Single-Molecule FRET Imaging for Observing the Conformational Dynamics of Dynamin-Like GTPase Atlastin

Summary

The functions of dynamin superfamily proteins depend on conformational changes coupled with GTP hydrolysis. A system is described using the single-molecule FRET (smFRET) technique to monitor the conformational dynamics of dynamin-like GTPase atlastin in different nucleotide-loading states.

Abstract

The rigid-body rotation of the three-helical middle domain (3HB) relative to the GTPase domain of dynamin-like protein atlastin (ATL) is a crucial driver of homotypic membrane fusion within the endoplasmic reticulum (ER). Disruptions in this process have been associated with hereditary spastic paraplegia (HSP), a neurodegenerative disorder. Structural and biochemical studies suggest that the conformational changes in ATL are linked to GTP hydrolysis, but real-time visualization of these conformational dynamics during the GTP hydrolysis cycle remains challenging. To better understand the mechanical mechanisms behind ATL function, single-molecule Förster resonance energy transfer (smFRET) was utilized. Three specific strategies were employed to immobilize the N-terminal cytosolic region of human ATL1 (ATL1cyto) in a streptavidin-coated microfluidic chamber, facilitating the application of intramolecular and intermolecular smFRET imaging. This allowed precise monitoring of protein conformations in various nucleotide-loading states, providing direct insights into individual molecular behaviors. This method can be applied to study other mechanochemical proteins as well.

Introduction

In eukaryotic cells, the dynamin superfamily proteins mediate the remodeling of biological membranes, including membrane tubulation, fission, and fusion^1,2,3. Mutations in these proteins lead to a variety of human diseases, such as neurodegenerative diseases. The members of the dynamin superfamily typically contain a GTPase domain, a middle domain consisting of helical bundles, a motif for membrane binding, and a GTPase effector domain (GED). One such dynamin-like GTPase is atlastin (ATL), which catalyzes homotypic membrane fusion of the endoplasmic reticulum (ER) to form a n....

Protocol

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

1. Preparation of modified coverslip for protein immobilization chamber

1. Preparation
   1. Prepare 50 mL of piranha solution (compose of concentrated sulfuric acid and hydrogen peroxide in a ratio of 3:1) by slowly adding hydrogen peroxide to concentrated sulfuric acid to prevent excessive temperatures. Place it carefully in a fume hood.
   <.......

Discussion

FRET technology is based on energy transfer between two fluorescent dyes (donor and acceptor). In the case of their proximity to each other (usually 1-10 nm), the excited donor transfers its energy to the acceptor, resulting in a decrease in the donor fluorescence intensity and an increase in the acceptor fluorescence intensity.

In smFRET experiments, the molecules are diluted to very low concentrations and immobilized on slides, and the FRET signals of individual molecules are detected by TIR.......

Materials

Name	Company	Catalog Number	Comments
10 K Centrifugal Filters	Amicon	UFC901096
3-Aminopropyltriethoxysilane (APTES)	Sigma-Aldrich	440140
45 Ti rotor
532-nm laser	Olympus
640-nm laser	Olympus
7 K MWCO spin desalting columns	Thermo Scientific	89882
Adenosine 5-triphosphate disodium salt (ATP)	Roche	11140965001
Benzoic acid	Sigma-Aldrich	242381
Biotin-PEG-SVA-5000	Laysan Bio	170-124
Biotinylated anti His antibody	Bioss Antibodies	bs-0287R-bio
d-biotin	Sigma-Aldrich	B4501
EDTA-free protease inhibitor cocktail	Roche	11873580001
EMCCD camera	Andor	IX897
Guanosine 5′-diphosphate sodium salt (GDP)	Sigma-Aldrich	G7127
Guanosine 5'-O-[gamma-thio]triphosphate (GTPγS)	Roche	10220647001
High numerical aperture oil immersion objective	Nikon		Nikon, 100x, N.A. 1.49, oil immersion
ImageJ	NIH		https://imagej.net/ij/
Isopropyl-Β-D-Thiogalactoside	Sigma-Aldrich	I5502
LD555-MAL	Lumidyne	4
LD655-MAL	Lumidyne	10
L-glutamic acid potassium	Sigma-Aldrich	G1501
Magnesium acetate tetrahydrate	Sigma-Aldrich	M0631
Matlab	The MathWorks, Natick, MA		https://www.mathworks.com/
Microscope cover glass	Fisherbrand	18834	(24 mm × 60 mm)
Microscope slides			Customized, (75 mm × 26 mm × 1 mm) with 6 pairs of 1.2 mm diameter through holes
mPEG-SVA-5000	Laysan Bio	170-106
Ni Sepharose 6 Fast Flow	Cytiva	17531802
Origin	Origin software		https://www.originlab.com/
Polystyrene particles	QDSphere	AG1265
Protocatechuate 3,4-dioxygenase	Sigma-Aldrich	P8279
Streptavidin	Sangon Biotech	A610492
Superdex 200 Increase (10/300 GL)	Cytiva	28990944
TIRF microscope	Nikon	Ti2
Tris(2-carboxyethyl)phosphine hydrochloride (TCEP)	Thermo	75259