egg phosphatidylcholine (PC) (Avanti Polar Lipids, catalog number: 840051C-25mg)

  23. Bio-beads SM2 absorbent media (Bio-Rad, catalog number: 152-3920)

  24. Protocatechuic acid (PCA) (Sigma-Aldrich, catalog number: 37580-25G-F)

  25. Protocatechuate-3,4-dioxygenase (PCD) (Sigma-Aldrich, catalog number: P8279)

  26. L-arabinose (GoldBio, catalog number: A-300-1)

  27. HEPES (Fisher Bioreagents, catalog number: BP310-100)

  28. Sodium chloride (NaCl) (Fisher Scientific, catalog number: S271-3)

  29. Potassium chloride (KCl) (EMD Millipore, catalog number: PX1405-1)

  30. Calcium chloride dihydrate (Sigma-Aldrich, catalog number: 223506)

  31. Tris (2-carboxyethyl) phosphine hydrochloride, 98% (TCEP) (Alfa Aesar, catalog number: 40587-09)

  32. Ethylenediaminetetraacetic acid disodium salt, dihydrate 100% (EDTA) (Alfa Aesar, catalog number: C1201100-500A5)

  33. Lysozyme, egg white (GoldBio, catalog number: L-040-10)

  34. Phenylmethylsulfonyl fluoride (PMSF) (MP Biomedicals, catalog number: 195381)

  35. Sucrose, ultrapure, RNase Free (MP Biomedicals, catalog number: 04821713)

  36. L-Aspartic acid, monopotassium salt (Research Products International, catalog number: 1115-63-5)

  37. n-Dodecyl-β-D-maltoside (DDM) (Anagrade, Anatrace, catalog number: D310 25 GM)

  38. Thrombin from bovine plasma (Sigma-Aldrich, catalog number: T4648)

  39. β-D-1-thiogalactopyranoside (IPTG) (GoldBio, catalog number: I2481C100)

  40. Bovine serum albumin (BSA) (Fraction V, OmniPur, catalog number: 2930)

  41. Streptavidin (Invitrogen, catalog number: S888)

  42. Thermo ScientificTM EZ-LinkTM Maleimide-PEG11-Biotin (Fisher Scientific, catalog number: PI21911)

  43. N-ethyl maleimide (Sigma-Aldrich, catalog number: E3876)

  44. QIAprep spin miniprep kit (50×) (QIAGEN, catalog number: 27104)

  45. T7 express lysY competent E. coli (High efficiency) (New England Biolabs, catalog number: C3010I)

  46. Thermo ScientificTM FastDigest DpnI (Fisher Scientific, catalog number: FERFD1704)

  47. PfuUltra II Hotstart PCR master mix (Agilent Technologies, catalog number: 600852)

  48. LB agar plates, ampicillin-100 (Teknova, catalog number: L1004)

  49. Pet21(+) vector (Genscript)

  50. Resuspension buffer (see Recipes)

  51. Wash buffer (see Recipes)

  52. Elution buffer (see Recipes)

  53. Size Exclusion Chromatography (SEC) buffer (see Recipes)

  54. cysGltPh SEC buffer (see Recipes)

  55. Resting buffer (see Recipes)

  56. Non-activating buffer (see Recipes)

  57. Activating buffer (see Recipes)

  58. T50 buffer (see Recipes)

  59. cysGltPh Resuspension buffer (see Recipes)

Equipment

  1. Spectrophotometer (Horiba Photon Technology PTI, Quantamaster 8000)

  2. NanoDrop 2000c (Fisher Scientific, catalog number: ND-2000C)

  3. 2,800 ml Fernbach-style culture flasks with baffles (Fisher Scientific, catalog number: 09-552-39)

  4. Glass beaker

  5. Magnetic stir bars

  6. Superdex 200 Increase 10/300 GL column (GE Healthcare, catalog number: 28-9909-44)

  7. AKTA pure chromatography system (GE Lifesciences)

  8. Aldrich® single-neck round-bottom flask, 100 ml capacity, Joint: ST/NS 24/40 (Sigma-Aldrich, catalog number: Z414492)

  9. BUCHI RotavaporTM R-300 Rotary Evaporator with Controller and V-300 Pump

  10. Dry-seal vacuum desiccator (Fisher Scientific, catalog number: 086427)

  11. Avanti Mini Extruder kit (Avanti Polar Lipids, catalog number: 610000)

  12. Filter Support (Avanti Polar Lipids, catalog number: 610014)

  13. MAXQ 8000 Incubated/Refrigerated Stackable Shaker (Thermo Fisher Scientific)

  14. Drummond Portable Pipet-Aid (Thomas Scientific, catalog number: 13-661-17E)

  15. Pipetman Kit, P1000, P200, P20, P10 (Gilson, catalog number: F167370)

  16. Avanti JXN-26 Centrifuge (Beckman Coulter)

  17. Avanti JXN-30 Centrifuge (Beckman Coulter)

  18. Optima MAX-XP Tabletop Ultracentrifuge (Beckman Coulter)

  19. Optima L-100XP Ultracentrifuge (Beckman Coulter)

  20. J-LITE JLA-8.1000 Fixed-Angle Aluminum Rotor, 6 × 1,000 ml, 8,000 rpm, 15,970 × g (Beckman Coulter, catalog number: 363688)

  21. JA-20 Fixed-Angle Aluminum Rotor, 8 × 50 ml, 20,000 rpm, 48,400 × g (Beckman Coulter, catalog number: 334831)

  22. Type 45 Ti Fixed-Angle Titanium Rotor (Beckman Coulter, catalog number: 339160)

  23. TLA-100.3 Fixed-Angle Rotor (Beckman Coulter, catalog number: 349481)

  24. 1 L (1,000 ml) Polycarbonate Bottle with Cap Assembly, 95 × 191 mm (Beckman Coulter, catalog number: A98812)

  25. 70 ml, Polycarbonate Bottle Assembly, 38 × 102 mm (Beckman Coulter, catalog number: 355622)

  26. 26.3 ml, Polycarbonate Bottle with Cap Assembly, 25 × 89 mm (Beckman Coulter, catalog number: 355618)

  27. 50 ml, Polycarbonate Bottle with Cap Assembly, 29 × 104 mm (Beckman Coulter, catalog number: 357000)

  28. Avestin EmulsiFlex C3 cell disruptor (Avestin)

  29. Dounce tissue grinder set

  30. XK 16/20 column (Cytivalifesciences, catalog number: 28988937)

  31. SpectrumTM Spectra/PorTM 4 RC Dialysis Membrane Tubing 12,000 to 14,000 Dalton MWCO (Fisher Scientific, catalog number: 08-667D)

  32. SpectrumTM Dialysis Tubing Closures: Spectra/PorTM Standard Type (Fisher Scientific, catalog number: 08-670-11A)

Software

  1. SPARTAN (Scott Blanchard laboratory, https://www.scottcblanchardlab.com/software)

  2. MATLAB (MathWorks, https://www.mathworks.com/products/matlab.html)

  3. Prism (GraphPad Software, Inc., https://www.graphpad.com/)

  4. Origin (OriginLab Corporation, Northampton, MA, USA, https://www.originlab.com/)

Procedure

  1. ccPEB1a sensor construction

    1. Clone PEB1a with C terminal 6x His-tag into pet21a (+) vector.

    2. Mutagenize PEB1a to remove native cysteine and introduce two cysteines for site-specific labeling with fluorophores (C18S, N73C, K149C), yielding ccPEB1a construct.

      C18S:

      Forward primer: GGGTGCGAGCGTTGCGTTTAG

      Reverse primer: CGCAACGCTCGCACCCAGCGC

      N73C:

      Forward primer: CTGGACTGTGGCAGCGTGGATGCGGTTATC

      Reverse primer: GATAACCGCATCCACGCTGCCACAGTCCAG

      K149C:

      Forward primer: CGGCATTGATGTGTGCTTCAGCGAATTTCC

      Reverse primer: GGAAATTCGCTGAAGCACACATCAATGCCG

    3. Introduce additional mutations at or near the aspartate/glutamate-binding site to tune affinity. ccPEB1a binds aspartate with a dissociation constant of ~79 nM. ccPEB1a Y198F variant has 5.6 µM affinity allowing visualization of up to 5 transport cycles in 100 nm liposomes.

    4. Mix 100 ng of DNA with the above mutations and 50 µl of T7 Express lysY competent E. coli cells in a 1.5 ml reaction tube and incubate on ice for 30 min.

    5. Heat shock at 42 °C for 45 s and immediately place the tube on ice for 5 min and add 750 µl of autoclaved LB.

    6. Incubate for 1 h at 37 °C with shaking at 250 rpm and plate on agar plates supplemented with 100 µg/ml ampicillin.

    7. Grow overnight for ~16 h at 37 °C with shaking at 250 rpm and prepare a glycerol stock by mixing 500 µl culture with 500 µl of autoclaved 50% glycerol, store at -80 °C.


  2. ccPEB1a purification

    1. Prepare 4 L of LB according to manufacturer’s protocol in two 2,800 ml Fernbach-style culture flasks with baffles and separately 300 ml in 1 L flask. Autoclave and let cool to 37 °C or room temperature.

