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Guanine Nucleotide Exchange Assay Using Fluorescent MANT-GDP
使用荧光MANT-GDP的鸟嘌呤核苷酸交换测定法   

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Developmental Cell
Jul 2017

Abstract

GTPases are molecular switches that cycle between the inactive GDP-bound state and the active GTP-bound state. GTPases exchange nucleotides either by its intrinsic nucleotide exchange or by interaction with guanine nucleotide exchange factors (GEFs). Monitoring the nucleotide exchange in vitro, together with reconstitution of direct interactions with regulatory proteins, provides key insights into how a GTPase is activated. In this protocol, we describe core methods to monitor nucleotide exchange using fluorescent N-Methylanthraniloyl (MANT)-guanine nucleotide.

Keywords: GTPase (GTP酶), N-Methylanthraniloyl (MANT) (N-甲基氨茴酰基(MANT)), Nucleotide exchange (核苷酸交换), GEF assay (GEF测定法), in vitro (体外), Fluorescence (荧光), Fluorescent nucleotides (荧光核苷酸), Fluorescent GDP (荧光GDP)

Background

GTPases are guanine nucleotide binding proteins that regulate a breadth of cellular processes, ranging from protein biosynthesis to cell-cycle progression and from cytoskeletal reorganization to membrane trafficking. GTPases can be thought of as molecular switches that cycle between a GDP-bound ‘off’ state and a GTP-bound ‘on’ state; upon GTP-binding via nucleotide exchange of GDP for GTP, GTPases become active and will bind to down-stream effector proteins to recruit and activate the biological function of these effectors. GTPases bind the γ-phosphate of GTP via interactions with a highly conserved threonine of the switch I loop (G2 domain) and a glycine within a DxxG motif of the switch II loop (G3 domain). Upon GTP hydrolysis, the loss of the interaction with the γ-phosphate causes a dynamic conformational change that turns the GTPase into an off state (Vetter and Wittinghofer, 2001). Typically, small GTPases have a very high affinity for guanine nucleotides, with dissociation constants in the nanomolar to picomolar range (Bos et al., 2007), and therefore require guanine nucleotide exchange factors (GEFs) to lower their nucleotide affinity to allow for rapid activation. Notable exceptions include several highly conserved centrosomal/ciliary small GTPases, such as Rabl2 (Kanie et al., 2017), ARL13B (Ivanova et al., 2017), Ift27/Rabl4 (Bhogaraju et al., 2011), and Arl6 (Price et al., 2012), and many large GTPases, such as the dynamin family (Gasper et al., 2009), which have lower affinities for GDP/GTP, with dissociation constants in the micromolar range. In this configuration, these GTPases can become activated without the use of GEFs via their intrinsic nucleotide exchange. Although small GTPases that show spontaneous exchange may have additional regulatory proteins, finding that a small GTPase has a micromolar affinity for GDP/GTP and is capable of spontaneous exchange may suggest the absence of a traditional GEF and suggest looking for other forms of GTPase regulatory factors (see Kanie et al. [2017] for a notable example).

Fluorescently-labeled guanine nucleotides are more suitable for monitoring nucleotide exchange than radioactive GDP/GTP, as they are safer and allow continuous spectroscopic monitoring and thus provide a more detailed analysis of kinetics. N-Methylanthraniloyl (MANT) is the most widely used fluorescent analog to label guanine nucleotides because it is smaller than most fluorophores and unlikely to cause major perturbations of protein-nucleotide interactions (Hiratsuka, 1983). Another attractive feature of this fluorescent nucleotide is that the emitted fluorescent signal increases dramatically upon binding to a GTPase (typically twice as high as the signal of unbound MANT-guanine nucleotide) (John et al., 1990), allowing one to directly monitor the association and dissociation of guanine nucleotides from GTPases. The most commonly used method to monitor nucleotide exchange is tracking the decrease in the fluorescence of protein-bound MANT-GDP upon addition of an excess amount of GppNHp, a non-hydrolyzable GTP analog. MANT-GDP-bound GTPase can be prepared by the incubation of nucleotide free GTPase with 1.5 fold excess of MANT-GDP (John et al., 1990; Eberth and Ahmadian, 2009). Although this protocol is described in great detail and allows us to save expensive MANT-GDP, it is time-consuming and limited by the accessibility to high performance liquid chromatography. Alternatively, we describe here a simpler protocol where MANT-GDP is loaded onto GTPase by incubating GTPase with 20-fold molar excess of MANT-GDP in the presence of ethylenediaminetetraacetic acid (EDTA). EDTA chelates magnesium ions, which form coordination bonds with the β and γ phosphates of GTP (or β phosphate of GDP) and with the GTPase (Pai et al., 1990; Tong et al., 1991). EDTA significantly lowers the affinity of the GTPase for guanine nucleotide. The loading reaction is stopped by the addition of excess magnesium chloride. Unbound MANT-GDP is removed by gel filtration using a NAP-5 prepacked column and the nucleotide exchange reaction is initiated by the addition of 100-fold molar excess of GppNHp. Exchange is monitored by a decrease in the fluorescent signal as recorded by a spectrometer with excitation wavelength at 360 nm and emission at 440 nm.

Variations on this method are readily accomplished by addition of biochemically pure nucleotide variants, drugs, or purified protein regulatory factors, allowing for a range of mechanistic experiments and definitive tests of biochemical mechanisms.

Materials and Reagents

  1. Microcentrifuge tube (Corning, Costar®, catalog number: 3621 )
  2. 50 ml conical tube (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339652 )
  3. Oak ridge tube (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3119-0050 )
  4. 15 ml conical tube (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 339650 )
  5. (Optional) Amicon ultra concentrator (Merck, catalog number: UFC901024 )
  6. Dialysis tubing (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 88242 )
  7. NAP-5 columns (GE Healthcare, catalog number: 17085301 )
  8. Aluminum foil
  9. 384-well microplate (Greiner Bio One International, catalog number: 784076 )
  10. (Optional) 96-well microplate (Corning, catalog number: 3686 )
  11. 0.2 µm disposable bottle top filter (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 597-4520 )
  12. 0.22 µm syringe filter (Merck, catalog number: SLGV033RS )
  13. Disposable cuvettes (Fisher Scientific, catalog number: 14-955-127 )
  14. Multichannel pipette tip (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 7421 )
  15. Rosetta2 bacterial cells (Merck, EMD Millipore, catalog number: 71403 )–Store at -80 °C
  16. Recombinant GTPase
    Note: Ideally, the concentration should be 100 µM or greater. See the protocol below for the expression and purification of recombinant GTPases. Store at -80 °C.
  17. Gateway-cloning compatible pGEX6p vector
  18. 4x LDS sample buffer (Thermo Fisher Scientific, InvitrogenTM, catalog number: NP0008 )
  19. 2-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )–Store at 4 °C
  20. GppNHp (Abcam, catalog number: ab146659 )
    Note: Dissolve in Milli-Q water to prepare a 50 mM stock solution. Store at -20 °C.
  21. Nu-PAGE gel (Thermo Fisher Scientific, InvitrogenTM, catalog number: NP0321BOX )
  22. Coomassie Brilliant Blue R-250 (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 20278 )
  23. (Optional) Classical Laemmli sodium dodecyl sulfate (SDS) sample buffer/SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gel
  24. Glutathione Sepharose 4B media (GE Healthcare, catalog number: 17075605 )–Store at 4 °C
  25. GST-PreScission (10 mg/ml)
    Note: We prepare GST tagged PreScission by ourselves. 1 µl of our protease cleaves approximately 1 mg of GST-ARL3, a test protein. Alternatively, you can purchase the protease from GE Healthcare, catalog number: 27084301 . Store at -80 °C.
  26. Bradford Reagent Concentrate (Bio-Rad Laboratories, catalog number: 5000006 )–Store at 4 °C
  27. Liquid nitrogen
  28. BSA standard, 2 mg/ml (Thermo Fisher Scientific, catalog number: 23209 )–Store at 4 °C
  29. TritonX-100 (Acros Organics, catalog number: 215682500 )
  30. Milli-Q water
  31. Protease inhibitor tablet (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: A32965 )–Store at 4 °C
  32. Glycerol (Sigma-Aldrich, catalog number: G5516 )
  33. MANT-GDP triethylammonium salt solution (Sigma-Aldrich, catalog number: 69244 )–Store at -20 °C protected from light
  34. Trizma base (Sigma-Aldrich, catalog number: T6066 )
  35. Concentrated HCl (Aqua Solutions, catalog number: H2505-500ML )
  36. Glacial acetic acid (Fisher Scientific, catalog number: A490-212 )
  37. Methanol (Fisher Scientific, catalog number: A412-4 )
  38. HEPES (Sigma-Aldrich, catalog number: H3375 )
  39. Sodium hydroxide (Fisher Scientific, catalog number: BP359-500 )
  40. Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: S3014-500G )
  41. Magnesium chloride hexahydrate (MgCl2·6H2O) (Sigma-Aldrich, catalog number: M2670 )
  42. Dithiothreitol (DTT) (Promega, catalog number: V3155 )
  43. Ethylenediaminetetraacetic acid (EDTA) (Fisher Scientific, catalog number: S311 )
  44. LB broth (Fisher Scientific, catalog number: BP9723 )
  45. LB agar (Thermo Fisher Scientific, InvitrogenTM, catalog number: 22700 )
  46. Terrific broth (Fisher Scientific, catalog number: BP24682 )
  47. Carbenicillin (Sigma-Aldrich, catalog number: C3416 )
  48. Ampicillin (Fisher Scientific, catalog number: BP1760 )
  49. Chloramphenicol (Sigma-Aldrich, catalog number: C0378 )
  50. IPTG (Thermo Fisher Scientific, InvitrogenTM, catalog number: 15529019 )–Store at -20 °C
  51. LB media (see Recipes)
  52. TB media (see Recipes)
  53. 100 mg/ml ampicillin (see Recipes)
  54. 50 mg/ml carbenicillin (see Recipes)
  55. 34 mg/ml chloramphenicol (see Recipes)
  56. LB agar plate containing 50 µg/ml carbenicillin and 34 µg/ml chloramphenicol (see Recipes)
  57. 1 M IPTG (see Recipes)
  58. 1 M Tris-HCl (pH 7.5) (see Recipes)
  59. 1 M HEPES-NaOH (pH 7.5) (see Recipes)
  60. 5 M NaCl (see Recipes)
  61. 1 M MgCl2 (see Recipes)
  62. 1 M DTT (see Recipes)
  63. 0.25 M EDTA (pH 8) (see Recipes)
  64. 10 M NaOH (see Recipes)
  65. Coomassie Brilliant Blue staining solution (see Recipes)
  66. Coomassie Brilliant Blue destaining solution (see Recipes)
  67. Lysis buffer (see Recipes)
  68. PreScission cleavage buffer (see Recipes)
  69. Storage buffer (see Recipes)
  70. Low magnesium buffer (see Recipes)
  71. 2x MANT-GDP loading buffer (see Recipes)
  72. Nucleotide exchange buffer (see Recipes)
    Note: Unless otherwise noted, materials are stored at room temperature. Frozen proteins and nucleotides can be maintained for at least a year if pure.

