FRET Dilution Assay for Analyzing Dynamic Exchange Between Protein Assemblies

Summary

This protocol describes the purification and covalent labeling of reflectin A1 as well as a FRET dilution assay for characterizing the dynamic exchange of reflectin protein between protein assemblies as a function of age.

Abstract

As the molecular driver for tunable iridescence in cephalopods, the tunable phase behavior of reflectin A1 protein continues to be a focus of biomaterial engineering. Modulating salt concentration and protein net charge density of reflectin A1 drives the protein to form dynamic assemblies as intermediates to liquid-liquid phase separation. Reflectin assemblies, while limited in size by the extent of charge neutralization of the protein's cationic, Coulombic repulsion, are initially in dynamic exchange with monomers or oligomers from the surrounding solution. A novel fluorescence resonance energy transfer (FRET) dilution assay was used, in conjunction with dynamic light scattering (DLS) and protein concentration assays, to characterize the two-way flux of protein between reflectin A1 assemblies and a dilute phase as a function of assembly age. This FRET dilution assay distinguishes between one-way and two-way flux of protein into and out of protein assemblies and, therefore, can be applied during assembly formation. Differentiating between dynamic and kinetically arrested protein assemblies is crucial to understanding their biophysical origins, and this novel FRET dilution assay can be adapted to supplement biophysical investigations of other protein assemblies.

Introduction

Tunable iridescence in loliginid squid is mediated by reflectin proteins in iridocyte cells^1,2,3. Iridocytes contain extensive and regular membrane invaginations that form stacks of protein-dense Bragg lamellae, which reflect light in an angle- and wavelength-dependent manner^1,4,5,6. An acetylcholine-triggered signal cascade culminates in the phosphorylation of cationic reflectin proteins^1,2,7, reducing intra- and inter-protein electrostatic repulsion and driving the condensation of reflectins in Bragg lamellae from 10-50 nm hydrodynamic radius particles to a dense liquid condensate^3,8,9. Reflectin A1 in vitro forms size-stable 10+ nm radius assemblies whose sizes are precisely tuned by protein net charge density^10,11,12, and this tunable colloidal behavior has garnered much bioengineering interest in designing reflectin-based tunable biophotonic materials^{13,14,15,16,17,18}. It was recently demonstrated that NaCl drives reflectin A1 assembly and LLPS (Liquid-Liquid phase separation) by entropically increasing the energetic contribution of the hydrophobic effect and by screening the repulsive charges of the alternating cationic linkers¹⁹.

For protein-dense liquid-like condensates, dynamic exchange between the dense phase and dilute phase can be readily analyzed using whole-droplet FRAP (Fluorescence Recovery After Photobleaching)²⁰; an analogous assay for nanometer-scale protein assemblies is needed. Recent reports of physiologically relevant protein assemblies as intermediates to LLPS^19,21,22 suggested the need for equivalent methods to interrogate assembly dynamics and dynamic exchange between assemblies and the coexisting dilute phase. Protein clusters can include kinetically arrested glass-like colloids produced by arrested spinodal decomposition, dynamic equilibrium assemblies that exchange monomers or oligomers from the dilute phase, and metastable clusters^{23,24,25,26,27}. Age-dependent protein assembly dynamics are therefore key to understanding the physical processes driving the formation of protein assemblies within the context of more complex phase behavior.

FRET-based approaches analyzing the interaction between assemblies of donor fluorophore-labeled protein and assemblies of acceptor-fluorophore labeled protein have been used to characterize the dynamics of metastable protein clusters²¹. Upon mixing these clusters, the emergence of FRET demonstrates exchange and transport of protein into the clusters²¹. However, for some proteins, such as reflectin A1, clusters continuously form and grow over the duration of the experiment¹⁹. For such proteins, an approach that deconvolutes one-way transport of protein into clusters, as might occur during the continual formation of dynamically arrested clusters, and two-way transport of protein between assemblies is needed.