    2. Supplement all LB with 100 µg/ml ampicillin before use.

    3. Inoculate 300 ml LB with bacterial glycerol stock of ccPEB1a and grow overnight for ~16 h at 37 °C with shaking at 250 rpm.

    4. Inoculate LB with the overnight culture from Step B3 to a final absorbance of 0.1 at 600 nm. Dilute 200 µl of cell culture 5 times with LB for absorbance measurements.

    5. Induce cells with 1 mM freshly prepared IPTG when the absorbance reaches 0.8.

    6. Let cultures grow for 3 h at 37 °C and 16 h at 18 °C with shaking at 250 rpm.

    7. Harvest cells by centrifuging for 15 min at 3,326 × g at 4 °C in Avanti JXN-26 centrifuge using JLA-8.1000 fixed-angle rotor. Use 10 ml of Resuspension buffer (see Recipe 1) per 2 L bacterial cell cultures to resuspend the pellets.

    8. Add 100 µg/ml lysozyme.

    9. Incubate on ice for 30 min and dilute 1:3 (v:v) with deionized water.

    10. Centrifuge for 30 min at 3,578 × g in Avanti JXN-26 centrifuge using JA-20 fixed-angle rotor at 4 °C to pellet the debris. The supernatant still has some particulate matter and is very viscous. A second identical centrifugation step can further clarify the supernatant, prevent clogging the column, and extend the life of the resin.

    11. Mix supernatant with 3-5 ml of Ni-NTA resin washed with 3 × 5 column volumes (CV) of water and 1 × 5 CV of wash buffer (see Recipe 2) in a beaker and stir gently on a stirrer plate for 1 h at 4 °C using a magnetic stir bar.

    12. Assemble the XK20/16 column as described in the manufacturer’s instructions and slowly pour the resin slurry in, pulling the flow-through with a syringe.

    13. Wash with 5 CV of Wash buffer by pulling the flow-through with a syringe.

    14. Elute proteins using 3 CV Elution buffer (see Recipe 3) by gravity.

    15. Concentrate the sample in an Amicon Ultra-15 Centrifugal filter with a 10K molecular weight cutoff until the volume reaches < 500 µl. Determine the protein concentration by measuring absorbance at 280 nm in a spectrophotometer. Calculate protein’s extinction coefficient and molecular weight using the online Expasy Protparam tool from Expasy Bioinformatics Resource Portal.

    16. Centrifuge the protein solution at 49,192 × g at 4 °C for 15 min in Optima MAX-XP Tabletop Ultracentrifuge using TLA-100.3 fixed-angle rotor. Aspirate the protein solution using a 1 ml pipette without disturbing the pellet of protein aggregates.

    17. Polish the protein by Size Exclusion Chromatography (SEC) using Superdex 200 Increase 10/300 GL column, following pre-equilibration of Superdex 200 Increase 10/300 GL column in 50 ml of SEC buffer (see Recipe 4). Set the flow rate and pressure limit of the column according to the manufacturer’s instructions. A flow rate of 0.3 ml/min and 2 MPa pressure limit are ideal for this column. Monitor protein elution by following absorbance at 280 nm and collect 0.4 ml fractions.

    18. Collect the peak fractions and immediately proceed with the labeling procedure. The protein should come out as a monomer (Figure 2).



      Figure 2. SEC profile of monomeric PEB1a eluting from the Superdex 200 Increase 10/300 GL column


  3. ccPEB1a labeling

    1. Resuspend 1,000 nmol dried LD555 or LD655 in 100 µl DMSO to a 10 mM final concentration. Vortex until the dye chunks disappear. Aliquot in 10 µl aliquots and store at -20 °C.

    2. Thaw aliquots of LD555 and LD655 dyes and mix with freshly SEC-purified ccPEB1a protein at 1:1:1 (mol:mol:mol) ratio. The molar ratio can be empirically optimized depending on the labeling efficiency. Note that there are no reducing agents in the SEC buffer because they can reduce the labeling efficiency. Thus, solvent-exposed cysteine residues may oxidize over time and it is critical to mix the protein solution with maleimide reagents immediately after elution from SEC.

    3. Wrap the Eppendorf tube in aluminum foil and incubate on a rotator for 45 min-1 h.

    4. Purify labeled monomeric ccPEB1a from the unreacted dye by SEC, following pre-equilibration of Superdex 200 Increase 10/300 GL as described in Step B17 of the ‘ccPEB1a purification’ section. Use the same flow rate and pressure limits as in Step B17.

    5. Concentrate labeled protein to ~5 µM using Amicon Ultra-4 Centrifugal filter with 10K molecular weight cutoff.

    6. If the ccPEB1a protein variant binds substrate with dissociation constant < 5 µM, dialyze the protein against 4,000-fold SEC buffer in a 4 L bucket using dialysis membrane tubing with a 12-14 kDa molecular weight cutoff to remove bound L-aspartate. Incubate 15 cm tubing in distilled water for 30 min. Rinse the tubing with distilled water and close one end with a dialysis tubing clip. After making sure the clip is tight and there are no leaks, load the protein solution. Fold the excess tubing over itself, leaving a 2 cm space on top of the protein solution, and close it with a clip. Invert the assembly 1-2 times to ensure no leaks and place it into a beaker filled with SEC buffer. Replace the buffer three times every 4 h.

    7. Measure the protein sample absorption at 280 nm, 555 nm (LD555), and 655 nm (LD655). Use the extinction coefficients of the protein and the dyes: 150,000 cm-1 M-1 (LD555) and 250,000 cm-1 M-1 (LD655) to calculate their respective concentrations and determine labeling efficiency.

    8. Aliquot labeled protein in 20 µl aliquots and flash freeze in liquid N2. Store in liquid N2 until use.


  4. Preparation of the liposome stocks

    1. Mix 4 ml (100 mg) of E. coli Polar Lipid Extract and 1.33 ml (33 mg) of Egg PC (stored at -80 °C in chloroform) at a final 3:1 (w:w) ratio in a 100 ml round bottom flask. Use glass pipettes to transfer chloroform solutions. Dry the mixture using a rotary evaporator, rotating the flask for ~30 min under vacuum; lipids will form a thin film on the flask’s walls.

    2. Cover the flask opening loosely with foil and leave in a dry-seal vacuum desiccator connected to vacuum overnight.

    3. To hydrate the lipid film, add 33.25 ml of Resting buffer (see Recipe 6) to the round bottom flask to achieve a final concentration of 4 mg/ml lipids. To facilitate the lipid film hydration process, perform freeze/thaw cycles as follows: freeze the lipid suspension by immersing the flask into the liquid nitrogen. You can prepare a liquid nitrogen bath in a thermal flask or an ice bucket. Use a thermal glove to hold the flask by its neck or use a vapor tube extender as you lower it into the liquid nitrogen. The lipid suspension will make a sound akin to an egg on a frying pan as it freezes. Once the noise subsides in 1-2 min, the suspension is fully frozen. Transfer the flask into a water bath at room temperature to thaw the suspension. Video 1 showcases the setup we use. Repeat freeze/thaw cycles 10 times until the lipid film on the flask sides disappears, and the suspension is turbid white color.


      Video 1. Preparation of liposomes stocks for protein reconstitution

    4. Aliquot liposomes in 1 ml aliquots, flash-freeze in liquid N2, and store at -80 °C until further use.


  5. Reconstitution of sodium/aspartate symporter, GltPh, into liposomes and encapsulation of ccPEB1a

    1. Purify N278C/C321A GltPh single cysteine mutant (cysGltPh), as previously described (Akyuz et al., 2013), with a few modifications below.

    2. Repeat Steps B1-B4 with 12 L of LB instead of 4 L and inoculate LB with a cysGltPh glycerol stock.

    3. Induce with 20 mM of L-arabinose for 3 h at 37 °C with shaking at 250 rpm.

    4. Lyse the cells in cysGltPh Resuspension buffer (see Recipe 10) using the EmulsiFlex C3 cell disruptor. 4 passes are usually enough to disrupt the majority of the cells.

    5. Centrifuge the cell suspension for 1 h at 100,000 × g and 4 °C in Optima L-100XP Ultracentrifuge using Type 45 Ti Fixed-Angle Titanium Rotor to pellet the membranes.

    6. Homogenize crude cell membranes in Solubilization buffer (see Recipe 11) supplemented with 40 mM n-dodecyl β-D-maltopyranoside (DDM) for 1 h at 4 °C and remove the insoluble material by centrifuging for 1 h at 100,000 × g and 4 °C in Optima L-100XP Ultracentrifuge using Type 45 Ti Fixed-Angle Titanium Rotor.

    7. Mix the supernatant with 3-5 ml of Ni-NTA resin and assemble the column as described in Steps B11-B12.

    8. Wash with 5 CV of Solubilization buffer supplemented with 1 mM DDM and 40 mM imidazole and elute in the presence of 250 mM imidazole.

    9. Incubate for ~16 h at 21 °C with thrombin at 10 units per mg protein to cleave the His-tag.

    10. Purify cleaved cysGltPh by SEC using Superdex 200 Increase 10/300 GL column pre-equilibrated in 50 ml cysGltPh SEC buffer (see Recipe 5).

    11. Thaw aliquots of maleimide-PEG11-biotin and NEM and mix with freshly purified cysGltPh protein at 1:2:4 (mol:mol:mol) protein:maleimide-PEG11-biotin:NEM ratio. Note that there are no reducing agents in the SEC buffer, and cysteine residues may oxidize over time. Thus, it is critical to mix the protein solution with maleimide reagents immediately after elution from SEC.