Equipment

  1. Milli-Q generator (Merck, model: Milli-Q® Advantage A10, catalog number: Z00Q0V0WW )
  2. 250 ml culture flask (Corning, PYREX®, catalog number: 4980-250 )
  3. 2,800 ml culture flask (Corning, PYREX®, catalog number: 4424-2XL )
  4. Incubator shaker (Eppendorf, New BrunswickTM, model: Excella® E25 , catalog number: M1353-0002)
  5. Refrigerated centrifuge (Eppendorf, model: 5424 R )
  6. 500 ml centrifuge bottle (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 3141-0500 )
  7. JA-10 rotor (Beckman Coulter, model: JA-10 , catalog number: 369687)
  8. Avanti J-25I refrigerated centrifuge (Beckman Coulter, model: Avanti J-25I , catalog number: 363106)
  9. JA-17 rotor (Beckman Coulter, model: JA-17 , catalog number: 369691)
  10. Allegra X-15R refrigerated centrifuge (Beckman Coulter, model: Allegra® X-15R , catalog number: 392932)
  11. BioSpectrometer (Eppendorf, catalog number: 6136000010 )
  12. Thermomixer (Fisher Scientific, catalog number: 05-412-401 )
    Note: This equipment has been discontinued. Thermomixer C (Fisher Scientific, catalog number: 05-412-503; Manufacturer: Eppendorf, catalog number: 5382000023 ) is available as an alternative. Any type of temperature-controlled mixer with cooling function should work for this experiment.
  13. TECAN Infinite M1000 Microplate reader (TECAN, part number: 30034301 )
    Note: There are a number of plates or cuvette-based fluorimeters that can be adapted to this procedure. Those include FluoroMax-4 (Horiba) (Price et al., 2012), Synergy H4 Hybrid Microplate reader (Bio Tek) (Ivanova et al., 2017), and Envision (Perkin-Elmer) (Maurer et al., 2012).
  14. Branson Digital Sonifier (Branson)
    Note: This equipment has been discontinued. Branson Ultrasonics Sonifier SFX250/SFX550 (Fisher Scientific, catalog number: 15-345-141; Manufacturer: EMERSON, Branson, catalog number: 101063969R ) is available as an alternative.
  15. 360° vertical rotator (Grant Instruments, model: PTR-30 )
    Note: This equipment has been discontinued. Grant bio 360° vertical rotator PTR-35 (Grant Instruments, model: PTR-35 ) is available as an alternative.
  16. Lab stand (Humboldt, catalog number: H-21207 )
  17. Vortex mixer (Scientific Industries, model: Vortex-Genie 2 , catalog number: SI-0236)
  18. Multichannel electronic pipette (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 2069MTRX )
    Note: This equipment has been discontinued. A newer model (Thermo Fisher Scientific, Thermo ScientificTM, catalog number: 4671020BT ) is available as an alternative.
  19. Autoclave

Software

  1. Microsoft Excel (Microsoft)
  2. GraphPad Prism 7 software (GraphPad Software)

Procedure

  1. Expression and purification of recombinant GTPases from E. coli
    Recombinant GTPases can be produced and purified from Escherichia coli, since the nucleotide exchange assay does not generally require post-translational modifications of the GTPase. Experimental conditions, such as temperature and culture time for inducing protein expression and Isopropyl β-D-1-thiogalactopyranoside (IPTG) concentration, need to be determined for each GTPase to obtain the maximal yield of soluble recombinant proteins. Conditions can be optimized in small scale (we typically perform a pilot experiment with 2 ml of LB media). We typically induce the protein expression with 0.1 mM IPTG at 18 °C overnight. A typical yield from 1 L culture is 10 mg of protein.
    1. Transform Rosetta2 bacteria cells with a vector containing gene of interest. Plate the cells onto an LB agar plate containing 50 µg/ml carbenicillin (or 100 µg/ml ampicillin) and 34 µg/ml chloramphenicol. Incubate the plate at 37 °C overnight.
      Note: We typically use Gateway-cloning compatible pGEX6p vector (Wright et al., 2011), which produces an N-terminally Glutathione S-transferase (GST) tagged protein with PreScission protease recognition site between GST and protein of interest. The vectors containing CDC42, a GEF-dependent GTPase, and IIGP1 and CrRabl2, GEF-independent GTPases, work as positive controls for the experiment and are provided by the authors upon request. Chloramphenicol is added into an agar plate because tRNAs for rare codons are maintained by the chloramphenicol-resistant plasmid in the Rossetta2 bacteria cells. Other antibiotics might have to be used depending on bacteria cells used.
    2. Inoculate 50 ml of LB media containing 100 µg/ml ampicillin and 34 µg/ml chloramphenicol with a single colony of the transformed cells in a 250 ml flask. Grow the starter culture at 37 °C at 250 rpm overnight.
    3. Pre-warm 1 L of autoclaved TB media without antibiotics in 2,800 ml flask at 37 °C overnight in an incubator shaker, before starting a large-scale culture.
    4. Inoculate 1 L of pre-warmed TB media with 25 ml of the starter culture. Shake the flask at 37 °C at 250 rpm until OD600 reaches to 0.6-0.7. It takes approximately 2 h.
    5. As soon as OD600 reaches to 0.6-0.7, set the flask on ice and chill culture for 15 min.
    6. Add 100 µl of 1 M IPTG (the final concentration is 0.1 mM) to induce protein expression.
    7. Shake the flask at 18 °C at 250 rpm in an incubator shaker overnight.
    8. Place the flask on ice before harvesting the bacteria cells.
    9. To confirm the expression level and the solubility of the protein, transfer 1 ml of the bacterial suspension into a microcentrifuge tube. Collect the cell pellet by centrifugation at 8,000 rpm (6,010 x g) at 4 °C for 1 min using a refrigerated centrifuge. Re-suspend the cell pellet in 300 µl of pre-chilled lysis buffer and lyse the cells by sonication for 1 min on ice (0.6 sec/0.4 sec on/off cycle at 30% amplitude), followed by centrifugation at 15,000 rpm (21,130 x g) at 4 °C for 10 min. Transfer the supernatant into a microcentrifuge tube and add 100 µl of 4x LDS sample buffer and 10 µl of 2-mercaptoethanol to prepare the soluble fraction. To prepare the insoluble fraction, add 290 µl of the lysis buffer, 100 µl of 4x LDS sample buffer and 10 µl of 2-mercaptoethanol to the pellet. Incubate the samples at 70 °C for 10 min and run them on Nu-PAGE gel at 200 V until the dye front reaches to the bottom of the gel. Stain the gel in Coomassie Brilliant Blue staining solution for 1 h at room temperature with gentle agitation, followed by destaining in Coomassie Brilliant Blue destaining solution overnight at room temperature with gentle agitation. Your protein of interest should appear in the soluble fraction.
      Note: Classical Laemmli sodium dodecyl sulfate (SDS) sample buffer/SDS-polyacrylamide gel electrophoresis (SDS-PAGE) can be used instead of 4x LDS sample buffer/Nu-PAGE system.
    10. Harvest bacteria by centrifugation in four 500 ml centrifuge bottles at 6,000 rpm (6,371 x g) in JA-10 rotor using Avanti J-25I refrigerated centrifuge at 4 °C for 10 min.
      Note: Put a maximum of 300 ml in each tube, to prevent spilling during centrifugation.
    11. Discard supernatant and freeze the cell pellet at -20 °C until you confirm the expression and the solubility of the protein in Step A9.
      Note: Bacterial pellet can be stored at -20 °C for at least one week. Alternatively, you may keep the bacteria pellet on ice for same day confirmation of the expression/solubility then proceed immediately to the next step.
    12. Re-suspend the 1 L cell pellet in 30 ml of pre-chilled lysis buffer and transfer suspension to a 50 ml conical tube.
    13. Sonicate the cells on ice until the suspension becomes watery (approximately 2 min; 0.6 sec/0.4 sec on/off cycle at 80% amplitude).
    14. Transfer the lysate into an oak ridge tube and clarify the lysate by centrifugation at 15,000 rpm (30,996 x g) at 4 °C in the JA-17 rotor using the Avanti J-25I refrigerated centrifuge for 30 min.
    15. During centrifugation, wash 2 ml bed volume of glutathione Sepharose 4B media twice in 20 ml of ice-cold lysis buffer in a 50 ml conical tube. To wash the beads, pellet the beads by centrifugation at 1,466 rpm (500 x g) at 4 °C for 5 min using the Allegra X-15R refrigerated centrifuge and aspirate the supernatant. Add 20 ml of the lysis buffer and mix by inverting the tube. All washes in the following steps will be done in the same manner.
      Note: 1 ml bed volume of beads bind approximately 5 mg of GST.
    16. Add clarified lysate into the washed glutathione Sepharose beads and rotate the 50 ml conical tube on a 360° vertical rotator at 4 °C for 2 h.
    17. Wash the beads three times with 40 ml (20 resin volumes) of lysis buffer without protease inhibitor.
    18. Wash the beads twice with 40 ml (20 resin volumes) of PreScission cleavage buffer.
    19. Add 4 ml (2 resin volumes) of PreScission cleavage buffer and transfer them into a 15 ml conical tube.
    20. Add 10 µl of 10 mg/ml GST-PreScission to elute the protein. Rotate the 15 ml conical tube on a 360° vertical rotator at 4 °C for 16 h.
      Note: 10 µl of 10 mg/ml GST-PreScission is added, because we expect the final yield 10 mg and 1 µl of our GST-PreScission cleaves 1 mg of GST tagged protein. The amount of GST-PreScission that should be added depends on the expression level of the protein and/or how much protein is desired. The amount of proteins contained in the lysate can be estimated based on the amount of protein in the test sample taken at Step A9.
    21. Pellet the beads by centrifugation at 1,466 rpm (500 x g) at 4 °C for 5 min using the Allegra X-15R refrigerated centrifuge and collect supernatant into a 15 ml conical tube.
    22. Wash the beads with 2 ml (one resin volume) of PreScission cleavage buffer and collect supernatant following centrifugation at 1,466 rpm (500 x g) at 4 °C for 5 min. Repeat wash one more time and combine with the previous supernatant.
    23. Measure the concentration of the protein using the Bradford assay (see Step B8 for detail).
      Note: If the concentration is less than 100 µM, concentrate the eluted protein using the Amicon ultra concentrator (Merck, catalog number: UFC901024).
    24. Check the quality of the protein by Nu-PAGE and Coomassie Brilliant Blue staining.
      Note: Classical Laemmli SDS sample buffer/SDS-PAGE can be used instead of 4x LDS sample buffer/Nu-PAGE system.
    25. Dialyze the protein using dialysis tubing in at least 100-fold protein volumes of storage buffer at 4 °C overnight.
    26. Prepare 200 µl aliquots of the purified protein (the concentration should be 100 µM or greater) and snap freeze the aliquots in liquid nitrogen. Store at -80 °C.

