High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

Summary

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

Abstract

A protocol on how to perform high-precision interdye distance measurements using Förster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all "dimensions" of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 Å in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 Å for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

Introduction

A fundamental goal of structural biology studies is to unravel the relationship between the structure and function of biomolecular machines. The first visual impression of biomolecules (e.g., proteins and nucleic acids) occurred in the 1950s through the development X-ray crystallography^1,2. X-ray crystallography provides high-resolution, static structural information constrained by the crystal packing. Therefore, the inherent immobility of X-ray structural models shuns the dynamic nature of biomolecules, a factor that impacts most biological functions^3,

Protocol

1. PBS Buffer Preparation and Chamber Treatment

NOTE: Wear a laboratory coat and disposable gloves when performing wet chemical experiments. Use eye protection when aligning the laser.

1. PBS buffer preparation
   1. Dissolve 4.5 g of Na₂HPO₄, 0.44 g of NaH₂PO₄, and 3.5 g of NaCl in 400 mL of distilled water. Ensure a pH of 7.5 and sterilize the solution by autoclaving on a liquid cycle for 1 h (depending on the autoclave system).
   2. Take 15 mL of the PBS solution and mix it with 0.1 g of charcoal. Filter the mix by using a regular 20 mL syringe filter with ....

Results

In typical smFRET experiments using an MFD setup (laser lines: 485 nm at 60 µW and 640 nm at 23 µW, section 5.1), the fluorescence sample is diluted to a low-picomolar concentration (10^-12 M = 1 pM) and placed in a confocal microscope, where a sub-nanosecond laser pulse excites labeled molecules freely diffusing through an excitation volume. A typical confocal volume is <4 femtoliters (fL). At such low concentrations, only single molecules are detected one at a time. The emi.......

Discussion

In this work, the protocol to align, calibrate, and measure interdye distances with high precision using PIE-MFD single-molecule FRET experiments is presented. By carefully calibrating all instrumental parameters, one can increase the accuracy of the measured distances and reach Angstrom accuracy. To do so, various multidimensional histograms are used to analyze and identify populations for further characterization. Using the mean macro time to verify the stability of the measured samples, it is possible to correct for d.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
charcoal	Merck KGaA	K42964486 320
syringe filter	Fisherbrand	09-719C	size: 0.20 µm
chambered coverglass	Fisher Scientific	155409	1.5 borosilicate glass, 8 wells
microscope cover glass	Fisher Scientific	063014-9	size: 24 x 60-1.5
Nuclease free water	Fisher Scientific	148859	nuclease free
tween-20	Thermo Scientific	28320	10% solution of Polysorbate 20
acceptor DNA strand (High FRET)	Integrated DNA Technologies	178124895	5´-d(CGG CCT ATT TCG GAG TTG TAA ACA GAG AT(Cy5)C GCC TTA AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)
acceptor DNA strand (Low FRET)	Integrated DNA Technologies	177956424	5´-d(CGG CCT ATT TCG GAG TTG TAA ACA GAG ATC GCC TT(Cy5)A AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)
donor DNA strand	Integrated DNA Technologies	177951437	5´ -d(GCA ATA CTT GGA CTA GTC TAG GCG AAC GTT TAA GGC GAT CTC TGT TT(Alexa488)A CAA CTC CGA AAT AGG CCG)
DNA strand (No FRET)	Integrated DNA Technologies		5´ -d(CGG CCT ATT TCG GAG TTG TAA ACA GAG ATC GCC TTA AAC GTT CGC CTA GAC TAG TCC AAG TAT TGC)
thermal cycler	Eppendorf	E6331000025	nexus gradient
Alexa Fluor 488 C5 Maleimide	Thermo Scientific	A10254	termed cyan-green fluorophore in the manuscript
Alexa Fluor 647 C2 Maleimide	Thermo Scientific	A20347	termed far-red fluorophore in the manuscript
Rhodamine 110	Sigma-Aldrich	83695-250MG
Rhodamine 101	Sigma-Aldrich	83694-500MG
LB Broth, Miller	Fisher Scientific	BP1426	For culture of E. coli
Ampicillin	Sigma-Aldrich	A0166	Used at 100 µg/mL final concentration in selective LB medium to maintain plasmid selection
Tetracyline	Calbiochem	58346	Used at 12.5 µg/mL final concentration in selective LB medium to maintain gor (flutathione reductase) mutation in Origami B(DE3) strains to facilitate disulfide bond oxidation
Kanamycin	Fisher Scientific	BP906-5	Used at 15 µg/mL final concentration in selective LB medium to maintain trxB (rhioredoxin reductase) mutation in B(DE3) stains to facilitate disulfide bond oxidation
Origami B(DE3) Competent Cells	Millipore	70837-3	Competent E. coli cells for expression of protein with disulfide bridges
Isopropyl-β-D-thiogalactopyranoside (IPTG)	Fisher Scientific	BP1755	For induction of E. coli protein expression
HiTrap Chelating HP	GE Life Sciences	17-0409-01	For Large-scale FPLC Purification of His-tagged protein
Imidazole	Sigma-Aldrich	56749
Ni-NTA Agarose	Qiagen	30210
PD-10 Desalting Column	GE Life Sciences	17-0851-01
AktaPurifier	GE Life Sciences	28406264	FPLC Instrument
Dialysis tubing	Spectrum labs	132562	15 kD MWCO 24 mm Flath width, 10 meters/roll
Dichroics	Semrock	FF500/646-Di01-25x36	500/646 BrightLight
50/50 Beam splitter polarizer	Qioptiq Linos	G33 5743 000	10 x 10 film polarizer
Green pass filer	Chroma	ET525/50m	ET525/50m 25 mm diameter mount
Red pass filter	Chroma	ET720/150m	ET720/150m 25 mm diameter mount
Power Meter	ThorLabd	PM200
UV-Vis spectrophotometer	Varian	Cary300Bio
Fluorolog 3 fluorometer	Horiba	FL3-22-R3
Fluorohub TCSPC controller	Horiba	Fluorohub-B	TCSPC electronics for ensemble measurements
NanoLed 485L	Horiba	485L	Blue diode laser
NanoLed 635L	Horiba	635L	Red diode laser
Olympus IX73 Microscope	Olympus	IX73P2F	Microscope frame
PMA 40 Hybrid Detector	PicoQuant GmbH	932200, PMA 40	Optimized for green detection
PMA 50 Hybrid Detector	PicoQuant GmbH	932201, PMA 50	Optimized for ed shifter sensitivity
485 nm laser	PicoQuant GmbH	LDH-D-C-485
640 nm laser	PicoQuant GmbH	LDH-D-C-640
Hydraharp 400 and TTTR acqusition software	PicoQuant	930021	Picosecond event timer and Time Correlated Single Photon Coutning Unit, includes TTTR acqusition software
SEPIA II SLM 828 and SEPIA software	PicoQuant	910028	Laser driver for picosecond pulses , includes SEPIA software controller.
computer	Dell	optiplex 7010	cpu: i7-3770 ram:16GB
FRET Positioning and Screening (FPS) software	Heinrich Heine Unviersity		It include the Accesibel Volume clacualtor available at http://www.mpc.hhu.de/software/fps.html
MFD suite	Heinrich Heine Unviersity		It includes the BIFL software package Paris; Margarita for visualization of the multiparameter hisotrams, and Probability Distribution Analysis software availabel at http://www.mpc.hhu.de/software/software-package.html