    12. Purify labeled cysGltPh from the unreacted reagents by SEC, using pre-equilibrated Superdex 200 Increase 10/300 GL column.

    13. Concentrate labeled transporter to 4 mg/ml using Amicon Ultra-4 Centrifugal filter with 100K molecular weight cutoff.

    14. Take out 1 ml liposome aliquot (4 mg/ml) and extrude through 400 nm membranes using a mini-extruder as shown in the video (https://www.sigmaaldrich.com/video/life-science/avanti-mini-extruder.html). 21 passes should yield uniformly sized unilamellar liposomes.

    15. Destabilize liposomes by adding Triton X-100 at 1:2 (w:w) detergent to lipid ratio.

    16. Add cysGltPh to liposomes at a 1:1000 (w:w) PLR and incubate on a rotator at room temperature for 30 min. Adjust PLR to enrich the liposome population with one transporter per vesicle. We detail calculations in Ciftci et al. (2020).

    17. Wash Bio-Beads extensively with 10 CV ethanol and 20 CV water before use in a 50 ml tube. Invert the slurry 2-3 times, let the beads settle at the bottom, and aspirate the supernatant with a pipettor. Add the beads at a 100 mg/ml final concentration to the liposome suspension and incubate on a rotator at room temperature for 2 h. It is critical not to add too many beads to avoid lipid loss, which would reduce the yield of proteoliposomes.

    18. Transfer the liposome solution from the beads using a 1 ml pipettor into a new cryovial. Repeat Steps C17 and C18 for5 more times using fresh beads at 4 °C.

    19. Pellet proteoliposomes by centrifugation at 49,192 × g at 4 °C for 1 h in Optima MAX-XP Tabletop Ultracentrifuge using TLA-100.3 fixed-angle rotor.

    20. Remove supernatant and resuspend proteoliposomes in an equal volume of the Resting buffer, freeze in liquid N2 and thaw in a water bath at room temperature.

    21. Repeat Steps C20 and C21 twice more.

    22. Pellet proteoliposomes as above and resuspend at 25 mg/ml in 160 µl of the Resting buffer.

    23. Add fluorescently labeled ccPEB1a from Step C8 to a final concentration of 0.6 µM and encapsulate by 2 freeze/thaw cycles as above. Two freeze/thaw cycles should result in up to 30 % encapsulation efficiency (Colletier et al., 2002; Chaize et al., 2004; Costa et al., 2014). Increasing the number of the freeze/thaw cycles might lead to improved encapsulation efficiency but might also result in an increased fraction of the denatured ccPEB1a. We found that the current procedure allows us to image up to 500 vesicles in a single sm-FRET recording, sufficient for efficient data collection. Proteoliposomes can be stored at -80 °C at this step until further use.

    24. Pellet proteoliposomes by centrifugation as above to remove unencapsulated PEB1a. Replace the supernatant with an equal volume of the Resting buffer.

    25. Extrude proteoliposomes (21 passages) through 100 nm membranes using a mini-extruder (Avanti Polar Lipids). Proteoliposomes can be stored at 4 °C for up to 5-6 h before use. You can verify the liposome integrity and size distribution by negative-stain or cryo-EM. Representative images are shown in Figure S7 in Ciftci et al. (2020).


  6. Single-molecule experiments

    1. The preparation of quartz microscope slides and glass coverslips functionalized with PEG-biotin for TIRF microscopy is based on published methods (Joo and Ha, 2012).

    2. We performed all single-molecule FRET imaging experiments using a home-built prism-based TIRF microscope constructed around a Nikon Eclipse Ti inverted microscope body, as described previously (Juette et al., 2016). Step-by-step guides on building a prism-based single-molecule TIRF microscopy setup are available (Selvin and Ha, 2008; Axelrod, 2008; Roy et al., 2008).

    3. Passivate the quartz slides using a solution of 1 µM BSA and 1 µM duplex DNA in 150 µl of T50 buffer (see Recipe 9) by flowing the mixture through the injection port and incubating for 5 min. Further details can be found in Blanchard et al. (2004) and Joo and Ha (2012). Wash away excess BSA and DNA by flowing 150 µl of T50 buffer through the injection port.

    4. To image non-specifically attached liposomes, flow no more than 10 µl of the proteoliposome suspension from Step E25. Keep a record of the sample quantity you have used in this step.

    5. Immediately wash the surface with 150 µl of Resting buffer to remove unattached proteoliposomes.

    6. Image attached molecules at 100 mW laser power at 10 Hz temporal resolution for a total of 300 s.

    7. Photobleach non-specifically attached molecules by imaging the entire immobilization surface at 600 mW laser power and wash the chamber with 150 µl of T50 buffer.

    8. Flow 150 µl of pre-mixed 0.8 µM streptavidin and 1 µM duplex DNA solution in T50 buffer and incubate for 5-10 min.

    9. Wash away excess streptavidin-duplex DNA with 150 µl of T50 buffer and 150 µl of the Resting buffer before attaching proteoliposomes.

    10. Flow the same volume of the liposomes as in Step F4. Immediately wash the surface with 150 µl Resting buffer to remove unattached proteoliposomes. If necessary, flow a more concentrated sample or increase the sample volume to reach the appropriate single-molecule density. At optimal density, the fraction of molecules rejected in gettraces module of SPARTAN should be ~40% (see Data analysis and the online SPARTAN manual). If injecting larger sample amounts, re-check non-specific attachment (Steps F4-F7) using a matching sample.

    11. Image attached molecules at 100 mW laser power at 10 Hz temporal resolution for a total of 300 s and determine the number of molecules that specifically and non-specifically attach to the surface (Data analysis). Ideally, the non-specifically attached fluorescent particles imaged in Step F6 should be < 25% of the specifically attached molecules.

    12. Supplement the Non-activating and Activating buffers (see Recipes 7-8) with an oxygen-scavenging system consisting of 2 mM PCA and 50 nM PCD (Aitken et al., 2008).

    13. Image proteoliposomes to test for leakage by flowing 50 µl of the Non-activating buffer approximately 3 s after the start of the movie. Trace amounts of detergent left after Steps E17-E18 can allow L-asp to spontaneously diffuse into liposomes and lead to changes in FRET efficiency of encapsulated ccPEB1a in the absence of the active sodium-driven uptake. If a significant number of single-molecule trajectories (>10%) show FRET efficiency changes at this step, discard the liposomes and increase the amount of bio-beads or the number of bio-beads incubation cycles in your next preparation. Do not exceed 200 mg/ml of bio-beads in each round. If 200 mg/ml of bio-beads is still not enough, increase the number of incubation rounds. Carefully observe the turbidity level of liposome preparations after each round. A significant reduction in turbidity indicates the adsorption of liposomes to the excess bio-beads. We have observed that 6 rounds using 100 mg/ml bio-beads are enough to remove Triton X-100 detergent from our liposome preparations. This number can differ depending on the nature of detergent used to solubilize liposomes and incubation temperature. Detergents with higher critical micelle concentration (CMC) are easier to remove. One can check the remaining detergent in liposome preparations using a colorimetric assay (Urbani and Warne, 2005). We have observed that slight temperature variations (by a temperature-controlled chamber) can lead to significant differences in the detergent removal efficiency.

    14. Image proteoliposomes to measure transport by flowing 50 µl of the Activating buffer approximately 3 s after the start of the movie. Adjust the time resolution and the total duration of movies depending on the transporter’s turnover rate. For cysGltPh variants with turnover times of ~30-100 s, we use the laser power of 20 mW at 2.5 Hz temporal resolution. At this laser power, the mean photobleaching time is ~300 s. For variants with turnover times of ~1 s, we use laser power of 75 mW at 10 Hz, resulting in the mean photobleaching time of ~60 s.

    15. Wash the sample chamber with 2 ml of Resting buffer and photobleach any remaining fluorescent molecules as in Step F7 before attaching new proteoliposomes for the next round of transport recordings. Repeat Steps F10- F15.

Data analysis

  1. Perform smFRET data analysis using SPARTAN, a freely available custom-built software package written in MATLAB (Juette et al., 2016). This provides a detailed explanation of particle detection and generation of fluorescence trajectories implemented in SPARTAN (https://www.scottcblanchardlab.com/software).

  2. Process data files collected in TIFF format using the ‘gettraces’ module to detect single molecules and extract time-dependent fluorescence changes. Correct for crosstalk. This module creates an output file with .rawtraces extension. The crosstalk value can be calculated using the ‘crosstalkcorrect’ function implemented in SPARTAN, which calculates the mean acceptor intensity observed after the acceptor photobleaches across all trajectories in a given dataset, where acceptor photobleached before the donor. This function requires a .rawtraces file and returns the crosstalk value along with the percentage of trajectories used to calculate it.

  3. Process the .rawtraces files from step 2 in ‘autotrace’ module. Apply the default parameters for FRET lifetime, background noise, number of donor blinking events, and signal-to-background ratio described in Supplementary Table 1 in Juette et al. (2016). We also apply a lower threshold of 0.4 and an upper threshold of 0.7 for FRET efficiency of the first frame of each single-molecule trajectory. We chose these thresholds based on the analysis of thousands of ccPEB1a single-molecule trajectories described in the ‘Engineering L-asp sensor’ section and shown in Figure 1B in Ciftci et al. (2020). FRET efficiency values outside of the 0.4-0.7 range likely correspond to denatured ccPEB1a.