  2. Buffer exchange to low magnesium buffer
    Loading MANT-GDP on GTPase is performed in the buffer containing 0.5 mM MgCl2 in the presence of 5 mM EDTA. Since our storage buffer contains 5 mM MgCl2 (see Recipes) for the stability of GTPase, we first exchange the storage buffer for the low magnesium buffer (see Recipes) using a gel filtration column.
    1. Thaw 200 µl of purified GTPase (100 µM or greater) on ice.
      Note: 100 µM of GTPase is generally a good starting point.
    2. Clamp a NAP-5 column onto a lab stand.
    3. Remove the top and bottom cap from the NAP-5 column to allow the excess manufacturer storage buffer to flow through.
    4. Equilibrate the NAP-5 gel filtration column with low magnesium buffer by three complete refills of the column. The volume of buffer used for equilibration is approximately 10 ml in total.
    5. Apply 200 µl of purified GTPase onto the center of the equilibrated NAP-5 column and allow the sample to enter the gel bed completely by gravity flow.
    6. Apply 500 µl of low magnesium buffer to the column and collect the flow through in a microcentrifuge tube.
      Note: This fraction should not contain any proteins.
    7. Apply 1 ml of low magnesium buffer to elute the protein. Collect eluate 100 µl at a time in microcentrifuge tubes.
      Note: Most of the protein should be eluted in the first 4 fractions.
    8. Measure the concentration of each fraction including the flow through by the Bradford assay.
      1. Briefly, dilute one part of Bradford Reagent Concentrate with 4 parts of Milli-Q water, and add 1 ml of diluted Bradford reagent to disposable cuvettes. Add 10 µl of Milli-Q water or BSA standards (at 0.1, 0.2, 0.4, and 0.8 mg/ml), or 1-2 µl of each fraction of buffer exchanged GTPase into each cuvette. Mix the cuvettes by vortexing and incubate at room temperature for 5 min. Measure the concentration of each fraction on the BioSpectrometer.
      2. Calculate the molar concentration from the measured concentration. Use the fraction containing the highest concentration of protein for further experiment. The protein concentration following buffer exchange is generally half as much as the starting concentration.
      Note: A detailed instruction manual from Bio-Rad can be found online. There are different methods available to measure a protein concentration, including the Bradford assay, bicinchoninic acid (BCA) assay, and spectrophotometric methods. We prefer the Bradford assay, because it is a fast and easy method and all of the buffer components used in this protocol are compatible with the Bradford reagent (reagent compatibility chart from Bio-Rad can be found online). BCA assay might be affected by a reducing reagent contained in the nucleotide exchange buffer. We do not recommend a spectrophotometric method because GTPases are often purified with guanine nucleotides, which absorb at the wavelength used for the measurement. We typically get more than half of the starting concentration in a peak fraction.

  3. Loading MANT-GDP on GTPase
    1. Mix 100 µl of buffer exchanged GTPase with 100 µl of 2x MANT-GDP loading buffer (see Recipes) in microcentrifuge tubes. The final reaction contains 20 mM HEPES-NaOH (pH 7.5), 50 mM NaCl, 0.5 mM MgCl2, 5 mM EDTA, 1 mM dithiothreitol (DTT), GTPase, and 20-fold molar excess of MANT-GDP.
      Note: The concentration of MANT-GDP depends on the protein concentration and must be calculated in every experiment.
    2. Incubate the reaction at 20 °C in a thermomixer without shaking for 90 min. Protect the reaction from the light with aluminum foil.
    3. Stop the reaction by adding 2 µl of 1 M MgCl2 (the final concentration is 10 mM). Mix the reaction by inverting the tubes, followed by centrifugation then incubate the tubes at 20 °C for 30 min.
    4. During the incubation, equilibrate a NAP-5 gel filtration column with three complete refills of nucleotide exchange buffer (see Recipes). The volume of buffer used for equilibration is approximately 10 ml in total.
    5. To remove unbound MANT-GDP, apply the entire volume (200 µl) of MANT-GDP loaded GTPase onto the NAP-5 column. Allow the sample to enter the gel bed completely by gravity flow.
    6. Apply 500 µl of nucleotide exchange buffer and collect flow through.
      Note: This fraction should not contain any proteins.
    7. Apply 1 ml of nucleotide exchange buffer to elute the protein. Collect 100 µl at a time in microcentrifuge tubes. Keep MANT-GDP loaded GTPases on ice, protected from light (by aluminum foil).
      Note: Most of the protein should be eluted in the first 4 fractions.
    8. Measure the concentration of each fraction by the Bradford assay as described above.

  4. Measure the loading efficiency on the fluorimeter
    1. Prepare MANT-GDP standards by making two-fold serial dilutions of free MANT-GDP from 40 µM to 0.15625 µM in nucleotide exchange buffer.
      Note: The minimal concentration of free MANT-GDP that can be detected on TECAN Infinite M1000 Microplate reader is around 0.1 µM.
    2. Pipette 15 µl of nucleotide exchange buffer, free MANT-GDP standards, or MANT-GDP loaded GTPase into 384-well microplate.
      Note: This measurement also works with 96-well format. If you use 96-well format, pipette 50 µl of each sample into a 96-well microplate (Corning).
    3. Read the fluorescent signal every 15 sec, 10 times in total, at room temperature (25 °C). The parameters used for the detection are listed (Table 1).

      Table 1. The parameters used for the detection of MANT fluorescence


    4. To calculate the loading efficiency, average the fluorescence intensity for each sample from all 10 time points, then calculate the normalized average by subtracting the background fluorescence value measured for the buffer-only control.
    5. Plot the free MANT-GDP concentration vs. measured fluorescence and fit the standard curve to a linear function using Microsoft Excel. Use the standard curve to determine the concentration of the MANT-GDP-loaded GTPase from its measured fluorescence; since MANT-GDP signal typically increases approximately two-fold upon binding to protein, first divide the fluorescent signal by two then estimate the concentration of loaded GTPase using the standard curve function. A representative example of this data (Table 2) and the standard curve of free MANT-GDP created from the data (Figure 1) are shown.
    Note: The loading efficiency calculated here might not be accurate, as the increase in the signal intensity of MANT-GDP upon binding to GTPases varies among GTPases. Examples of the loading efficiency can be found in the original paper. (Figure S4E in Kanie et al., 2017).

    Table 2. An example of the data obtained from loading of MANT-GDP on GTPase



    Figure 1. A standard curve of free MANT-GDP created using the data shown in Table 2

  5. Nucleotide exchange assay
    1. Add 14 µl of nucleotide exchange buffer or MANT-GDP loaded GTPase into an appropriate number (the number of conditions x 4 technical replicates) of wells of a 384-well microplate.
      Note: The concentration of protein that is added to the well depends on the loading efficiency calculated above, since the starting fluorescence intensity of the sample should be high enough to detect dynamic changes in the fluorescence. If the loading efficiency is high (more than 50%), 1 µM or less GTPase can be used. If the loading efficiency is low (less than 10%), the highest concentration of GTPase should be added.
    2. Before measuring the fluorescence, prepare a working solution of GppNHp, which has 15 x 100 times higher concentration than that of the GTPase, with nucleotide exchange buffer. If you use 1 µM of GTPase, prepare 1.5 mM GppNHp.
    3. Read the fluorescence for 150 sec at room temperature (25 °C) and confirm the fluorescent signal is stable.
      Note: The same detection parameters used in the Step D3 (see Table 1) can be used in the nucleotide exchange assay.
    4. Press the ‘Pause’ button on the display to eject the plate, and then add 1 µl of nucleotide exchange buffer or GppNHp working solution (the final concentration is 100 times higher than that of GTPase) to respective wells. If GEF mediated nucleotide exchange is to be measured, add purified GEF in addition to GppNHp.
      Note: A multichannel pipet should be used to simultaneously add buffer or GppNHp to start all reactions simultaneously. To ensure rapid addition of the buffer or GppNHp, we pre-fill a multichannel pipette with 1 µl of the buffer/GppNHp solution from another 384-well plate before ejecting the assay plate, and then pipette reagent into the assay plate immediately after ejection of the plate. To avoid making any bubble, we set the fill volume to 1.5 µl and the dispense volume to 1 µl.
    5. Press the ‘Continue’ button to insert the plate and continue reading fluorescence every 15 sec at room temperature (25 °C) for at least 30 min. 