A FRET dilution approach accomplishes this via fluorophore dilution (Figure 1), in which a decrease in FRET signifies two-way transport of protein between assemblies. Protein assemblies of reflectin protein were prepared that contained equal amounts of protein labeled with either acceptor-fluorophore or donor-fluorophore. These co-labeled reflectin assemblies were diluted with a solution containing unlabeled reflectin assemblies, and dynamic exchange of protein between assemblies was analyzed as a function of assembly age. This method distinguishes the continual formation of kinetically arrested protein clusters from dynamic clusters, including but not limited to metastable clusters, clusters that grow due to Ostwald ripening or similar coarsening processes, clusters that grow by fusion, and clusters that are not intermediates to LLPS. It does not solely distinguish between these different mechanisms of dynamic exchange, which requires orthogonal methods such as dynamic light scattering, nanoparticle tracking analysis (NTA), and Bradford assays^19,21. This protocol describes methods for the purification of reflectin A1 wild-type and the single cysteine-containing mutant A1-C232S, the covalent labeling of A1-C232S, and the FRET dilution analysis.

Protocol

The reagents and the equipment used are listed in the Table of Materials.

1. Protein purification and covalent labeling

1. Transformation
   1. Clone reflectin A1 and reflectin A1 C232S genes (GenBank KF661517.1) into the pj411 plasmids.
   2. Transfect Rosetta 2 (DE3) cells with plasmids using heat shock treatment²⁸.
   3. Plate transfected cells onto agar containing 50 µg/mL kanamycin.
   4. Select an isolated colony and add to 5 mL TB (Terrific Broth) containing 50 µg/mL kanamycin and incubate at 37° C on a shaker at 250 rpm overnight.
   5. Isolate plasmids using the plasmid isolation kit protocol for sequencing.
2. Protein expression, lysis, and inclusion body purification
   1. Add 5 mL of culture to 1 L LB (Lysogeny Broth) with 50 µg/mL kanamycin, and incubate at 37° C while shaking. At an A₆₀₀ of 0.6-0.7, induce expression by adding IPTG (isopropyl-β-D-thiogalactopyranoside) to a final concentration of 5 mM.
   2. After 16 h, pellet cells were centrifuged at 12,000 x g for 10 min. Decant supernatant and freeze cell pellets in a -80° C freezer.
   3. Lyse cells and purify inclusion bodies using inclusion body purification reagent per manufacturer's protocol, including 20 min treatment with 1 µL (25-29 units) of benzonase and 3-5 KU of lysozyme per mL inclusion body purification reagent after initial resuspension of cell pellet. Purified inclusion bodies will appear as dark brown pellets.
3. FPLC (Fast Protein Liquid Chromatography)
   1. Prepare 2 L of 8 M urea: 5% v/v acetic acid solution (Buffer A) and 1 L of 8 M guanidinium hydrochloride: 5% v/v acetic acid solution (Buffer B). Filter both using a 0.22 µm vacuum flask filter.
      CAUTION: Avoid inhalation, eye, and skin contact with guanidinium hydrochloride and wear proper PPE (Personal Protective Equipment) when handling. Dispose of in a dedicated waste container.
   2. Resuspend inclusion body pellets in 8 M urea: 5% acetic acid solution by vigorous stirring with a magnetic stir bar. The solution will appear turbid. Pre-chill centrifuge to 4 °C, centrifuge at 18,000 x g at 4 °C for 70 min, and collect supernatant for FPLC. The supernatant will appear brown and transparent.
   3. Using a flow rate of 2 mL/min for all following steps, equilibrate 10 mL of the cation exchange column with 5 column volumes (CVs) of 95% Buffer A, 5% Buffer B, and load the sample. Wash out unbound sample with 5 CVs, then perform the step gradient: 5 CVs of 94% Buffer A/6% Buffer B, then 5 CVs 10% Buffer B. Reflectin A1 elutes at 10% Buffer B.
   4. Assess the purity of fractions corresponding to the initial step change to 10% Buffer B by SDS-PAGE and pool if similar. Pre-chill a centrifuge to 4 °C and concentrate fractions to a final volume of 4-5 mL using a 15 mL 10 kDa MWCO spin concentrator.
4. HPLC (High Performance Liquid Chromatography)
   1. Prepare 2 L of 18 mΩ water with 0.1% trifluoroacetic acid (TFA) and 2 L of acetonitrile with 0.1% TFA. Filter both using a 0.22 µm vacuum flask filter.
      CAUTION: TFA is a strong fuming acid. Handle in a fume hood, wearing appropriate PPE, and transfer only using glass. Avoid inhalation, eye, and skin contact with acetonitrile, and wear proper PPE when handling. Dispose of in a dedicated waste container.