  4. Manually inspect the selected trajectories from ‘autotrace’ module using ‘sorttraces’ module. Discard trajectories with multiple donor or acceptor photobleaching steps which indicates the presence of more than one sensor per vesicle or non-specific labeling of the sensor. Discard also trajectories that show a visually apparent lack of anti-correlation between donor and acceptor intensities (Figure 4).

  5. Classify trajectories into responding and nonresponding in ‘sorttraces’ by using the bins designated as ‘No’, ‘All’, or ‘Best’. Save responding and nonresponding trajectories separately. Figure S5 in Supplemental Information in Ciftci et al. (2020), shows examples of responding trajectories (Figure 4).

  6. Manually record the time points at which FRET efficiency starts increasing in each single-molecule trajectory in the first column of an EXCEL file response_times.xlsx such that the order of the trajectories in responding_molecules.traces file matches the order of response time entries in response_times.xlsx. Thus, the response time detected in the 1st trajectory is in the 1st row, the response time detected in the 2nd trajectory is in the 2nd row, etc. Record the shortest response time observed in any trajectory to the first row of the second column; this number is a good approximation for the Activating buffer injection time. Tracking the order is only important if one uses the MATLAB script TurnoverTime.m in step 7.

  7. Fit the gradual increases in FRET efficiencies following the initial step-wise increases to a time-dependent binding equation to obtain mean turnover times. Materials and Methods section ‘Analysis of the smFRET transport trajectories’ in Ciftci et al. (2020) explains the derivation of this equation. Optionally, use a MATLAB script TurnoverTime.m (https://github.com/hdciftci/SingleMoleculeTransportAssay.git) to fit the data. This script is modified based on avgFretTime.m script found in SPARTAN software (https://www.scottcblanchardlab.com/software) and requires two input files; the responding_molecules.traces and response_times.xlsx. Rate_constants.csv outputted by this script will have fitted turnover times along with other optimized parameters used in the equation.

  8. Plot the distributions of the response times after subtracting the Activating buffer injection time, which correspond to the first half-cycle times, and the mean turnover times. We used PRISM (GraphPad Software, Inc.) for all histograms and Origin (OriginLab Corp., Northampton, MA, USA) for population contour plots in Ciftci et al. (2020). Data in Ciftci et al. (2020) are averages of at least 3 independent experiments.

  9. For a more detailed explanation of the analysis approach, please refer to the Materials and Methods ‘Analysis of the smFRET trajectories’ section in Ciftci et al. (2020). A summarized view of steps and example data are shown in Figure 3 and Figure 4, respectively.



    Figure 3. Flowchart of the data analysis



    Figure 4. Example data generated from steps described in ‘Data Analysis’. (Steps 1-2) Fluorescence intensities are corrected for crosstalk, and single molecules are picked using ‘gettraces’ module. (Step 3) Fluorescence intensities of donor and acceptor molecules and the corresponding FRET efficiencies of picked molecules are extracted using ‘autotrace’ module. (Steps 4-5) Fluorescence trajectories are inspected for anti-correlation and response pattern using ‘sorttraces’ module and classified accordingly as responding or non-responding molecules. Trajectories without anti-correlation and/or showing multiple donor or acceptor photobleaching steps are excluded from further analysis. (Step 6) Response times are manually detected and recorded in an excel sheet. (Step 7) Average turnover times are calculated using the ‘Turnover Time’ script.

Recipes

  1. Resuspension buffer

    10 mM Tris-HCl, pH 8.0

    2 mM EDTA

    0.5 M sucrose

  2. Wash buffer

    10 mM Tris-HCl, pH 7.4

    200 mM NaCl

    1 mM Tris(2-carboxyethyl) phosphine (TCEP)

    40 mM imidazole

  3. Elution buffer

    10 mM Tris-HCl, pH 7.4

    200 mM NaCl

    1 mM Tris(2-carboxyethyl) phosphine (TCEP)

    250 mM imidazole

  4. Size Exclusion Chromatography (SEC) buffer

    50 mM HEPES/Tris, pH 7.4

    200 mM KCl

  5. cysGltPh SEC buffer

    20 mM HEPES/Tris, pH 7.4

    200 mM NaCl

    0.1 mM L-aspartic acid

    1 mM DDM

  6. Resting buffer

    50 mM HEPES/Tris, pH 7.4

    200 mM KCl

  7. Non-activating buffer

    50 mM HEPES/Tris pH 7.4

    200 mM KCl

    1 µM L-aspartic acid

  8. Activating buffer

    50 mM HEPES/Tris, pH 7.4

    200 mM NaCl

    1 µM L-aspartic acid

  9. T50 buffer

    10 mM Tris/HCl, pH 7.4

    50 mM KCl

  10. cysGltPh Resuspension buffer

    20 mM HEPES/Tris, pH 7.4

    200 mM NaCl

    1 mM EDTA, pH 7.4

  11. Solubilization buffer

    50 mM HEPES/Tris, pH 7.4

    200 mM NaCl

    1 mM TCEP

    1 mM L-asp

Acknowledgments

We thank Roger Altman for the preparation of smFRET devices. Funding: NINDS R37NS085318 to O.B. and S.C.B., AHA 19PRE34380215 to H.D.C. 7R01GM098859 to S. C. B. This project has received funding from the European Union's Horizon 2020 research and innovation program under the Maire Sklodowska-Curie grant agreement MEMDYN No 660083 (G.H.M.H). This work is originally published as Ciftci et al. (2020).

Competing interests

S. C. B holds equity interest in Lumidyne Technologies.

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简介

[摘要]次级活性转运蛋白驻留在细胞膜中,利用离子的电化学梯度作为能量源,可针对陡峭的浓度梯度转运极性氨基酸(如氨基酸)。通常,基于集合的放射性标记底物摄取或转运电流的测量可确定转运蛋白的动力学参数。在这里,我们描述了一种基于荧光的谷氨酸和天冬氨酸转运蛋白功能测定方法,该方法使用古细菌升降剂型钠和天冬氨酸共转运蛋白Glt Ph作为模型系统,提供了单转运蛋白,单转运周期的分辨率。我们准备包含重组的纯化的Glt Ph转运蛋白和封装的周质谷氨酸/天冬氨酸结合蛋白,PEB1a,用供体和受体荧光团标记的蛋白脂质体。然后,我们将蛋白脂质体表面固定化,并使用单分子全内反射荧光(TIRF)显微镜测量随时间变化的运输依赖性荧光共振能量转移(FRET)效率变化。与放射性配体摄取测定法相比,该测定法在时间分辨率上提高了10-100倍。它还可以对不同转运周期步骤进行动力学表征,并识别转运蛋白种群内的动力学异质性。


[背景]膜驻留的二级主动转运蛋白或溶质载体(SLC)介导氨基酸,激素,神经递质,维生素和药物等溶质的细胞摄取。他们将集中的底物摄取与主要通过Na + / K + ATPases的作用维持的离子电化学梯度的能量上有利的耗散结合在一起(Lingrel and Kuntzweiler ,1994)。在过去的几十年中,许多次要主动转运蛋白的结构机制得到了揭示(Grewer和Rauen,2005;Shi ,2013 ;Vandenberg和Ryan ,2013 ; Drew和Boudker,2016 )。

测量细胞,膜囊泡或蛋白脂质体中放射性配体的摄取是测定转运的传统方法(Nimigean,2006;Geertsma等,2008 ;Volpe ,2016)。细胞和膜囊泡提供了最天然的环境。相比之下,蛋白脂质体可以精确控制膜和腔溶液的组成。广义上讲,纯化的转运蛋白通过内部和外部缓冲液可重构为脂质体,从而提供必要的离子梯度。然后测量放射性标记的底物在脂质体中的梯度依赖性积累。具体而言,将放射性标记的底物添加到外部缓冲液中,在不同的时间点取等分试样并过滤。最后,测量囊泡内过滤器中捕获的放射性。初始转运速率根据底物浓度,脂质组成或温度的函数进行测量。这些基于集合的度量提供了在运输人群中和随时间推移的平均平均摄取率。该方法的缺点在于估计以活性形式重构转运蛋白的哪一部分。因此,准确的周转时间可能难以确定。此外,如使用各种通道和转运蛋白(包括一级和二级溶质转运蛋白和钾离子通道)的研究所报道的那样,手动移取等分试样将时间分辨率限制为5 s或更长(Knol等,1996 ;Heginbotham等,1998 ;Jung等人,1998 ;van der Heide和Poolman ,2000 ;Enkvetchakul等人,2004 ;Borths等人,2005 ;Yamashita等人,2005 ;Boudker等人,2007)。

我们描述了一种单分子荧光共振能量转移(smFRET)系摄取脂质体的纯化重组转运蛋白(图测定URE 1)。与c urrent该测定的实施例提供小号一个时间分辨率的大约50毫秒,100倍的增加相对于传统的放射性配体摄取测定。囊泡中转运蛋白的数量和方向可以控制。所述分析检测所花费的时间为每个TR ansporter到第一衬底分子输送(在这段时间内,该蛋白质所结合,易位和释放衬底进入囊泡腔),并允许的各转运的平均周转时间估计。一次测量遵循ca的活动。可以同时运送100-500个单独的运输车。消除了对放射性材料的需求,因为这种材料的采购可能困难而昂贵,并且对研究人员和环境都有害。