Data analysis

  1. Export the fluorimeter data to a Microsoft Excel spreadsheet.
  2. Subtract the background intensity of the nucleotide exchange buffer from each fluorescence intensity.
  3. Set the fluorescence intensity at the first time point after the addition of either buffer or GppNHp to 1 and calculate the relative fluorescence of each of the later time points.
  4. A pseudo first order rate constant (observed rate constant or Kobs) can be calculated from the data, since the reaction contains a large excess of GppNHp over GTPase (100 times or more).
    Because the fluorescence of MANT-bound GTPase decreases exponentially with time, software programs that are capable of fitting data with nonlinear regression can be used for calculating Kobs. Those programs include, but are not limited to, GraphPad Prism, KaleidaGraph and Grafit. We describe here how to do the analysis by using GraphPad Prism 7 as an example.
    1. Open GraphPad Prism 7 and create a new XY table.
    2. Enter the values into each column, click the ‘Analyze’ button and choose Nonlinear regression from the list of XY analyses.
    3. Select ‘Dissociation-One phase exponential decay’ to obtain the Kobs value.
    Note: The data should be confirmed by at least three independent experiments.

Notes

In our hand, the loading efficiency of GTPases with low guanine nucleotide affinity was much lower than that of typical small GTPases, which have a high affinity for guanine nucleotides. Presumably that reflects the higher dynamics of the low affinity GTPase. In the former case, the fluorescence signal is largely affected by dilution of the protein, probably because dilution itself changes the equilibrium.

Recipes

  1. LB media (1 L)
    Dissolve 25 g of LB broth in 900 ml of Milli-Q water
    Adjust pH to 7.2 with 10 M NaOH and bring volume with Milli-Q water to 1 L
    Sterilize by autoclave and store at room temperature
  2. TB media (1 L)
    Dissolve 50.8 g of terrific broth in 900 ml of Milli-Q water and bring volume with Milli-Q water to 1 L
    Transfer into a 2.8 L flask and rotate the flask at 250 rpm at 28 °C in an incubator shaker until the powder is completely dissolved
    Note: It may take less than 30 min.
    Sterilize by autoclave
  3. 100 mg/ml ampicillin (10 ml)
    Dissolve 1 g of ampicillin in 8 ml of Milli-Q water and bring volume with Milli-Q water to 10 ml
    Sterilize with a 0.22 µm syringe filter
    Dispense into 1 ml aliquots, and store at -20 °C
  4. 50 mg/ml carbenicillin (10 ml)
    Dissolve 500 mg of carbenicillin in 8 ml of Milli-Q water and bring volume with Milli-Q water to 10 ml
    Sterilize with a 0.22 µm syringe filter
    Dispense into 1 ml aliquots
    Store at -20 °C
  5. 34 mg/ml chloramphenicol (10 ml)
    Dissolve 340 mg of chloramphenicol in 8 ml of 100% ethanol and bring volume with 100% ethanol to 10 ml
    Sterilize with a 0.22 µm syringe filter
    Dispense into 1 ml aliquots
    Store at -20 °C
  6. LB agar plate containing 50 µg/ml carbenicillin and 34 µg/ml chloramphenicol
    Dissolve 32 g of LB agar in 900 ml of Milli-Q water and bring volume with Milli-Q water to 1 L
    Sterilize by autoclave and cool down to 50 °C
    Add 1 ml of 50 mg/ml carbenicillin and 1 ml of 34 mg/ml chloramphenicol
    Pour into plates and store at 4 °C
  7. 1 M IPTG (10 ml)
    Dissolve 2.383 g of IPTG in 8 ml of Milli-Q water and bring volume with Milli-Q water to 10 ml
    Sterilize with a 0.22 µm syringe filter
    Dispense into 1 ml aliquots
    Store at -20 °C
  8. 1 M Tris-HCl (pH 7.5, 1 L)
    Dissolve Trizma base in 800 ml Milli-Q water
    Adjust pH with concentrated HCl to 7.5 and bring volume with Milli-Q water to 1 L
    Autoclave or filter to sterilize
  9. 1 M HEPES-NaOH (pH 7.5, 1 L)
    Dissolve 238.30 g of HEPES base in 800 ml of Milli-Q water
    Adjust pH to 7.5 with 10 M NaOH and bring volume with Milli-Q water to 1 L
    Filter sterilize through a 0.2 µm disposable bottle top filter
    Store at 4 °C
  10. 5 M NaCl (1 L)
    Dissolve 292.2 g of NaCl in 700 ml of Milli-Q water and bring volume with Milli-Q water to 1 L
    Sterilize by autoclave and store at room temperature
  11. 1 M MgCl2 (100 ml)
    Dissolve 20.33 g of MgCl2·6H2O in 70 ml of Milli-Q water and bring volume with Milli-Q water to 100 ml
    Sterilize by autoclave and store at room temperature
  12. 1 M DTT (30 ml)
    Dissolve 4.63 g of DTT in 20 ml of Milli-Q and bring volume with Milli-Q water to 30 ml
    Dispense into 1 ml aliquots
    Store at -20 °C
  13. 0.25 M EDTA (pH 8.0, 1 L)
    Dissolve 93.06 g of EDTA·2H2O in 800 ml of Milli-Q water
    Adjust pH to 8.0 with NaOH
    Note: EDTA will not dissolve unless pH is ~8.
    Bring volume with Milli-Q water to 1 L
    Sterilize by autoclave and store at room temperature
  14. 10 M NaOH (500 ml)
    Slowly add 200 g of sodium hydroxide into 400 ml of Milli-Q water while stirring continuously
    Bring volume with Milli-Q water to 500 ml
  15. Coomassie Brilliant Blue staining solution (1 L)
    Dissolve 2.5 g Coomassie Brilliant Blue R-250 in 500 ml methanol
    Add 400 ml of Milli-Q water and 100 ml of glacial acetic acid
    Filter through a 0.2 µm disposable bottle top filter
  16. Coomassie Brilliant Blue destaining solution (1 L)
    Add 500 ml Milli-Q H2O and 400 ml methanol to a 1 L bottle
    Add 100 ml glacial acetic acid slowly to solution
  17. Lysis buffer (500 ml)

    One tablet of a protease inhibitor is added in 50 ml of lysis buffer immediately before use
    Make fresh each time
  18. PreScission cleavage buffer

    Make fresh each time
  19. Storage buffer (500 ml)

    Make fresh each time
  20. Low magnesium buffer (200 ml)

    Store at room temperature
  21. 2x MANT-GDP loading buffer (100 µl)

    Make fresh each time. The concentration of MANT-GDP should be calculated from the GTPase concentration
  22. Nucleotide exchange buffer (100 ml)

    Make fresh each time

Acknowledgments

This protocol was adapted from the previous work (Kanie et al., 2017). The authors would like to thank Keene Abbott and Henrietta Bennett for careful reading of the manuscript. This work was support by funds from the Baxter Laboratory for Stem Cell Research, the Stanford Department of Research, the Stanford Cancer Center, NIH grants R01GM114276 and R01GM121565 to PKJ, and postdoctoral support from the Uehara memorial foundation and Human Frontier Science Program to TK. The authors declare no conflict of interest.

References

  1. Bhogaraju, S., Taschner, M., Morawetz, M., Basquin, C. and Lorentzen, E. (2011). Crystal structure of the intraflagellar transport complex 25/27. EMBO J 30(10): 1907-1918.
  2. Bos, J. L., Rehmann, H. and Wittinghofer, A. (2007). GEFs and GAPs: critical elements in the control of small G proteins. Cell 129(5): 865-877.
  3. Eberth, A. and Ahmadian, M. R. (2009). In vitro GEF and GAP assays. Curr Protoc Cell Biol Chapter 14: Unit 14.9.
  4. Gasper, R., Meyer, S., Gotthardt, K., Sirajuddin, M. and Wittinghofer, A. (2009). It takes two to tango: regulation of G proteins by dimerization. Nat Rev Mol Cell Biol 10(6): 423-429.
  5. Hiratsuka, T. (1983). New ribose-modified fluorescent analogs of adenine and guanine nucleotides available as substrates for various enzymes. Biochim Biophys Acta 742(3): 496-508.
  6. Ivanova, A. A., Caspary, T., Seyfried, N. T., Duong, D. M., West, A. B., Liu, Z. and Kahn, R. A. (2017). Biochemical characterization of purified mammalian ARL13B protein indicates that it is an atypical GTPase and ARL3 guanine nucleotide exchange factor (GEF). J Biol Chem 292(26): 11091-11108.
  7. John, J., Sohmen, R., Feuerstein, J., Linke, R., Wittinghofer, A. and Goody, R. S. (1990). Kinetics of interaction of nucleotides with nucleotide-free H-ras p21. Biochemistry 29(25): 6058-6065.
  8. Kanie, T., Abbott, K. L., Mooney, N. A., Plowey, E. D., Demeter, J. and Jackson, P. K. (2017). The CEP19-RABL2 GTPase complex binds IFT-B to initiate intraflagellar transport at the ciliary base. Dev Cell 42(1): 22-36 e12.
  9. Maurer, T., Garrenton, L. S., Oh, A., Pitts, K., Anderson, D. J., Skelton, N. J., Fauber, B. P., Pan, B., Malek, S., Stokoe, D., Ludlam, M. J., Bowman, K. K., Wu, J., Giannetti, A. M., Starovasnik, M. A., Mellman, I., Jackson, P. K., Rudolph, J., Wang, W. and Fang, G. (2012). Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity. Proc Natl Acad Sci U S A 109(14): 5299-5304.
  10. Pai, E. F., Krengel, U., Petsko, G. A., Goody, R. S., Kabsch, W. and Wittinghofer, A. (1990). Refined crystal structure of the triphosphate conformation of H-ras p21 at 1.35 A resolution: implications for the mechanism of GTP hydrolysis. EMBO J 9(8): 2351-2359.
  11. Price, H. P., Hodgkinson, M. R., Wright, M. H., Tate, E. W., Smith, B. A., Carrington, M., Stark, M. and Smith, D. F. (2012). A role for the vesicle-associated tubulin binding protein ARL6 (BBS3) in flagellum extension in Trypanosoma brucei. Biochim Biophys Acta 1823(7): 1178-1191.
  12. Tong, L. A., de Vos, A. M., Milburn, M. V. and Kim, S. H. (1991). Crystal structures at 2.2 A resolution of the catalytic domains of normal ras protein and an oncogenic mutant complexed with GDP. J Mol Biol 217(3): 503-516.
  13. Vetter, I. R. and Wittinghofer, A. (2001). The guanine nucleotide-binding switch in three dimensions. Science 294(5545): 1299-1304.
  14. Wright, K.J., Baye, L.M., Olivier-Mason, A., Mukhopadhyay, S., Sang, L., Kwong, M., Wang, W., Pretorius, P.R., Sheffield, V.C., Sengupta, P., et al. (2011). An ARL3-UNC119-RP2 GTPase cycle targets myristoylated NPHP3 to the primary cilium. Genes Dev 25: 2347-2360.