   2. Using a 1 mL/min flow rate for all following steps, equilibrate the C4 column with 5 CVs of 85% 18 mΩ water, 0.1% TFA, and 15% acetonitrile, 0.1% TFA.
   3. Begin flowing and inject the sample. Flow 2 CVs, then begin constant gradient to 80% acetonitrile, 0.1% TFA over 5 CVs. Reflectin A1 typically elutes at 73% acetonitrile.
   4. Shell-freeze fractions that contain reflectin protein in 5 mL tubes using an ethanol and dry ice bath. Lyophilize, and store at - 80 °C. Protein will appear as thin white sheets.
5. Unlabeled protein stock preparation
   1. Prepare 4 L of pH 4, 25 mM acetic acid buffer (21.05 mM acetic acid, 3.95 mM sodium acetate trihydrate) using glacial acetic acid and sodium acetate trihydrate. Adjust pH if needed with 4 M HCl or 4 M NaOH. Filter using a 0.22 µm vacuum filter.
      CAUTION: Handle strong acids/bases wearing appropriate PPE. Never add water to concentrated strong acid or base.
   2. Thaw the tube containing lyophilized reflectin protein, and with the tube in an ice bath, resolubilize with pH 4, 25 mM acetic acid buffer. A completely resolubilized protein solution will appear colorless with no visible turbidity or precipitate.
      1. Using a 10 kDa molecular weight cut off dialysis cassette, dialyze resolubilized reflectin protein in 1000x sample volume of pH 4, 25 mM acetic acid buffer pre-chilled to 4 °C using two 12 h changes at 4 °C.
   3. Dilute the sample with a pH 4, 25 mM acetic acid buffer to obtain a final protein concentration of 100 µM and a final volume of at least 150 µL. With a UV-VIS spectrometer, measure the absorbance at 280 nm and use the molar absorption coefficient²⁹ of reflectin A1 (123,685 M^-1 cm^-1) to determine protein concentration.
   4. Transfer the reflectin protein stock to a 0.5 mL snap-cap microcentrifuge tube and store at 4 °C up to 7 days. Immediately prior to experiments, centrifuge reflectin protein stock at 4 °C in a pre-chilled microcentrifuge at 18,000 x g for 15 min. Prepare reflectin A1 C232S stock similarly using the molar absorption coefficient of 123,540 M^-1 cm^-1.
6. Protein labeling
   1. Separately prepare 10 mg/mL stocks of FMTS (fluorescein-methanethiosulfonate ) and SRMT (sulforhodamine methanethiosulfonate) by adding 1 mL of dry dimethyl-sulfoxide (DMSO) to a vial containing 10 mg of fluorescent probe and pipette mixing.
      CAUTION: Avoid inhalation, eye, and skin contact with methanethiosulfonate reagents.
   2. Add 7.7 µL of the 10 mg/mL FMTS stock or 10.4 µL of the 10 mg/mL SRMT stock to a pH 4, 25 mM acetic acid buffer to a final volume of 150 µL and final concentration of 500 µM FMTS or SRMT. The solution will appear turbid. Aliquot and freeze the remainder of fluorescent probe stocks at -80 °C.
   3. Combine with 150 µL of 100 µM reflectin protein stock and thoroughly pipette mix. Final protein concentration is 50 µM, and final fluorescent probe concentration is 500 µM. The turbidity of the solution will decrease.
   4. Wrap in foil, and incubate upright with gentle orbital stirring at room temperature for 4 h, then overnight at 4 °C. The turbidity of the solution should not change.
   5. If any precipitate is present, resuspend by vigorous pipette mixing. The solution will be turbid. Concentrate reflectin using a 10,000 kDa MWCO centrifugal filter to a target volume of 100 µL and a concentration of 150 µM.
   6. Use the same HPLC and lyophilization method as for protein purification to remove unbound fluorescent probe and store protein at - 80 °C wrapped in foil. Resolubilize the labeled protein using the same method for the unlabeled protein. Determine the degree of labeling.
      NOTE: Resolubilized fluorescently labeled reflectin protein will appear yellow-green (C199-F) or dark red (C199-R) with no turbidity or precipitation.
7. Co-labeled protein stock preparation
   1. Add 27 µL of 100 µM A1 protein stock to a 0.5 mL snap-cap microcentrifuge.
   2. Add 1.5 µL of fluorescein-labeled reflectin A1-C232S (referred to as C199-F to denote the amino acid position of fluorescein) to the same tube.
   3. Add 1.5 µL of sulforhodamine-labeled reflectin A1-C232S (referred to as C199-R to denote the amino acid position of fluorescein) to the same tube. Final concentrations are 90 µM A1, 5 µM C199-F, and 5 µM C199-R.
   4. Wrap in foil and store at 4 °C up to 7 days.