我们最近使用了该测定法来研究古细菌钠和天冬氨酸的同向转运蛋白Glt Ph (Ciftci et al。,2020)。我们通过引入两个半胱氨酸突变体以供体和受体荧光团(ccPEB1a)进行标记,基于周质谷氨酸/天冬氨酸结合蛋白PEB1a设计了谷氨酸/天冬氨酸FRET传感器。标记的ccPEB1a结合氨基酸后,其FRET效率会提高。我们进一步突变了ccPEB1a,以调整其天冬氨酸亲和力,以追踪单次或最多5至30轮转运。GLT博士包含在运输机上的外侧上的单个半胱氨酸突变被生物素化并复原到脂质体中以1:1000蛋白质与脂质比(PLR),以丰富含有一个转运(图脂蛋白体的人口URE 1A)。ccPEB1a通过冷冻-解冻循环包封,和脂蛋白是通过0.1微米的过滤器,固定在smFRET成像室,配备有快速灌注系统中挤出,并使用TIRF显微镜(图成像URE 1B)。从将基质注入成像室后,单分子轨迹中的FRET效率提高开始,手动确定每个转运蛋白的前半个循环时间。在FRET效率随之不断增加装配到时间依赖性天冬氨酸结合方程(Ciftci等人,2020) ,以获得平均转运周转时间(图URE 1C)。

该测定法的启发是并且部分地是基于利用周质亮氨酸,异亮氨酸,缬氨酸结合蛋白LIV-BP为中性氨基酸转运蛋白MhsT开发的单分子测定法(Fitzgerald et al。,2019)。我们期望利用周质结合蛋白(Berntsson等,2010)或其他类型的传感器蛋白(Deuschle等,2005;Hou e )的自然多样性,为其他各种转运蛋白开发类似的单分子转运分析方法。牛逼人,2011 ;陈等人,2013;刘等人,2015年)。


图1.单分子转运测定。一。样品制备流程图。将生物素化的转运蛋白重构为脂质体,将FRET传感器封装,并将蛋白脂质体固定在成像室中。B.首先在静止缓冲液(K + )中成像蛋白脂质体,然后在非激活缓冲液(K + / L-Asp)中成像以测试渗漏,然后在激活缓冲液(Na + / L-Asp,蓝色圆圈)中成像。开始运输。橙色和紫色箭头分别表示前半个周期和连续的营业额。C.代表性的单分子FRET效率轨迹。

关键字:单分子荧光共振能量转移, 谷氨酸转运蛋白, 转运试验, 全内反射荧光显微镜, 氨基酸的传感器



材料和试剂


50 ml注射器(BD,货号:309654)
Amicon Ultra-15离心过滤器100K(EMD Millipore,目录号:UFC910096)
Amicon Ultra-4离心过滤器100K(EMD Millipore,货号:UFC810096)
Amicon Ultra-15离心过滤器10K(EMD Millipore,目录号:UFC901024)
Amicon Ultra-4离心过滤器10K(EMD Millipore,目录号:UFC801024)
Whatman核孔刻蚀膜,0.4 µm,19 mm(Sigma-Aldrich,目录号:WHA800282)
Whatman核孔刻蚀膜,0.1 µm,19毫米(Sigma-Aldrich,目录号:WHA800309)
1.5 ml反应管的离心管/旋转器
通用的长期存储低温管(Nalgene TM ,Thermo Fisher Scientific,目录号:5000-0020)
50毫升试管(Fisher Scientific,目录号:1443222)
甘油(Fisher Scientific,目录号:G334)
液态氮2
氯仿(Fisher Scientific,目录号:507517453)
海卫一X-100 (西格玛奥德里奇,目录号:T9284)
LB肉汤,米勒(Fisher Bioreagents,货号:BP1426-2)
氨苄西林钠盐(GoldBio,目录号:A-301-100)
Ni-NTA超级流量(QIAGEN,目录号:30430)
硫醇活性染料(LD555,LD655)(Lumidyne Technologies,目录号:LD555-MAL,LD655-MAL)
二甲基亚砜(DMSO)(Fisher Bioreagents,货号:BP231-100)
天冬氨酸-谷氨酸结合蛋白,PEB1a(UniProt ID:M9QLL2。位点特异性标记的突变:C18S / N73C / K149C;亲和力突变:A64F,D102L,Y198F,R89L)
大肠埃希氏菌(大肠杆菌)极性脂质提取物(Avanti极性脂质,目录号:100600C-100毫克)
卵磷脂酰胆碱(PC)(Avanti极性脂质,目录号:840051C-25mg)
Bio-beads SM2吸收介质(Bio - Rad,目录号:152-3920)
原儿茶酸(PCA)(Sigma-Aldrich,目录号:37580-25G-F)
原儿茶酸-3,4-二加氧酶(PCD)(Sigma-Aldrich,目录号:P8279)
L-阿拉伯糖(GoldBio,目录号:A-300-1)
HEPES(Fisher生物试剂,目录号:BP310-100)
氯化钠(NaCl)(Fisher Scientific,目录号:S271-3)
氯化钾(KCl)(EMD密理博,目录号:PX1405-1)
二水合氯化钙(Sigma - Aldrich,目录号:223506)
三(2-羧乙基)膦盐酸盐,98%(TCEP)(Alfa Aesar,目录号:40587-09)
乙二胺四乙酸二钠盐,二水合物100%(EDTA)(Alfa Aesar,目录号:C1201100-500A5)
溶菌酶,蛋清(GoldBio,目录号:L-040-10)
苯甲基磺酰氟(PMSF)(MP Biomedicals,目录号:195381)
超纯蔗糖,无RNase(MP Biomedicals,目录号:04821713)
L-天冬氨酸一钾盐(国际研究产品,目录号:1115-63-5)
n-十二烷基-β-D-麦芽糖苷(DDM)(Anagrade,Anatrace,目录号:D310 25 GM)
牛血浆中的凝血酶(Sigma - Aldrich,目录号:T4648)
β-D-1-硫代吡喃半乳糖苷(IPTG)(GoldBio,目录号:I2481C100)
牛血清白蛋白(BSA)(组分V,OmniPur,目录号:2930)
链霉亲和素(Invitrogen,货号:S888)
的Thermo Scientific TM EZ-Link的TM马来酰亚胺PEG11生物素(Fisher Scientific公司,目录号:PI21911)
N-乙基马来酰亚胺(Sigma - Aldrich,目录号:E3876)
QIAprep旋转微量制备试剂盒(50 × )(QIAGEN,目录号:27104)
T7特快lysY主管大肠杆菌(高效率)(新英格兰生物实验室,目录号:C3010I)
Thermo Scientific TM FastDigest DpnI(Fisher Scientific,目录号:FERFD1704)
PfuUltra II Hotstart PCR预混液(Agilent Technologies,目录号:600852)
LB琼脂板,氨苄西林100(Teknova,目录号:L1004)
Pet21(+)vector(Genscript)
重悬缓冲液(请参见配方)
洗涤缓冲液(请参见食谱)
洗脱缓冲液(请参见配方)
体积排阻色谱(SEC)缓冲液(请参见配方)
cysGlt Ph SEC缓冲区(请参见食谱)
休息缓冲液(请参见食谱)
非激活缓冲区(请参见食谱)
激活缓冲区(请参阅食谱)
T50缓冲液(请参阅配方)
cysGlt Ph重悬缓冲液(请参见食谱)


设备


分光光度计(Horiba Photon Technology PTI,Quantamaster 8000)
NanoDrop 2000c(Fisher Scientific,目录号:ND-2000C)
2,800 ml带挡板的芬巴赫式培养瓶(Fisher Scientific,目录号:09-552-39)
玻璃烧杯
磁力搅拌棒
Superdex 200增加10/300 GL色谱柱(GE Healthcare,目录号:28-9909-44)
AKTA纯色谱系统(GE生命科学)
Aldrich公司®单颈圆底烧瓶中,将100ml的容量,联合:ST / NS 24/40(Sigma-Aldrich公司,目录号:Z414492)
BUCHI旋转蒸发仪TM的R-300旋转蒸发仪具有控制器和V-300泵
干式密封真空干燥器(Fisher Scientific,目录号:088427)
Avanti迷你挤出机套件(Avanti极性脂质,目录号:610000)
过滤器支持(Avanti Polar Li pids,目录号:610014)
MAXQ 8000冷藏/冷藏可堆叠摇床(Thermo Fisher Scientific)
德拉蒙德便携式移液器(Thomas Scientific,目录号:13-661-17E)
移液器套件,P1000,P200,P20,P10(Gilson,目录号:F167370)
Avanti JXN-26离心机(贝克曼库尔特)
Avanti JXN-30离心机(贝克曼库尔特)
Optima MAX-XP台式超速离心机(贝克曼库尔特)
Optima L-100XP超速离心机(贝克曼库尔特)
J-LITE JLA-8.1000定角铝制转子,6 × 1,000 ml,8,000 rpm,15,970 ×g (Beckman Coulter,目录号:363688)
JA-20定角铝质转子,8 × 50毫升,20,000 rpm,48,400 ×g (贝克曼库尔特(Beckman Coulter),目录号:334831)
45型Ti固定角度钛转子(Beckman Coulter,目录号:339160)
TLA-100.3定角转子(贝克曼库尔特(Beckman Coulter),目录号349481)
1 L(1 ,000个毫升)聚碳酸酯瓶盖组装,95 × 191毫米(Beckman Coulter公司,目录号:A98812)
70 ml,聚碳酸酯瓶组件,38 × 102 mm(Beckman Coulter,货号:355622)
26.3毫升,带盖组件的聚碳酸酯瓶,25 × 89毫米(贝克曼库尔特(Beckman Coulter),目录号:355618)
50 ml,带盖组件的聚碳酸酯瓶,29 × 104 mm(Beckman Coulter,目录号:357000)
Avestin EmulsiFlex C3细胞分裂剂(Avestin)
杜恩纸巾研磨机套装
XK 16/20色谱柱(Cytivalifesciences,目录号:28988937)
Spectrum TM Spectra / Por TM 4 RC透析膜管12,000至14,000道尔顿MWCO(Fisher Scientific,目录号:08-667D)
Spectrum TM透析管密封件:Spectra / Por TM标准类型(Fisher Scientific,目录号:08-670-11A)