简介

GTP酶是分子开关,在无效GDP结合状态和活性GTP结合状态之间循环。 GTP酶通过其内在的核苷酸交换或通过与鸟嘌呤核苷酸交换因子(GEF)的相互作用来交换核苷酸。 监测体外核苷酸交换,以及与调节蛋白直接相互作用的重构,为GTP酶如何被激活提供了重要见解。 在该协议中,我们描述了使用荧光N-甲基呋喃酰基(MANT) - 鸟嘌呤核苷酸来监测核苷酸交换的核心方法。

【背景】GTPase是鸟嘌呤核苷酸结合蛋白,调节细胞过程的广度,从蛋白质生物合成到细胞周期进展,从细胞骨架重组到膜运输。 GTPases可以被认为是分子开关,它在GDP结合“关闭”状态和GTP结合“开启”状态之间循环;在通过GTP的GDP核苷酸交换结合GTP时,GTP酶变得活跃并且将结合下游效应蛋白以招募和激活这些效应子的生物学功能。 GTP酶通过与开关I环(G2结构域)的高度保守苏氨酸和开关II环(G3结构域)的DxxG基序内的甘氨酸的相互作用结合GTP的γ-磷酸。 GTP水解后,与γ-磷酸相互作用的丧失导致动态构象变化,从而使GTPase变为关闭状态(Vetter and Wittinghofer,2001)。通常,小GTP酶对鸟嘌呤核苷酸具有非常高的亲和力,解离常数在纳摩尔到皮摩范围内(Bos等人,2007),因此需要鸟嘌呤核苷酸交换因子(GEF)降低它们的核苷酸亲和力允许快速激活。值得注意的例外包括几种高度保守的中心体/睫状体小GTP酶,如Rabl2(Kanie等人,2017),ARL13B(Ivanova等人,2017),Ift27 / Rabl4(Bhogaraju等人,2011)和Arl6(Price等人,2012)以及许多大的GTP酶,如dynamin家族(Gasper等,等人,2009),其对GDP / GTP具有较低的亲和力,解离常数在微摩尔范围内。在这种构型中,这些GTP酶可以通过其内在的核苷酸交换而不使用GEF而被激活。尽管显示自发交换的小GTP酶可能具有额外的调节蛋白,发现小GTP酶对GDP / GTP具有微摩尔亲和力且能够自发交换可能暗示缺乏传统GEF并且暗示寻找其他形式的GTP酶调节因子(参见Kanie 等人 [2017]的一个显着的例子)。

荧光标记的鸟嘌呤核苷酸比放射性GDP / GTP更适合监测核苷酸交换,因为它们更安全,可以连续进行光谱监测,从而提供更详细的动力学分析。 N-Methylanthraniloyl(MANT)是最广泛使用的标记鸟嘌呤核苷酸的荧光类似物,因为它比大多数荧光团小,并且不太可能造成蛋白质 - 核苷酸相互作用的主要干扰(Hiratsuka,1983)。该荧光核苷酸的另一个有吸引力的特征是发射的荧光信号在与GTP酶结合时显着增加(通常是未结合的MANT-鸟嘌呤核苷酸信号的两倍)(John等人,1990) ,允许人们直接监测来自GTP酶的鸟嘌呤核苷酸的缔合和解离。监测核苷酸交换的最常用方法是追踪加入过量的GppNHp(一种不可水解的GTP类似物)后蛋白结合的MANT-GDP的荧光下降。通过将无核苷酸GTP酶与1.5倍过量的MANT-GDP温育(John等,1990; Eberth和Ahmadian,2009),可以制备结合MANT-GDP的GTP酶。尽管该协议详细描述并使我们能够节省昂贵的MANT-GDP,但是由于高效液相色谱的可访问性,其耗时且受到限制。或者,我们在这里描述了一个更简单的方案,通过在乙二胺四乙酸(EDTA)存在下将GTP酶与20倍摩尔过量的MANT-GDP温育,将MANT-GDP加载到GTP酶上。 EDTA螯合镁离子,其与GTP(或GDP的β磷酸)的β和γ磷酸盐和GTP酶形成配位键(Pai等人,1990; Tong等人, ,1991)。 EDTA显着降低GTP酶对鸟嘌呤核苷酸的亲和力。通过加入过量的氯化镁停止加载反应。使用NAP-5预装柱通过凝胶过滤除去未结合的MANT-GDP,并通过加入100倍摩尔过量的GppNHp引发核苷酸交换反应。通过荧光信号的减少监测交换,如通过激发波长在360nm和在440nm发射的光谱仪记录的。

通过添加生物化学纯的核苷酸变体,药物或纯化的蛋白质调节因子,容易实现该方法的变化,从而允许进行一系列机制实验和生化机制的确定性测试。

关键字:GTP酶, N-甲基氨茴酰基(MANT), 核苷酸交换, GEF测定法, 体外, 荧光, 荧光核苷酸, 荧光GDP

材料和试剂

  1. 微量离心管(Corning,Costar ,目录号:3621)
  2. 50ml锥形管(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:339652)
  3. 橡树脊管(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:3119-0050)
  4. 15ml锥形管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:339650)
  5. (可选)Amicon超浓缩器(Merck,目录号:UFC901024)
  6. 透析管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:88242)
  7. NAP-5色谱柱(GE Healthcare,产品目录号:17085301)
  8. 铝箔
  9. 384孔微孔板(Greiner Bio One International,目录号:784076)
  10. (可选)96孔微孔板(Corning,目录号:3686)
  11. 0.2μm一次性瓶顶过滤器(Thermo Fisher Scientific,Thermo Scientific TM,目录号:597-4520)
  12. 0.22μm注射器过滤器(Merck,产品目录号:SLGV033RS)
  13. 一次性比色皿(Fisher Scientific,目录号:14-955-127)
  14. 多道移液管吸头(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:7421)
  15. Rosetta2细菌细胞(Merck,EMD Millipore,目录号:71403) - 在-80°C储存
  16. 重组GTPase
    注:理想情况下,浓度应为100μM或更高。关于重组GTP酶的表达和纯化,参见下面的方案。存放在-80°C。
  17. 网关克隆兼容pGEX6p载体
  18. 4×LDS样品缓冲液(Thermo Fisher Scientific,Invitrogen TM,目录号:NP0008)
  19. 2-巯基乙醇(Sigma-Aldrich,目录号:M3148) - 在4℃储存
  20. GppNHp(Abcam,目录号:ab146659)
    注意:用Milli-Q水溶解以制备50mM储备溶液。在-20°C储存。
  21. Nu-PAGE凝胶(Thermo Fisher Scientific,Invitrogen TM,目录号:NP0321BOX)
  22. 考马斯亮蓝R-250(Thermo Fisher Scientific,Thermo Scientific TM,目录号:20278)
  23. (可选)经典Laemmli十二烷基硫酸钠(SDS)样品缓冲液/ SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)凝胶
  24. 谷胱甘肽琼脂糖4B培养基(GE Healthcare,产品目录号:17075605) - 在4°C储存
  25. GST-PreScission(10毫克/毫升)
    注:我们自己编写了GST标记的PreScission。 1μl我们的蛋白酶切割约1mg的测试蛋白质GST-ARL3。或者,您可以购买GE Healthcare的蛋白酶,产品目录号为27084301. -80°C储存。
  26. Bradford试剂浓缩液(Bio-Rad Laboratories,产品目录号:5000006) - 在4°C储存
  27. 液氮
  28. BSA标准,2 mg / ml(赛默飞世尔科技,产品目录号:23209) - 在4°C储存。
  29. TritonX-100(Acros Organics,目录号:215682500)
  30. Milli-Q水
  31. 蛋白酶抑制剂片剂(Thermo Fisher Scientific,Thermo Scientific TM,目录号:A32965) - 在4℃储存
  32. 甘油(Sigma-Aldrich,目录号:G5516)
  33. MANT-GDP三乙基铵盐溶液(Sigma-Aldrich,目录号:69244) - 储存于-20℃避光保存
  34. Trizma碱(Sigma-Aldrich,目录号:T6066)
  35. 浓HCl(Aqua Solutions,目录号:H2505-500ML)
  36. 冰醋酸(Fisher Scientific,目录号:A490-212)
  37. 甲醇(Fisher Scientific,目录号:A412-4)
  38. HEPES(Sigma-Aldrich,目录号:H3375)
  39. 氢氧化钠(Fisher Scientific,目录号:BP359-500)
  40. 氯化钠(NaCl)(Sigma-Aldrich,目录号:S3014-500G)
  41. 氯化镁六水合物(MgCl 2·6H 2 O)(Sigma-Aldrich,目录号:M2670)
  42. 二硫苏糖醇(DTT)(Promega,目录号:V3155)
  43. 乙二胺四乙酸(EDTA)(Fisher Scientific,目录号:S311)
  44. LB肉汤(Fisher Scientific,目录号:BP9723)
  45. LB琼脂(Thermo Fisher Scientific,Invitrogen TM,产品目录号:22700)
  46. 棒极了肉汤(Fisher Scientific,目录号:BP24682)
  47. 羧苄青霉素(Sigma-Aldrich,目录号:C3416)
  48. 氨苄西林(Fisher Scientific,目录号:BP1760)
  49. 氯霉素(Sigma-Aldrich,目录号:C0378)
  50. IPTG(Thermo Fisher Scientific,Invitrogen TM,产品目录号:15529019) - 储存于-20℃。
  51. LB媒体(请参阅食谱)
  52. 结核病媒体(见食谱)
  53. 100毫克/毫升氨苄青霉素(见食谱)
  54. 50毫克/毫升羧苄青霉素(见食谱)
  55. 34毫克/毫升氯霉素(见食谱)
  56. 含有50μg/ ml羧苄青霉素和34μg/ ml氯霉素的LB琼脂平板(参见食谱)
  57. 1 M IPTG(见食谱)
  58. 1M Tris-HCl(pH 7.5)(见食谱)
  59. 1 M HEPES-NaOH(pH 7.5)(见食谱)
  60. 5 M NaCl(见食谱)
  61. 1 M MgCl 2(见食谱)
  62. 1 M DTT(见食谱)
  63. 0.25 M EDTA(pH 8)(见食谱)
  64. 10 M NaOH(见食谱)
  65. 考马斯亮蓝染色液(见食谱)
  66. 考马斯亮蓝脱色液(见食谱)
  67. 裂解缓冲液(见食谱)
  68. PreScission分裂缓冲液(见食谱)
  69. 存储缓冲区(请参阅食谱)
  70. 低镁缓冲液(见食谱)
  71. 2个MANT-GDP加载缓冲区(请参阅食谱)
  72. 核苷酸交换缓冲液(见食谱)
    注:除非另有说明,否则材料在室温下储存。如果纯净的话,冷冻蛋白质和核苷酸可以保存至少一年。