2. FRET time series

1. Preparing buffers and filtration
   1. Prepare 500 mL of 25 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer. Adjust the pH of the solution to 7 by slowly adding 4 M sodium hydroxide. Filter 10 mL using a 10 mL syringe and a 0.22 µm syringe filter.
2. Centrifuging protein
   1. Pre-chill tabletop microcentrifuge to 4 °C. Centrifuge unlabeled and co-labeled reflectin A1 stocks at 18,000 x g for 15 min at 4 °C.
3. Preparing labeled A1 assemblies
   1. To prepare three replicates, pipette 9.6 µL of MOPS buffer (pH 7, 25 mM) into three 0.5 mL snap-cap microcentrifuge tubes.
   2. Add 0.4 µL of labeled protein stock to each tube already containing 9.6 µL of MOPS buffer to a final protein concentration of 4 µM. Slowly pipette mix twice by aspirating and dispensing the entire sample volume.
   3. Immediately wrap in tin foil and place in the incubator at 25 °C. This and all following solutions of assemblies lack apparent turbidity or precipitation before or after incubation.
4. Preparing unlabeled A1 assemblies
   1. To prepare three replicates, pipette 38.4 µL of MOPS buffer into three 0.5 mL snap-cap microcentrifuge tubes.
   2. Add 1.6 µL of reflectin A1 protein stock to each tube already containing 38.4 µL of MOPS buffer to a final protein concentration of 4 µM. Slowly pipette mix twice by aspirating and dispensing the entire sample volume.
   3. Immediately wrap in tin foil and place in the incubator at 25 °C.
5. Timing and mixing of co-labeled and unlabeled A1 assemblies
   1. Order the preparation of assemblies as follows: simultaneously and separately prepare assemblies of experimental one co-labeled reflectin and experimental one unlabeled reflectin. Repeat steps 2.3 and 2.4.
   2. Mix by adding 40 µL of unlabeled A1 assembly experimental to 10 µL of respective co-labeled experimental, and gently pipette mix by aspirating and dispensing the entire volume twice. Measurements will be performed 24 h after this step.
   3. The time between driving assembly of reflectin A1 by dilution into MOPS buffer (pH 7, 25 mM) and mixing of labeled and unlabeled assemblies is the assembly age. Assemblies' ages used for this data were 0 min, 1 min, 10 min, 30 min, 60 min, 120 min, 180 min, 540 min, 1080 min and 1620 min.
6. Controls
   1. Positive control
      1. From the A1 and labeled protein stocks, prepare 20 µL of protein stock that is 80 µM A1 and 20 µM co-labeled protein stock.
      2. Add 38.4 µL of MOPS buffer to a 0.5 mL snap-cap microcentrifuge tubes, then add 1.6 µL of 80 µM A1/20 µM labeled protein and pipette mix twice by slowly aspirating and dispensing entire volume. Immediately cover in foil and place in the incubator at 25 °C. Repeat for a total of 9 samples.
   2. Negative control
      1. Add 0.4 µL of labeled stock to a 0.5 mL snap-cap microcentrifuge tube already containing 9.6 µL of MOPS buffer. Slowly pipette mix twice by aspirating and dispensing the entire sample volume.
      2. Add 40 µL of MOPS buffer and pipette mix twice by aspirating and dispensing the entire sample volume. Immediately cover in foil and place in the incubator at 25 °C. Repeat for a total of 9 samples.
7. Cuvette and buffer blank
   1. Set the fluorimeter to excite at 488 nm and record continuous spectra from 520-700 nm.
   2. Fill a clean quartz sub-micro fluorimeter cuvette with 50 µL of MOPS buffer (pH 7, 25 mM) and measure.
   3. Remove buffer.
8. Experimental procedure
   NOTE: All experimentals should be recorded 24 h after mixing co-labeled and unlabeled reflectin A1 assemblies. Gently pipette 50 µL of the experimental solution into the cuvette and measure.
   1. Remove the sample, rinse with 500 µL acetic acid buffer, then with 500 µL 0.22 µm-filtered 18 mΩ water. Perform buffer blank.
9. Controls
   1. Measure at 1440 min, 1960 min, and 3060 min after preparing controls (corresponding to assembly ages of 0 min, 520 min, and 1620 min). These measurements conclude the experimental portion of the protocol.

