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,

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.<....

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