软件


SPARTAN(斯科特·布兰查德实验室,https: //www.scottcblanchardlab.com/software )
MATLAB(MathWorks,https://www.mathworks.com/products/matlab.html )
Prism(GraphPad Software,Inc.,https://www.graphpad.com/)
来源(美国马萨诸塞州北安普顿市的OriginLab Corporation,https://www.originlab.com/)


程序


ccPEB1a传感器结构
将带有C末端6x His-tag的PEB1a克隆到pet21a(+)载体中。
对PEB1a进行诱变以去除天然的半胱氨酸,并引入两个半胱氨酸,用荧光团(C18S,N73C,K149C)进行位点特异性标记,从而生成ccPEB1a构建体。
C18S :


正向引物:GGGTGCGAGCGTTGCGTTTAG


反向引物:CGCAACGCTCGCACCCAGCGC


N73C :


正向引物:CTGGACTGTGGCAGCGTGGATGCGGTTATC


反向引物:GATAACCGCATCCACGCTGCCACAGTCCAG


K149C :


正向引物:CGGCATTGATGTGTGCTTCAGCGAATTTCC


反向引物:GGAAATTCGCTGAAGCACACATCAATGCCG


在天冬氨酸/谷氨酸结合位点或附近引入其他突变,以调节亲和力。ccPEB1a以约79 nM的解离常数结合天冬氨酸。ccPEB1a Y198F变体具有5.6 µM的亲和力,可在100 nm脂质体中显示多达5个转运周期。
在1.5 ml反应管中混合100 ng具有上述突变的DNA和50 µl T7 Express lysY感受态大肠杆菌细胞,并在冰上孵育30分钟。
在42°C下热震45 s,立即将管在冰上放置5分钟,然后添加750 µl高压灭菌的LB。
在37°C下以250 rpm摇动孵育1 h,然后在琼脂板上添加100 µg / ml氨苄青霉素。
生长过夜为在37℃〜16小时在250摇动转一次通过混合用500μl高压灭菌50%甘油的500μl的培养制备甘油储备,°存储在-80℃。


ccPEB1a纯化
根据制造商的规程,在两个带挡板的2,800 ml芬巴赫式培养瓶中分别制备4 L LB,并在1 L烧瓶中分别制备300 ml。高压灭菌,冷却至37°C或室温。
使用前,在所有LB中补充100 µg / ml氨苄青霉素。
接种300毫升LB与ccPEB1a的细菌甘油原液并生长过夜为在37℃下以250〜振摇16小时转。
用来自S tep B 3的过夜培养物接种LB,使其在600 nm的最终吸光度为0.1。用LB稀释200 µl细胞培养物5次以进行吸光度测量。 
当吸光度达到0.8时,用1 mM新鲜制备的IPTG诱导细胞。
让培养物生长3小时,在37℃和16小时在18℃下以250rpm振荡培养。
通过使用JLA-8.1000定角转子在Avanti JXN-26离心机中于4°C以3,326 × g离心15分钟来收获细胞。每2 L细菌细胞培养物使用10 ml重悬缓冲液(请参见配方1)重悬沉淀。
加入100 µg / ml溶菌酶。
在冰上孵育30分钟,然后用去离子水稀释1:3(v:v)。
在4°C下使用JA-20固定角转子在Avanti JXN-26离心机中以3,578 × g的速度离心30分钟,以沉淀碎片。上清液仍然具有一些颗粒物质,并且非常粘。第二个相同的离心步骤可以进一步澄清上清液,防止色谱柱堵塞,并延长树脂的使用寿命。 
在烧杯中,将上清液与3-5 ml的Ni-NTA树脂(用3 × 5柱体积(CV)的水和1 × 5 CV的洗涤缓冲液(请参见配方2)洗涤)混合,并在搅拌板上轻轻搅拌1 h在4°C下使用磁力搅拌棒搅拌。
按照制造商的说明组装XK20 / 16色谱柱,然后慢慢倒入树脂浆液,并用注射器将其通过。
用注射器拉动流通管,用5 CV的洗涤缓冲液洗涤。
使用3 CV洗脱缓冲液(请参见第3条)通过重力洗脱蛋白质。 
在截留分子量为10K的Amicon Ultra-15离心过滤器中浓缩样品,直到体积达到< 500 µl。通过在分光光度计中测量280 nm处的吸光度来确定蛋白质浓度。使用Expasy生物信息学资源门户网站的在线Expasy Protparam工具计算蛋白质的消光系数和分子量。
使用TLA-100.3固定角转子在Optima MAX-XP台式超速离心机中在4°C下以49192 × g离心蛋白质溶液15分钟。使用1 ml移液管吸出蛋白质溶液,而不会干扰蛋白质聚集物的沉淀。 
在50 ml的SEC缓冲液中对Superdex 200增加10/300 GL色谱柱进行预平衡后,通过大小排阻色谱(SEC)使用Superdex 200增加10/300 GL色谱柱抛光蛋白质。根据制造商的说明设置色谱柱的流速和压力极限。0.3 ml / min的流速和2 MPa的压力极限是该色谱柱的理想选择。通过跟踪在280 nm处的吸光度监控蛋白质洗脱,并收集0.4 ml馏分。
收集峰馏分并立即进行标记程序。蛋白质应该出来作为单体(图URE 2)。




图2.从Superdex 200增加10/300 GL色谱柱洗脱的单体PEB1a的SEC谱图


ccPEB1a标记
重悬于100 µl DMSO中,将1000 nmol干燥的LD555或LD655重悬至最终浓度为10 mM。涡旋直至染料块消失。分装成10 µl等分试样,并储存在-20°C下。
解冻LD555和LD655染料的等分试样,并与新鲜SEC纯化的ccPEB1a蛋白按1:1:1(mol:mol:mol)的比例混合。可以根据标记效率根据经验优化摩尔比。请注意,SEC缓冲区中没有还原剂,因为它们会降低标记效率。因此,暴露于溶剂的半胱氨酸残基可能会随着时间而氧化,从SEC洗脱后立即将蛋白质溶液与马来酰亚胺试剂混合至关重要。
将Eppendorf管包裹在铝箔中,并在旋转器上孵育45分钟至1小时。
净化标记的单体ccPEB1a从未反应的染料通过SEC,以下的Superdex 200增加10/300 GL的预平衡如上述小号吨EP乙的“ccPEB1a纯化”部分的17。使用相同的流速和压力限制在小号TEP乙17。
使用截留分子量为10K的Amicon Ultra-4离心过滤器将标记的蛋白质浓缩至〜5 µM。
如果ccPEB1a蛋白质变体以解离常数< 5 µM结合底物,则使用截留分子量为12-14 kDa的透析膜管将4 L桶中的4,000倍SEC缓冲液透析该蛋白质,以去除结合的L-天冬氨酸。在蒸馏水中孵育15厘米管30分钟。用蒸馏水冲洗管道,并用透析管夹封闭一端。确保夹子紧紧且没有泄漏后,装入蛋白质溶液。将多余的管道折叠起来,在蛋白质溶液的顶部留出2厘米的空间,然后用夹子将其封闭。将组件翻转1-2次以确保没有泄漏,然后将其放入装有SEC缓冲液的烧杯中。每4小时更换缓冲液3次。
在280 nm,555 nm(LD555)和655 nm(LD655)处测量蛋白质样品的吸收率。使用蛋白质和染料的消光系数:150,000 cm -1 M -1 (LD555)和250,000 cm -1 M -1 (LD655)来计算它们各自的浓度并确定标记效率。
将等份标记的蛋白质分成20微升等分试样,并在液体N 2中快速冷冻。储存在液态N 2中直至使用。