3. Data analysis

1. Subtract the spectra of the buffer blank from the experimental spectra.
2. Determine the ratio of Em₅₈₈/Em₅₂₀.
3. For each replicate, then determine the mean and standard deviation of each group of three replicates. Plot Em₅₈₈/Em₅₂₀ as a function of assembly age.

Results

After diluting reflectin A1 assemblies containing both donor fluorophore-labeled protein and acceptor fluorophore-labeled protein with unlabeled reflectin A1 assemblies, an initial decrease in FRET emission at 588 nm relative to undiluted assemblies was observed (Figure 2A). FRET for assemblies of 0 min age was similar to the positive control, and FRET for assemblies of 1620 min age (the time between assembly formation and the mixing of unlabeled and labeled reflectin assemblies) was less th...

Discussion

A common point of protein loss during purification is incomplete resuspension of inclusion body pellets for all steps in the inclusion body purification protocol, and final resuspension of purified inclusion bodies in 8 M urea 5% v/v acetic acid. Inclusion body resolubilization can take up to 24 h of vigorous stirring with a small magnetic stir bar in a 40 mL conical tube. To avoid significant protein loss to filters, filtration preceding FPLC and HPLC was replaced with centrifugation at 15,000 x g for 70 min to...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10 μL capacity micropipette
2 μL capacity micropipette
2-[(5-Fluoresceinyl)aminocarbonyl]ethyl Methanethiosulfonate	Santa Cruz Biotechnologies	sc-216155	CAS 351330-42-2
20 μL capacity micropipette
200 μL capacity micropipette
3-(N-Morpholino)propanesulfonic acid, 4-Morpholinepropanesulfonic acid	Sigma-Aldrich	M1254	CAS 1132-61-2
Acetic acid	Sigma-Aldrich	A6283	CAS 64-19-7
Acetonitrile	Sigma-Aldrich	34851-1L	CAS 75-05-8
Agar powder	Carolina Biological	842133	CAS 9002-18-0
Aluminum Foil
Amicon Ultra Centrifugal Filter, 10 kDa MWCO	Millipore Sigma	UFC501008	0.5 ml capacity
Amicon Ultra Centrifugal Filter, 10 kDa MWCO	Millipore Sigma	UFC901008	15 ml capactity
analytical balance
Benzonase	Millipore-Sigma	70664-3	CAS 9025-65-4
BugBuster Protein Extraction Reagent	Millipore Sigma	70584-3
Cary Eclipse Fluorescence Spectrometer	Cary-Eclipse
Cary Eclipse Software
Corning 50 ml centrifuge tubes	Millipore-Sigma	CLS430829
Disposable syringes, Luer-Lok 10 mL	Cole-Parmer	UX-12915-02