设备

  1. Milli-Q发生器(Merck,型号:Milli-Q Advantage A10,目录号:Z00Q0V0WW)
  2. 250ml培养瓶(Corning,PYREX®,目录号:4980-250)
  3. 2800ml培养瓶(Corning,PYREX®,目录号:4424-2XL)
  4. 培养箱振荡器(Eppendorf,New Brunswick TM,型号:Excella E25,目录号:M1353-0002)
  5. 冷冻离心机(Eppendorf,型号:5424 R)
  6. 500ml离心瓶(Thermo Fisher Scientific,Thermo Scientific TM,产品目录号:3141-0500)
  7. JA-10转子(Beckman Coulter,型号:JA-10,目录号:369687)
  8. Avanti J-25I冷冻离心机(Beckman Coulter,型号:Avanti J-25I,目录号:363106)
  9. JA-17转子(Beckman Coulter,型号:JA-17,目录号:369691)
  10. Allegra X-15R冷冻离心机(Beckman Coulter,型号:Allegra X-15R,目录号:392932)
  11. BioSpectrometer(Eppendorf,目录号:6136000010)
  12. Thermomixer(Fisher Scientific,目录号:05-412-401)
    注:此设备已停产。 Thermomixer C(Fisher Scientific,目录号:05-412-503;制造商:Eppendorf,目录号:5382000023)可作为替代品。任何类型的具有冷却功能的温度控制混合器都应该适用于本实验。
  13. TECAN Infinite M1000微孔板读数器(TECAN,部件号:30034301)
    注:有许多平板或基于比色皿的荧光计可以适应这一程序。其中包括FluoroMax-4(Horiba)(Price等,2012),Synergy H4 Hybrid Microplate reader(Bio Tek)(Ivanova等,2017)和Envision(Perkin-Elmer)(Maurer等,2012)
  14. 布兰森数字声波器(布兰森)
    注:此设备已停产。 Branson Ultrasonics Sonifier SFX250 / SFX550(Fisher Scientific,目录号:15-345-141;制造商:EMERSON,Branson,目录号:101063969R)可作为替代品。
  15. 360°垂直旋转器(Grant Instruments,型号:PTR-30)
    注:此设备已停产。格兰特生物360°垂直旋转器PTR-35(Grant Instruments,型号:PTR-35)可作为替代品。
  16. 实验室支架(Humboldt,目录号:H-21207)
  17. 涡旋混合器(Scientific Industries,型号:Vortex-Genie 2,目录号:SI-0236)
  18. 多道电子移液管(Thermo Fisher Scientific,Thermo Scientific TM,目录号:2069MTRX)
    注:此设备已停产。更新的型号(赛默飞世尔科技Thermo Scientific TM TM,产品目录号:4671020BT)可作为替代产品。
  19. 高压灭菌器

软件

  1. Microsoft Excel(微软)
  2. GraphPad Prism 7软件(GraphPad软件)

程序

  1. 来自大肠杆菌的重组GTP酶的表达和纯化
    重组GTP酶可以从大肠杆菌产生和纯化,因为核苷酸交换测定通常不需要GTP酶的翻译后修饰。需要确定每种GTP酶的实验条件,例如诱导蛋白质表达的温度和培养时间以及异丙基β-D-1-硫代半乳糖吡喃糖苷(IPTG)浓度,以获得可溶性重组蛋白的最大产量。可以对条件进行小规模优化(我们通常使用2毫升LB培养基进行试验性实验)。我们通常在18℃下用0.1mM IPTG诱导蛋白质表达过夜。 1L培养物的典型产量是10mg蛋白质。
    1. 用含有感兴趣基因的载体转化Rosetta2细菌细胞。将细胞铺在含有50μg/ ml羧苄青霉素(或100μg/ ml氨苄青霉素)和34μg/ ml氯霉素的LB琼脂平板上。
      在37°C孵育过夜。
      注意:我们通常使用Gateway-cloning兼容的pGEX6p载体(Wright et al。,2011),其产生具有GST和目的蛋白之间的PreScission蛋白酶识别位点的N-末端谷胱甘肽S-转移酶(GST)标记蛋白。含有CDC42(一种GEF依赖性GTP酶)和IIGP1和CrRabl2(不依赖GEF的GTP酶)的载体作为实验的阳性对照,由作者根据要求提供。氯霉素被添加到琼脂平板中,因为稀有密码子的tRNA被Rossetta2细菌细胞中的氯霉素抗性质粒维持。根据使用的细菌细胞,可能需要使用其他抗生素。
    2. 用250ml烧瓶中的单个菌落的转化细胞接种50ml含有100μg/ ml氨苄青霉素和34μg/ ml氯霉素的LB培养基。
      在37℃以250转/分钟培养起始培养基

    3. 在启动大规模培养之前,在2,800ml烧瓶中预热1升无抗生素的高压灭菌TB培养基,37℃培养箱振荡器中过夜。
    4. 用25ml发酵剂培养物接种1L预热的TB培养基。在37℃以250rpm摇动烧瓶直至OD 600达到0.6-0.7。大约需要2小时。
    5. 当OD <600>达到0.6-0.7时,将烧瓶置于冰上并冷冻培养15分钟。

    6. 加入100μl1 M IPTG(终浓度为0.1 mM)以诱导蛋白质表达

    7. 在培养箱振荡器中以180转/分的速度摇动培养瓶

    8. 。收集细菌细胞之前,将烧瓶置于冰上
    9. 为了确认蛋白质的表达水平和溶解度,将1ml细菌悬浮液转移到微量离心管中。使用冷冻离心机以8,000rpm(6,010×g克)在4℃离心1分钟收集细胞沉淀。将细胞沉淀重新悬浮于300μl预冷的裂解缓冲液中,并通过在冰上超声处理1分钟(以30%振幅进行0.6秒/ 0.4秒开/关循环)裂解细胞,接着以15,000rpm(21,130 xg )在4℃下10分钟。将上清液转移到微量离心管中,加入100μl4×LDS样品缓冲液和10μl2-巯基乙醇以制备可溶性级分。为了制备不溶级分,将290μl裂解缓冲液,100μl4×LDS样品缓冲液和10μl2-巯基乙醇加入到沉淀中。将样品在70°C孵育10分钟,然后在200 V的Nu-PAGE凝胶上运行,直到染料前端到达凝胶底部。在室温下,在温和搅拌下将凝胶在考马斯亮蓝染色溶液中染色1小时,随后在考马斯亮兰染色脱水溶液中在室温下轻微脱色并温和搅拌。您感兴趣的蛋白质应该出现在可溶性部分。
      注意:可以使用经典的Laemmli十二烷基硫酸钠(SDS)样品缓冲液/ SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)代替4×LDS样品缓冲液/ Nu-PAGE系统。
    10. 通过使用Avanti J-25I冷冻离心机在4℃下在JA-10转子中以6,000rpm(6,371gxg)在四个500ml离心瓶中离心10分钟来收获细菌。
      注意:每个试管中最多放置300ml,以防止离心过程中溢出。
    11. 弃去上清液并在-20°C冷冻细胞团块,直到您确认蛋白质在步骤A9中的表达和溶解度。
      注意:细菌沉淀可以在-20°C储存至少一周。或者,您可以将细菌沉淀物保存在冰上以确认表达/溶解度,然后立即进行下一步操作。
    12. 将1L细胞沉淀重新悬浮在30ml预冷的裂解缓冲液中,并将悬浮液转移到50ml锥形管中。
    13. 在冰上超声处理细胞,直到悬浮液变成水样(大约2分钟;在80%幅度下0.6秒/ 0.4秒开/关循环)。
    14. 将溶胞产物转移到橡木脊管中,并通过使用Avanti J-25I冷冻离心机在JA-17转子中在4℃以15,000rpm(30,996×gg)离心30分钟来澄清裂解物。
    15. 在离心过程中,将2ml床体积的谷胱甘肽琼脂糖4B培养基在50ml锥形管中的20ml冰冷的裂解缓冲液中洗涤两次。为了洗涤珠子,使用Allegra X-15R冷冻离心机在4℃以1,466rpm(500×g)离心5分钟使珠粒沉淀并吸出上清液。加入20ml的裂解缓冲液并颠倒管混合。以下步骤中的所有洗涤都将以相同的方式完成。
      注意:1毫升床体积的珠粒约束5毫克GST。
    16. 将澄清的裂解液加入洗过的谷胱甘肽琼脂糖珠中,并在4°C的360°垂直旋转器上旋转50ml锥形管2小时。
    17. 用40ml(20树脂体积)不含蛋白酶抑制剂的裂解缓冲液洗珠3次。
    18. 用40毫升(20树脂体积)的PreScission裂解缓冲液洗两次珠。
    19. 加入4 ml(2树脂体积)的PreScission裂解缓冲液,并将它们转移到15 ml锥形管中。
    20. 加入10μl10 mg / ml GST-PreScission洗脱蛋白质。
      在4°C的360°垂直旋转器上旋转15 ml锥形管16小时。
      注意:加入10μl10mg / ml GST-PreScission,因为我们预计最终产量10mg和1μl我们的GST-PreScission切割1mg GST标记的蛋白质。应该添加的GST-PreScission的量取决于蛋白质的表达水平和/或需要多少蛋白质。根据步骤A9中获取的测试样品中的蛋白质量,可以估计裂解物中包含的蛋白质的量。
    21. 通过使用Allegra X-15R冷冻离心机在4℃以1,466rpm(500×g)离心5分钟使珠粒团粒化并将上清液收集到15ml锥形管中。
    22. 用2ml(一种树脂体积)的PreScission切割缓冲液洗涤珠粒,并在4℃以1,466rpm(500×g)离心5分钟后收集上清液。重复洗一次,并结合之前的上清液。