脂质体储备的制备
混合4毫升(100毫克)的大肠杆菌极性脂质提取物和蛋PC(储存在-80℃下以1.33毫升(33毫克)在氯仿中)在一个最终3:1(重量:重量)的比率在100毫升圆底烧瓶。使用玻璃移液器转移氯仿溶液。使用旋转蒸发仪干燥混合物,将烧瓶在真空下旋转〜30分钟;脂质会在烧瓶壁上形成薄膜。
用铝箔将烧瓶的开口松散地盖上,然后放在连接真空的干密封真空干燥器中过夜。
要水合脂质膜,请在圆底烧瓶中加入33.25 ml的Resting缓冲液(请参见第6条),以使最终浓度达到4 mg / ml脂质。为促进脂质膜的水化过程,请执行以下冷冻/解冻循环:通过将烧瓶浸入液氮中来冷冻脂质悬浮液。您可以在保温瓶或冰桶中准备液氮浴。在将烧瓶降低到液氮中时,请用防热手套将烧瓶握住其瓶颈,或使用蒸气管扩展器。脂质悬浮液在冻结时会发出类似于鸡蛋在煎锅上的声音。一旦噪音在1-2分钟内消退,悬浮液就会完全冻结。将烧瓶转移至室温的水浴中以解冻悬浮液。视频1展示了我们使用的设置。重复冷冻/解冻循环10次,直到烧瓶侧面的脂质膜消失,并且悬浮液呈浑浊白色。




视频1.制备用于蛋白质重建的脂质体原液


分装成1毫升等分的脂质体,在液体N 2中速冻,并保存在-80°C直至进一步使用。


将钠/天冬氨酸共转运蛋白Glt Ph重组为脂质体并包裹ccPEB1a
净化N278C / C321A GLT博士单个半胱氨酸突变体(cysGlt博士),先前所描述的(Akyuz等人,2013) ,具有低于一些修改。
用12 L的LB(而不是4 L)重复S teps B1-B4,并用cysGlt Ph甘油储备液接种LB。
在37°C下以250 rpm摇动,用20 mM L-阿拉伯糖诱导3 h 。
使用EmulsiFlex C3细胞裂解液将细胞溶解在cysGlt Ph重悬缓冲液中(请参见第10条)。4次通过通常足以破坏大多数细胞。
使用45型Ti固定角度钛转子在Optima L-100XP超速离心机中以100,000 × g和4°C的温度将细胞悬液离心1小时,以使膜沉淀。
在加有40 mM正十二烷基β-D-麦芽吡喃糖苷(DDM)的增溶缓冲液中匀浆粗细胞膜(请参见第11条),在4°C下放置1 h,然后通过在100,000 × g和4下离心1 h去除不溶物质在使用45 Ti固定角度钛转子的Optima L-100XP超速离心机中,以°C为单位。
将上清液与3-5 ml Ni-NTA树脂混合,然后按照步骤B11- B 12中所述组装色谱柱。
用5 CV增溶缓冲液(添加1 mM DDM和40 mM咪唑)洗涤,并在250 mM咪唑存在下洗脱。
与凝血酶以每毫克蛋白质10个单位的温度在21°C下孵育〜16小时,以裂解His-tag。
使用Superdex 200通过SEC纯化裂解的cysGlt Ph ,在50 ml cysGlt Ph SEC缓冲液中预先平衡10/300 GL色谱柱(请参见配方5)。
解冻等分的马来酰亚胺-PEG 11-生物素和NEM,并以1:2:4(mol:mol:mol)蛋白质:马来酰亚胺-PEG 11-生物素:NEM的比例与新鲜纯化的cysGlt Ph蛋白混合。请注意,SEC缓冲液中没有还原剂,半胱氨酸残基可能会随着时间而氧化。因此,从SEC洗脱后立即将蛋白质溶液与马来酰亚胺试剂混合是至关重要的。
使用预先平衡的Superdex 200增加10/300 GL色谱柱,通过SEC从未反应的试剂中纯化标记的cysGlt Ph 。
使用截留分子量为100K的Amicon Ultra-4离心过滤器将标记的转运蛋白浓缩至4 mg / ml。
取出1 ml脂质体等分试样(4 mg / ml),并使用微型挤出机挤出400 nm膜,如视频所示(https://www.sigmaaldrich.com/video/life-science/avanti-mini-extruder .html)。2 1次通过应产生大小均一的单层脂质体。
通过以1:2(w:w)洗涤剂/脂质比率添加Triton X-100,使脂质体不稳定。
将cysGlt Ph以1:1000(w:w)PLR加入脂质体中,并在室温下在旋转器上孵育30分钟。调整PLR,以每个小泡一个转运蛋白的形式富集脂质体。我们在(Ciftci et al.2020 )中详细介绍了计算。
在50毫升试管中使用之前,请先用10 CV乙醇和20 CV水彻底洗涤Bio-Beads 。颠倒浆液2-3次,让珠子沉淀在底部,然后用移液器吸出上清液。将最终浓度为100 mg / ml的珠子添加到脂质体悬浮液中,并在室温下在旋转器上孵育2小时。至关重要的是,不要添加太多的珠子以避免脂质流失,因为脂质流失会降低蛋白脂质体的产量。
使用1毫升移液器将脂质体溶液从微珠中转移到新的冷冻管中。重复小号TEPS Ç 17和Ç 18为使用新鲜的珠子在4℃下5次以上。
使用TLA-100.3固定角转子在Optima MAX-XP台式超速离心机中于4°C在49,192 × g下离心1 h制成球状蛋白脂质体。
除去上清液并将蛋白脂质体重悬于等体积的静止缓冲液中,冷冻在液氮2中,并在室温下于水浴中融化。
重复小号TEPS Ç 20 Ç 21的两倍多。
按上述方法沉淀蛋白脂质体,以25 mg / ml的浓度重悬于160 µl的静止缓冲液中。
添加来自S tep C8的荧光标记的ccPEB1a,使其终浓度为0.6 µM,并如上所述进行2次冷冻/解冻循环进行封装。两个冷冻/解冻循环应导致高达30%的封装效率(Colletier等,2002 ;Chaize等,2004 ;Costa等,2014)。冷冻/解冻循环次数的增加可能会提高封装效率,但也可能导致变性ccPEB1a的分数增加。我们发现,当前程序允许我们在单个sm-FRET记录中成像多达500个囊泡,足以进行有效的数据收集。P roteoliposomes可以储存在-80℃下在此步骤,直到进一步使用。
通过如上所述的离心方法沉淀蛋白脂质体,以除去未包封的PEB1a。用等体积的静止缓冲液替换上清液。
使用小型挤出机(Avanti Polar Lipids)通过100 nm膜挤出蛋白脂质体(21个通道)。亲teoliposomes可以储存于4℃多达5-6小时后使用。您可以通过阴性染色或cryo-EM验证脂质体的完整性和大小分布。代表性图像示于图URE S7中Ciftci等。(2020年)。


单分子实验
基于已公开的方法(Joo和Ha ,2012年),制备了用PEG-生物素功能化的TIRF显微镜用石英显微镜载玻片和玻璃盖玻片。
如前所述(Juette et al。,2016),我们使用围绕尼康Eclipse Ti倒置显微镜机身构建的基于棱镜的TIRF显微镜进行了所有单分子FRET成像实验。提供了有关建立基于棱镜的单分子TIRF显微镜设置的分步指南(Selvin和Ha ,2008 ;Axelrod ,2008 ;Roy等人,2008)。
通过使混合物流过进样口并孵育5分钟,使用1 µM BSA和1 µM双链DNA在150 µl T50缓冲液(请参见第9条)中的溶液对石英玻片进行钝化。可以在Blanchard等人的文章中找到更多详细信息。(2004年)以及Joo和Ha (2012年)。使150 µl T50缓冲液流过进样口,以洗去多余的BSA和DNA 。
要使非特异性连接的脂质体成像,请从S tep E 25中流出不超过10 µl的蛋白脂质体悬液。记录您在此步骤中使用的样品数量。
立即用150 µl的静止缓冲液清洗表面,以除去未附着的蛋白脂质体。
以100 mW激光功率以10 Hz的时间分辨率将图像附着的分子合计300 s。
通过以600 mW的激光功率对整个固定化表面进行成像,使漂白剂非特异性附着的分子发生光漂白,并将其置于装有150 µl T50缓冲液的室内。 
在T50缓冲液中加入150 µl预混合的0.8 µM链霉亲和素和1 µM双链DNA溶液,孵育5-10分钟。
在连接蛋白脂质体之前,用150 µl T50缓冲液和150 µl静置缓冲液冲洗掉多余的链霉亲和素双链体DNA 。
流动脂质体的相同体积的在小号TEP ˚F 4.立即清洗表面用150μl缓冲液休息以除去未附着的蛋白脂质体。如有必要,流动更浓缩的样品或增加样品量以达到合适的单分子密度。在最佳密度,在拒绝分子的分数gettraces模块SPARTAN的应为〜40%(请参阅数据一nalysis和在线SPARTAN手册)。如果进样量较大,请使用匹配的样品重新检查非特定附件(S teps F 4- F 7)。
以100mW激光功率图像附分子在总共300秒10赫兹时间分辨率,并确定特异性和非特异性附着到表面(数据分子数一nalysis)。理想情况下,在S tep F 6中成像的非特异性附着的荧光颗粒应小于特异性附着分子的25%。
用由2 mM PCA和50 nM PCD组成的除氧系统补充非激活和激活缓冲液(请参见第7-8节)(Aitken等,2008)。
影像蛋白脂质体通过在电影开始后约3 s流入50 µl非活化缓冲液来测试渗漏情况。在S teps E 17 - E 1 8后残留的痕量洗涤剂可以使L-asp自发扩散到脂质体中,并导致在没有活性钠驱动的摄取的情况下,封装的ccPEB1a的FRET效率发生变化。如果此步骤中有大量单分子轨迹(> 10%)显示FRET效率发生变化,请丢弃脂质体,并在下一个制备中增加生物珠的数量或生物珠孵育周期的数量。每个回合中的生物珠不得超过200毫克/毫升。如果200 mg / ml的生物珠仍然不够,请增加孵育轮数。每轮后仔细观察脂质体制剂的浊度水平。浊度的显着降低表明脂质体被过量的生物珠吸附。我们已经观察到,使用100 mg / ml生物珠进行6轮电泳足以从脂质体制剂中除去Triton X-100洗涤剂。该数目可以根据用于增溶脂质体的去污剂的性质和孵育温度而有所不同。具有较高临界胶束浓度(CMC)的洗涤剂更容易去除。可以使用比色测定法检查脂质体制剂中的残留洗涤剂(Urbani和Warne ,2005)。我们已经观察到(通过温控室)轻微的温度变化会导致去污剂去除效率的显着差异。
在电影开始后大约3秒钟,通过使50 µl激活缓冲液流动来对蛋白脂质体进行成像,以测量转运。根据运输商的周转率,调整时间分辨率和电影的总时长。对于周转时间约为30-100 s的cysGlt Ph变体,我们使用20 mW的激光功率,时间分辨率为2.5 Hz。在这种激光功率下,平均光漂白时间约为300 s。对于周转时间约为1 s的变体,我们使用10 Hz的75 mW激光功率,因此平均光漂白时间约为60 s。
在连接新的蛋白脂质体用于下一轮运输记录之前,用2 ml的缓冲液洗涤样品室,并按照S tep F 7的方法漂白所有残留的荧光分子。重复小号TEPS ˚F 10 ˚F 15。


