Related Experiment Video
Updated: Dec 18, 2025
10:19
Single-Molecule FRET Imaging for Observing the Conformational Dynamics of Dynamin-Like GTPase Atlastin
Published on: January 24, 2025
881
Nucleotide-Dependent Dimerization and Conformational Switching of Atlastin
John P O'Donnell1,2, Carolyn M Kelly1, Holger Sondermann3
1Department of Molecular Medicine, College of Veterinary Medicine, Cornell University, Ithaca, NY, USA.
Methods in Molecular Biology (Clifton, N.J.)
|June 13, 2020
Summary
Dynamin-related proteins like atlastin use GTP to drive ER membrane fusion. New methods, SEC-MALS and FRET, reveal how nucleotide binding changes protein structure and timing of catalytic events.
Area of Science:
- Biochemistry
- Cell Biology
- Structural Biology
Background:
- Dynamin-related proteins (DRPs) regulate membrane dynamics using GTP.
- Atlastin (ATL), an ER-resident protein, utilizes GTP binding and hydrolysis for ER tubule fusion and network formation.
Purpose of the Study:
- To describe two methods, SEC-MALS and FRET, for analyzing nucleotide-dependent structural changes in atlastin (ATL).
- To elucidate the mechanism of GTP-driven conformational changes and quaternary structure alterations in ATL.
Main Methods:
- Size-exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to assess equilibrium oligomeric states.
- Förster resonance energy transfer (FRET) assays to determine the temporal order of molecular events in the catalytic cycle.
Main Results:
- SEC-MALS provides equilibrium structural information.
- FRET offers high temporal resolution to order catalytic steps.
- Combined methods reveal ATL's oligomeric states and catalytic cycle timing.
Conclusions:
- SEC-MALS and FRET are complementary techniques for studying ligand-induced protein dimerization and conformational changes.
- These methods provide insights into the mechanism of ER network formation by atlastin.
Related Concept Videos
01:58Mechanical Protein Functions
5.4K
Proteins perform many mechanical functions in a cell. These proteins can be classified into two general categories- proteins that generate mechanical forces and proteins that are subjected to mechanical forces. Proteins providing mechanical support to the structure of the cell, such as keratin, are subjected to mechanical force, whereas proteins involved in cell movement and transport of molecules across cell membranes, such as an ion pump, are examples of generating mechanical force.
5.4K
01:42Actin Polymerization
8.1K
Actin polymerization occurs through the head-to-tail association of binding sites on monomeric actin or G-actin to form filamentous or F-actin. The polymerization can be divided into three phases ̶ nucleation, elongation, and steady-state phase.
The nucleation phase involves forming a stable nucleus consisting of three actin monomers to form a new actin filament. Actin-binding proteins such as formins and Arp2/3 complex help filament growth post-nucleation. The Formins form straight...
The nucleation phase involves forming a stable nucleus consisting of three actin monomers to form a new actin filament. Actin-binding proteins such as formins and Arp2/3 complex help filament growth post-nucleation. The Formins form straight...
8.1K
01:19Allosteric Proteins-ATCase
6.3K
Binding sites linkages can regulate a protein's function. For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis...
6.3K
01:14Generation of Straight or Branched Actin Filaments
3.6K
The straight or branched structure formation of actin filaments is controlled by nucleating proteins such as the formins and Arp2/3 complex. Formin-mediated assembly results in straight filaments, whereas Arp2/3 protein complex-mediated assembly results in branched actin filaments.
Arp2/3 Complex
Arp2/3 complex is a seven-subunit complex consisting of two proteins similar to actin- Arp2 and Arp3, and five other subunits that help keep Arp2 and Arp3 inactive. When required, the complex is...
Arp2/3 Complex
Arp2/3 complex is a seven-subunit complex consisting of two proteins similar to actin- Arp2 and Arp3, and five other subunits that help keep Arp2 and Arp3 inactive. When required, the complex is...
3.6K
00:49Ligand Binding and Linkage
5.4K
Allosteric proteins have more than one ligand binding site; the binding of a ligand to any of these sites influences the binding of ligands to the other sites. When a protein is allosteric, its binding sites are called coupled or linked. In the case of enzymes, the site that binds to the substrate is known as the active site and the other site is known as the regulatory site. When a ligand binds to the regulatory site, this leads to conformational changes in the protein that can influence...
5.4K
01:48ATP Synthase: Mechanism
16.3K
In animals, the mitochondrial F1F0 ATP synthase is the key protein that synthesizes ATP molecules through a complex catalytic mechanism. While the nuclear genome encodes the majority of ATP synthase subunits, the mitochondrial genome encodes some of the enzyme's most critical components. The formation of this multi-subunit enzyme is a complex multi-step process regulated at the level of transcription, translation, and assembly. Defects in one or more of these steps can result in decreased...
16.3K