    23. 使用Bradford检测法测量蛋白质浓度(详见步骤B8)。
      注意:如果浓度小于100μM,使用Amicon超浓缩器(Merck,目录号:UFC901024)浓缩洗脱的蛋白质。
    24. 通过Nu-PAGE和考马斯亮蓝染色检查蛋白质的质量。
      注意:可以使用经典的Laemmli SDS样品缓冲液/ SDS-PAGE代替4x LDS样品缓冲液/ Nu-PAGE系统。

    25. 使用透析管在至少100倍蛋白体积的储存缓冲液中透析4°C过夜
    26. 准备200微升纯化蛋白质的等分试样(浓度应为100μM或更高),并在液氮中快速冻结等分试样。存放在-80°C。

  2. 缓冲液交换到低镁缓冲液
    在5mM EDTA存在下,在含有0.5mM MgCl 2的缓冲液中进行GTP酶载荷MANT-GDP。由于我们的储存缓冲液含有5mM MgCl 2(参见食谱)以确保GTPase的稳定性,我们首先使用凝胶过滤柱将缓冲液换成低镁缓冲液(参见食谱)。

    1. 解冻200μl纯化的GTPase(100μM或更高) 注意:100μM的GTP酶通常是一个很好的起点。
    2. 将NAP-5柱夹在实验室支架上。

    3. 移除NAP-5色谱柱的顶部和底部盖子,让多余的制造商储存缓冲液流过。
    4. 用三次完全填充色谱柱,用低镁缓冲液平衡NAP-5凝胶过滤柱。
      。用于平衡的缓冲液总量大约为10毫升。
    5. 将200μl纯化的GTPase加到平衡的NAP-5柱的中心,让样品通过重力流动完全进入凝胶床。
    6. 将500μl低镁缓冲液加到柱子上,收集微量离心管中的流量。
      注意:该部分不应含有任何蛋白质。
    7. 用1毫升低镁缓冲液洗脱蛋白质。
      在微量离心管中收集100μl洗脱液 注意:大部分蛋白质应该在前4个部分洗脱。
    8. 通过Bradford分析测量每个部分的浓度,包括流量。
      1. 简言之,用4份Milli-Q水稀释Bradford试剂浓缩物的一部分,并将1ml稀释的Bradford试剂加入到一次性比色杯中。加入10μlMilli-Q水或BSA标准品(0.1,0.2,0.4和0.8 mg / ml)或1-2μl缓冲液交换GTP酶到每个比色杯中。通过涡旋混合比色杯并在室温下孵育5分钟。测量BioSpectrometer上每个部分的浓度。
      2. 从测量的浓度计算摩尔浓度。使用含有最高浓度蛋白质的部分进一步实验。缓冲液交换后的蛋白质浓度通常是起始浓度的一半。
      注意:Bio-Rad的详细使用说明可以在网上找到。有不同的方法可用于测量蛋白质浓度,包括Bradford测定法,bicinchoninic酸(BCA)测定法和分光光度法。我们更喜欢Bradford测定法,因为它是一种快速简便的方法,此协议中使用的所有缓冲液组分都可与Bradford试剂(Bio-Rad的试剂兼容性图表可在线查找)兼容。 BCA分析可能受到核苷酸交换缓冲液中所含还原剂的影响。我们不推荐使用分光光度法,因为GTP酶通常用鸟嘌呤核苷酸纯化,鸟嘌呤核苷酸在用于测量的波长处吸收。我们通常在峰值分数中获得起始浓度的一半以上。

  3. 在GTPase上加载MANT-GDP
    1. 在微量离心管中将100μl缓冲液交换的GTPase与100μl2x MANT-GDP上样缓冲液混合(见配方)。最终的反应物含有20mM HEPES-NaOH(pH7.5),50mM NaCl,0.5mM MgCl 2,5mM EDTA,1mM二硫苏糖醇(DTT),GTP酶和20倍摩尔过量的MANT-GDP。
      注:MANT-GDP的浓度取决于蛋白质浓度,必须在每次实验中计算。
    2. 在温度不超过20分钟的条件下,将反应液置于恒温混匀器中90分钟。用铝箔保护光线下的反应。
    3. 通过加入2μl1M MgCl 2(终浓度为10mM)终止反应。
      混匀反应管倒置,然后离心,然后在20°C孵育管30分钟。
    4. 在孵育过程中,用三次完整的核苷酸交换缓冲液补充平衡NAP-5凝胶过滤柱(见配方)。
      。用于平衡的缓冲液总量大约为10毫升。
    5. 为了去除未结合的MANT-GDP,将整个体积(200μl)MANT-GDP加载的GTP酶加到NAP-5色谱柱上。
      允许样品通过重力流动完全进入凝胶床。
    6. 应用500μl核苷酸交换缓冲液并收集流过。
      注意:该部分不应含有任何蛋白质。
    7. 应用1毫升核苷酸交换缓冲液洗脱蛋白质。在微量离心管中一次收集100μl。将MANT-GDP加载的GTP酶保存在冰上,避光(通过铝箔)。
      注意:大部分蛋白质应该在前4个部分洗脱。
    8. 如上所述,通过Bradford检测来测量每个部分的浓度。

  4. 测量荧光计上的加载效率
    1. 通过在核苷酸交换缓冲液中将自由MANT-GDP从40μM到0.15625μM的两倍连续稀释制备MANT-GDP标准。
      注:TECAN Infinite M1000微孔板读板器上可检测到的最低自由MANT-GDP浓度约为0.1μM。
    2. 吸取15μl核苷酸交换缓冲液,免费的MANT-GDP标准或MANT-GDP加载的GTP酶到384孔微量培养板中。
      注:此测量也适用于96孔格式。如果您使用96孔板,请将50μl每个样品吸入96孔微孔板(Corning)中。
    3. 在室温(25°C)下,每15秒读取一次荧光信号,共10次。列出了用于检测的参数(表1)。

      表1.用于检测MANT荧光的参数


    4. 为了计算加载效率,对来自所有10个时间点的每个样品的荧光强度进行平均,然后通过减去仅对缓冲液对照测量的背景荧光值来计算标准化平均值。
    5. 绘制免费的MANT-GDP浓度与测量的荧光的关系,并使用Microsoft Excel将标准曲线拟合成线性函数。使用标准曲线从其测量的荧光中确定MANT-GDP加载的GTP酶的浓度;由于MANT-GDP信号在与蛋白质结合后通常增加约两倍,因此首先将荧光信号除以二,然后使用标准曲线函数估计负载的GTP酶的浓度。这个数据的一个代表性例子(表2)和从数据中创建的免费MANT-GDP的标准曲线(图1)被显示出来。
    注意:这里计算的加载效率可能不准确,因为与GTPase结合后MANT-GDP的信号强度的增加在GTP酶之间不同。装载效率的例子可以在原稿中找到。 (Kanie等人,2017中的图S4E)。

    表2.以GTPase加载MANT-GDP获得的数据示例



    图1.使用表2中显示的数据创建的免费MANT-GDP的标准曲线

  5. 核苷酸交换测定
    1. 加入14μl核苷酸交换缓冲液或装载MANT-GDP的GTP酶至适当数量(条件数×4个技术重复)384孔微孔板的孔中。
      注意:添加到孔中的蛋白质浓度取决于上面计算的加载效率,因为样品的起始荧光强度应足够高以检测荧光的动态变化。如果加载效率很高(超过50%),则可以使用1μM或更少的GTP酶。如果装载效率低(小于10%),则应添加最高浓度的GTPase。
    2. 在测量荧光之前,用核苷酸交换缓冲液制备GppNHp的工作溶液,其浓度比GTP酶高15倍。如果您使用1μMGTP酶,请准备1.5 mM GppNHp。
    3. 在室温(25°C)下读取荧光150秒,确认荧光信号稳定。
      注意:在步骤D3中使用的相同检测参数(参见表1)可用于核苷酸交换测定。
    4. 按下显示器上的“Pause”按钮弹出平板,然后向各孔中加入1μl核苷酸交换缓冲液或GppNHp工作溶液(终浓度比GTPase高100倍)。如果要测量GEF介导的核苷酸交换,除了GppNHp之外,还要添加纯化的GEF。
      注意:应使用多通道移液管同时添加缓冲液或GppNHp以同时开始所有反应。为了确保缓冲液或GppNHp的快速添加,我们在排出分析板之前用另一个384孔板中的1μl缓冲液/ GppNHp溶液预先填充多通道移液管,然后在射出后立即将试剂吸入分析板中碟子。为避免产生任何气泡,我们将填充体积设定为1.5μl,分配体积设定为1μl。
    5. 按下“继续”按钮插入板,并在室温(25°C)下每隔15秒继续读荧光至少30分钟。&nbsp;