数据一nalysis


使用SPARTAN执行smFRET数据分析,SPARTAN是用MATLAB编写的可免费获得的定制软件包(Juette等,2016)。这提供了在SPARTAN (https://www.scottcblanchardlab.com/software)中实现的粒子检测和荧光轨迹生成的详细说明。
使用“ gettraces”模块处理以TIFF格式收集的数据文件,以检测单个分子并提取随时间变化的荧光变化。纠正串扰。此模块创建带有.rawtraces扩展名的输出文件。可以使用SPARTAN中实现的“ crosstalkcorrect”函数来计算串扰值,该函数计算在给定数据集中的所有轨迹上的受体发生光漂白之后,受体的光漂白后观察到的平均受体强度,其中受体在供体之前被光漂白。此功能需要一个。rawtraces文件,并返回串扰值以及用于计算它的轨迹百分比。
处理。来自“自动跟踪”模块中第2步的原始跟踪文件。应用FRET寿命,背景噪声,施主眨眼事件的次数以及Juette等人的补充表1中所述的信噪比的默认参数。(2016)。对于每个单分子轨迹的第一帧的FRET效率,我们还应用了0.4的下限阈值和0.7的上限阈值。我们选择基于十万ccPEB1a单分子轨迹的分析这些阈值在“描述的工程L-ASP传感器”部分,示于图URE在1B Ciftci等。(2020年)。FRET效率值超出0.4-0.7范围可能对应于变性的ccPEB1a。
手工检查从“所选择的轨迹自动跟踪”使用“模块sorttraces”模块。丢弃轨迹具有多个供体或受体光漂白步骤,其indicat上课多于一个的传感器每囊泡的存在或非特异性标记的传感器。丢弃还轨迹,显示在视觉上明显缺乏供体和受体强度之间的反相关性(图URE 4) 。
通过使用指定为' No' ,' All'或' Best'的垃圾箱,在' sorttraces '中将轨迹分为响应和非响应。分别保存响应轨迹和非响应轨迹。图。Ciftci等人的补充信息中的S5 。(2020) ,示出了响应轨迹的示例(图URE 4) 。
手动记录在该FRET效率开始于在一个EXCEL文件的第一列中的每个单分子的轨迹增大时间点response_times.xlsx使得在轨迹的顺序responding_molecules.traces文件在响应时间条目的顺序匹配response_times。 xlsx 。因此,响应时间在1个检测到的第一轨迹是1个第一行中,响应时间检测到2次轨迹是在2次行,等等记录在任何轨迹观察到的第一行的最短响应时间第二栏;这个数字非常适合激活缓冲液的注入时间。仅在步骤7中使用MATLAB脚本TurnoverTime.m时,跟踪订单才是重要的。
在初始逐步增加之后,将FRET效率的逐渐增加拟合为与时间有关的绑定方程式,以获得平均周转时间。Ciftci等人的“材料和方法”部分“ smFRET运输轨迹的分析” 。(2020)解释了这个方程的推导。(可选)使用MATLAB脚本TurnoverTime.m (https://github.com/hdciftci/SingleMoleculeTransportAssay.git)拟合数据。该脚本是根据SPARTAN软件(https://www.scottcblanchardlab.com/software)中的avgFretTime.m脚本进行修改的,并且需要两个输入文件。在responding_molecules.traces和response_times.xlsx 。此脚本输出的Rate_constants.csv将具有适合的周转时间以及方程式中使用的其他优化参数。
在减去激活缓冲液注入时间(对应于前半个循环时间)和平均周转时间后,绘制响应时间的分布。在Ciftci等人中,我们将PRISM(GraphPad Software,Inc.)用于所有直方图,并将Origin(OriginLab Corp.,Northampton,MA,USA)用于人口等高线图。(2020年)。Ciftci等人的数据。(2020)是至少3个独立实验的平均值。
有关分析方法的更详细说明,请参见Ciftci等人的材料和方法“ smFRET轨迹分析”部分。(2020年)。的步骤和实例数据A概括视图显示在图URE 3和图URE分别为4。




图3 。数据分析流程图




图4.从“数据分析”中描述的步骤生成的示例数据。(步骤1-2)针对串扰校正荧光强度,并使用“ gettraces”模块拾取单个分子。(第3步)使用“自动跟踪”模块提取供体和受体分子的荧光强度以及所提取分子的相应FRET效率。(步骤4-5)荧光轨迹被检查其反相关和响应模式使用“sorttraces”模块,并相应地分类为应答或无应答的分子。没有反相关和/或显示多个施主或受主光漂白步骤的轨迹被排除在进一步分析之外。(步骤6)的响应时间被手动检测并记录在Excel工作表。(步骤7)的平均周转时间是使用计算出的“周转时间”脚本。



菜谱


重悬缓冲
10 mM的Tris-HCl ,pH 8.0


2毫米EDTA


0.5 M蔗糖


洗涤缓冲液
10 mM Tris-HCl,pH 7.4


200毫米氯化钠


1 mM三(2-羧乙基)膦(TCEP)


40毫米咪唑


洗脱缓冲液
10 mM Tris-HCl,pH 7.4


200毫米氯化钠


1 mM三(2-羧乙基)膦(TCEP)


250 mM咪唑


体积排阻色谱(SEC)缓冲区
50 mM HEPES / Tris ,pH 7.4


200毫米氯化钾


cysGlt Ph SEC缓冲区
20 mM HEPES / Tris,pH 7.4


200毫米氯化钠


0.1 mM L-天门冬氨酸


1毫米DDM


休息缓冲
50 mM HEPES / Tris,pH 7.4


200毫米氯化钾


非激活缓冲区
50 mM HEPES / Tris pH 7.4


200毫米氯化钾


1 µM L-天冬氨酸


激活缓冲区
50 mM HEPES / Tris,pH 7.4


200毫米氯化钠


1 µM L-天冬氨酸


T50缓冲器
10 mM Tris / HCl,pH 7.4


50毫米氯化钾


cysGlt Ph重悬缓冲液
20 mM HEPES / Tris,pH 7.4


200毫米氯化钠


1 mM EDTA,pH 7.4


增溶缓冲液
50 mM HEPES / Tris,pH 7.4


200毫米氯化钠


1毫米TCEP


1 mM L -asp


致谢


我们感谢Roger Altman为smFRET设备所做的准备。资金:OB和SCB的NINDS R37NS085318,SCB的AHA 19PRE34380215到HDC 7R01GM098859的SCHA该项目已获得Maire Sklodowska-Curie拨款协议MEMDYN No 660083(GHMH)的欧盟Horizon 2020研究与创新计划的资助。这项工作最初以Ciftci等人的身份出版。(2020)。


利益争夺


渣打银行持有Lumidyne Technologies的股权。


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Copyright: © 2021 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Ciftci, D., Huysmans, G. H., Wang, X., He, C., Terry, D., Zhou, Z., Fitzgerald, G., Blanchard, S. C. and Boudker, O. (2021). FRET-based Microscopy Assay to Measure Activity of Membrane Amino Acid Transporters with Single-transporter Resolution. Bio-protocol 11(7): e3970. DOI: 10.21769/BioProtoc.3970.
  2. Ciftci, D., Huysmans, G. H. M., Wang, X., He, C., Terry, D., Zhou, Z., Fitzgerald, G., Blanchard, S. C. and Boudker, O. (2020). Single-molecule transport kinetics of a glutamate transporter homolog shows static disorder. Sci Adv 6(22): eaaz1949.
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