DMSO	Sigma-Aldrich	D8418	CAS 67-68-5
Dry Ice
Dual-pump FPLC system
Dual-pump HPLC system
Ethanol	Millipore Sigma	EX0280-3	CAS 64-17-5
Floor standing centrifuge rotor 500 ml vial capacity
Floor-standing centrifuge
Freezer, -80° C
Fume hood
Guanidine hydrochloride	Thermo Scientific	AAA135430B	50-01-1
HiTrap SP HP cation exchange chromatography column	Cytiva	17115101
Hydrochloric Acid	Sigma-Aldrich	258148-25ML	CAS 7647-01-0
Incubator
Isopropyl β-D-1-thiogalactopyranoside	Sigma-Aldrich	I6758-1G
Kanamycin sulfate	Sigma-Aldrich	K1637	CAS 25389-94-0
Lyophilizer
Lysozyme	Thermo Scientific	90082
Magnetic stir bar
Magnetic stir plate
Micro pH electrode S7	Metler Toledo	51343160
Microcentrifuge with temperature control
Millex PVDF syringe filter	Millipore	SLGVR33RS	0.22 μm, 33mm diam.
Milli-Q Millipak Filter	Millipore Sigma	MPGP002A1
Milli-Q UltraPure Water Purification System	Millipore Sigma
NaCl	Thermo Scientific	AA12314A9	CAS 7647-14-5
Nalgene centrifuge bottles, style 3120	Millipore-Sigma	B1283-4EA	For pelleting bacteria
Orbital lab  shaker with incubator
PC
Petri dishes	Sigma-Aldrich	P5731-500EA
pj411 plasmid	DNA 2.0
Protein LoBind  Conical Tubes	Eppendorf	EP30108302	5 ml capacity
Protein LoBind  Microcentrifuge Tubes	Eppendorf	EP022431102	0.5 ml capacity
Pyrex baffled Erlenmeyer flask 1,000 ml	Millipore-Sigma	CLS44501	For incubating bacterial cultures
Pyrex Griffin beakers 2,000 mL	Millipore-Sigma	CLS10002L	For all buffer and solution prep
Pyrex round media storage bottles 1,000 ml	Millipore-Sigma	CLS13951L	For storage of all buffer and solution prep, use with vacuum flask filters
Qiaprep Spin Miniprep Kit	Qiagen	27104
Refridgerator
Rosetta 2(DE3) Competent Cells	Novagen	71397-3
Slide-a-lyzer Dialysis Cassettes 10K MWCO	Thermo Scientific	PI66383	0.5 ml capacity
Sodium acetate trihydrate	Sigma-Aldrich	S8625	CAS 6131-90-4
Sodium hydroxide	Sigma-Aldrich	567530-250GM	CAS 1310-73-2
Steritop Vacuum Bottle Top Filter	Millipore Sigma	S2GPT05RE	0.22 μm sterile filter
Sub-microcuvette, 50 uL capacity	Starna Cells	16.40F-Q-10/Z15
sulforhodamine methanethiosulfonate	Santa Cruz Biotechnologies	sc-220172	CAS 386229-71-6
Trifluoroacetic acid	Sigma-Aldrich	106232-25G	CAS 407-25-0
Tryptone	Spectrum Chemical	T1333
Urea	Thermo Scientific	AAA123600E	CAS 57-13-6
UV-VIS spectrometer
Yeast Extract	Gibco	211929