数据分析

  1. 将荧光计数据导出到Microsoft Excel电子表格。

  2. 每个荧光强度减去核苷酸交换缓冲液的背景强度。
  3. 在添加缓冲液或GppNHp至1之后的第一时间点设置荧光强度,并计算每个后续时间点的相对荧光。
  4. 由于反应含有比GTPase大100倍或更多的GppNHp,因此可以从数据中计算出一级假定速率常数(观察到的速率常数或K obs)。
    由于MANT结合GTP酶的荧光随着时间呈指数下降,所以能够用非线性回归拟合数据的软件程序可用于计算K obs。这些程序包括但不限于GraphPad Prism,KaleidaGraph和Grafit。我们在这里描述如何使用GraphPad Prism 7作为例子进行分析。
    1. 打开GraphPad Prism 7并创建一个新的XY表格。
    2. 在每列中输入数值,单击“分析”按钮并从XY分析列表中选择“非线性回归”。
    3. 选择'Dissociation-One phase exponential decay'来获得K obs 值。
    注意:数据应至少通过三次独立实验进行确认。

笔记

在我们的手中,具有低鸟嘌呤核苷酸亲和力的GTP酶的加载效率远低于对鸟嘌呤核苷酸具有高亲和力的典型小GTP酶的加载效率。推测这反映了低亲和力GTP酶的更高的动力学。在前一种情况下,荧光信号很大程度上受到蛋白质稀释的影响,可能是因为稀释本身会改变平衡。

食谱

  1. LB媒体(1升)
    将25克LB肉汤溶解在900毫升Milli-Q水中
    用10 M NaOH将pH调节至7.2,并用Milli-Q水将体积调至1 L

    高压灭菌器灭菌并在室温下储存
  2. TB媒体(1升)
    将50.8克极好的肉汤溶于900毫升Milli-Q水中,用Milli-Q水将体积调至1升
    转移到一个2.8L烧瓶中,并在培养箱振荡器中于28℃以250rpm旋转烧瓶,直到粉末完全溶解。
    注:可能需要不到30分钟
    通过高压灭菌器消毒

  3. 100毫克/毫升氨苄青霉素(10毫升) 将1克氨苄青霉素溶于8毫升Milli-Q水中,用Milli-Q水定容至10毫升。
    用0.22μm注射器过滤器消毒
    分装成1毫升等分试样,并储存在-20°C。

  4. 50毫克/毫升羧苄青霉素(10毫升) 将500毫克羧苄青霉素溶于8毫升Milli-Q水中,用Milli-Q水定容至10毫升。
    用0.22μm注射器过滤器消毒
    分装成1毫升等分试样。
    在-20°C储存

  5. 34毫克/毫升氯霉素(10毫升) 将340毫克氯霉素溶于8毫升100%乙醇中,并用100%乙醇将体积加至10毫升。
    用0.22μm注射器过滤器消毒
    分装成1毫升等分试样。
    在-20°C储存
  6. 含有50μg/ ml羧苄青霉素和34μg/ ml氯霉素的LB琼脂平板
    将32克LB琼脂溶解于900毫升Milli-Q水中,并将Milli-Q水定容至1升。

    高压灭菌器消毒并冷却至50°C 加入1毫升50毫克/毫升羧苄青霉素和1毫升34毫克/毫升氯霉素
    倒入盘子并在4°C储存
  7. 1个IPTG(10毫升)
    将2.383克IPTG溶解在8毫升Milli-Q水中,并将Milli-Q水定容到10毫升。
    用0.22μm注射器过滤器消毒
    分装成1毫升等分试样。
    在-20°C储存
  8. 1M Tris-HCl(pH 7.5,1L)

    溶解800毫升Milli-Q水中的Trizma碱 用浓盐酸调节pH至7.5,用Milli-Q水将体积调至1升
    高压灭菌器或过滤器来消毒
  9. 1M HEPES-NaOH(pH 7.5,1L)

    将238.30克HEPES碱溶于800毫升Milli-Q水中 用10 M NaOH调节pH至7.5,用Milli-Q水将体积调至1 L
    过滤通过一个0.2微米的一次性瓶顶过滤器消毒
    在4°C储存
  10. 5 M NaCl(1 L)
    将292.2克NaCl溶于700毫升Milli-Q水中,并用Milli-Q水将体积加至1升。

    高压灭菌器灭菌并在室温下储存
  11. 1 M MgCl 2(100毫升)
    将20.33克MgCl 2·6H 2 O溶于70毫升Milli-Q水中,用Milli-Q水定容到100毫升。

    高压灭菌器灭菌并在室温下储存
  12. 1 M DTT(30毫升)
    将4.63克DTT溶于20毫升Milli-Q中,用Milli-Q水将体积加至30毫升。
    分装成1毫升等分试样。
    在-20°C储存
  13. 0.25M EDTA(pH 8.0,1L)

    在800毫升Milli-Q水中溶解93.06克EDTA·2H2O 用NaOH调节pH至8.0
    注意:除非pH值大于8,否则EDTA不会溶解。
    用Milli-Q水将体积调至1升

    高压灭菌器灭菌并在室温下储存
  14. 10 M NaOH(500毫升)

    在不断搅拌下,将200克氢氧化钠缓慢加入400毫升Milli-Q水中 用Milli-Q水将容量调至500毫升
  15. 考马斯亮蓝染色液(1 L)
    在500毫升甲醇中溶解2.5克考马斯亮蓝R-250

    加400ml Milli-Q水和100ml冰醋酸
    过滤0.2μm一次性瓶顶过滤器
  16. 考马斯亮蓝去污溶液(1 L)
    将500ml Milli-Q H 2 O和400ml甲醇加入到1L瓶中。

    缓慢加入100 ml冰醋酸
  17. 裂解缓冲液(500毫升)

    立即在使用前将一片蛋白酶抑制剂加入到50ml裂解缓冲液中

    每次都要新鲜
  18. PreScission分裂缓冲液


    每次都要新鲜
  19. 储存缓冲液(500毫升)


    每次都要新鲜
  20. 低镁缓冲液(200毫升)

    在室温下储存
  21. 2个MANT-GDP上样缓冲液(100μl)

    每次都要新鲜。 MANT-GDP的浓度应该根据GTPase浓度计算
  22. 核苷酸交换缓冲液(100毫升)

    每次都要新鲜

致谢

该协议是从以前的工作改编的(Kanie et。,2017)。作者要感谢Keene Abbott和Henrietta Bennett仔细阅读手稿。这项工作得到了巴克斯特干细胞研究实验室,斯坦福大学研究部,斯坦福癌症中心,美国国立卫生研究院拨款给PKJ的R01GM114276和R01GM121565以及上原纪念基金会和人类前沿科学计划给TK的博士后支持。作者宣称没有利益冲突。

参考

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  2. Bos,J.L.,Rehmann,H。和Wittinghofer,A。(2007)。 GEFs和GAPs:控制小G蛋白的关键因素 Cell 129(5):865-877。
  3. Eberth,A。和Ahmadian,M.R。(2009)。 体外 GEF和GAP分析。 Curr Protoc Cell Biol Chapter 14:Unit 14.9。
  4. Gasper,R.,Meyer,S.,Gotthardt,K.,Sirajuddin,M.和Wittinghofer,A。(2009)。 需要两个探戈:通过二聚化调节G蛋白。 Nat Rev Mol Cell Biol 10(6):423-429。
  5. Hiratsuka,T。(1983)。 可用作各种酶底物的腺嘌呤和鸟嘌呤核苷酸的新核糖修饰荧光类似物 Biochim Biophys Acta 742(3):496-508。
  6. Ivanova,A.A.,Caspary,T.,Seyfried,N.T.,Duong,D.M.,West,A.B.,Liu,Z。和Kahn,R.A。(2017)。 纯化哺乳动物ARL13B蛋白的生物化学表征表明它是一种非典型的GTPase和ARL3鸟嘌呤核苷酸交换因子( GEF)。 J Biol Chem 292(26):11091-11108。
  7. John,J.,Sohmen,R.,Feuerstein,J.,Linke,R.,Wittinghofer,A。和Goody,R. S.(1990)。 核苷酸与无核苷酸H-ras p21相互作用的动力学 生物化学 29(25):6058-6065。
  8. Kanie,T.,Abbott,K.L.,Mooney,N.A.,Plowey,E.D。,Demeter,J。和Jackson,P.K。(2017)。 CEP19-RABL2 GTPase复合物结合IFT-B以启动在睫状体基底处的鞭毛内运输。 a> Dev Cell 42(1):22-36 e12。
  9. Maurer,T.,Garrenton,LS,Oh,A.,Pitts,K.,Anderson,DJ,Skelton,NJ,Fauber,BP,Pan,B.,Malek,S.,Stokoe,D.,Ludlam,MJ, Bowman,KK,Wu,J.,Giannetti,AM,Starovasnik,MA,Mellman,I.,Jackson,PK,Rudolph,J.,Wang,W。和Fang,G。(2012)。 小分子配体结合Ras中的一个独特口袋并抑制SOS介导的核苷酸交换活性。 / a> Proc Natl Acad Sci USA 109(14):5299-5304。
  10. Pai,E.F.,Krengel,U.,Petsko,G.A.,Goody,R.S.,Kabsch,W。和Wittinghofer,A。(1990)。 H-ras p21三磷酸构象精确晶体结构,分辨率1.35 A:对机制的影响GTP水解。 EMBO J 9(8):2351-2359。
  11. Price,H.P.,Hodgkinson,M.R.,Wright,M.H.,Tate,E.W.,Smith,B.A.,Carrington,M.,Stark,M.and Smith,D.F。(2012)。 囊泡相关微管蛋白结合蛋白ARL6(BBS3)在鞭毛延伸中的作用 Trypanosoma brucei 。 Biochim Biophys Acta 1823(7):1178-1191。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Kanie, T. and Jackson, P. K. (2018). Guanine Nucleotide Exchange Assay Using Fluorescent MANT-GDP. Bio-protocol 8(7): e2795. DOI: 10.21769/BioProtoc.2795.
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