Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay

Neelima Boora; Vibha Verma; Ridhi Khurana; Gautam Gawande; Sanchi Bhimrajka; Komal Chaprana; Meenu Kapoor; Sanjay Kapoor

doi:10.3791/62791

Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor a...

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Determination of Tripartite Interaction between Two Monomers of a MADS-box Transcription Factor and a Calcium Sensor Protein by BiFC-FRET-FLIM Assay

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14:34 min

December 25, 2021

DOI: 10.3791/62791-v

Neelima Boora*¹, Vibha Verma*¹, Ridhi Khurana¹, Gautam Gawande¹, Sanchi Bhimrajka¹, Komal Chaprana¹, Meenu Kapoor², Sanjay Kapoor¹

¹Interdisciplinary Centre for Plant Genomics, Department of Plant Molecular Biology,University of Delhi South Campus, ²University School of Biotechnology,Guru Gobind Singh Indraprastha University

Overview

This study presents a novel method to visualize ternary complex formation between three proteins using bimolecular fluorescence complementation (BiFC) combined with fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM). The approach allows for the observation of protein-protein interactions within live cells, enhancing our understanding of complex biological processes.

Key Study Components

Research Area

Protein-protein interactions
Fluorescent imaging technologies
Cell biology

Background

Understanding protein interactions is crucial for studying various biological functions.
This method builds on previous work utilizing BiFC for examining protein complexes.
The addition of FLIM provides a more detailed analysis of interactions.

Methods Used

BiFC-FRET-FLIM assay for visualizing protein interactions
Nicotiana benthamiana as the biological model system
Fluorescence lifetime measurements to assess interactions

Main Results

Successful demonstration of tripartite interactions between selected proteins.
Visualization of reconstituted YFP and its application in FRET analysis.
Quantification of interaction through changes in fluorescence lifetime.

Conclusions

The study effectively illustrates the use of BiFC-FRET-FLIM for examining complex protein interactions.
This method is significant for advancing research in molecular biology and protein signaling pathways.

Frequently Asked Questions

What is the significance of using BiFC with FRET-FLIM?

This combination allows researchers to visualize complex protein interactions and quantify them in vivo, improving our understanding of cell signaling.

Which organism was used in this study?

The study utilized Nicotiana benthamiana, a common model in plant molecular biology.

What are the critical steps in carrying out the method?

Key steps include cloning genes of interest, transforming agrobacteria, and performing agroinfiltration into plant tissues.

How does the method validate the tripartite interactions?

The method validates interactions by measuring the fluorescence lifetime changes of the donor molecule in the presence of acceptors.

What potential applications does this method have?

This method can be applied to study various protein interactions, signaling pathways, and cellular processes in live cells.

How long does it take to prepare plants for the analysis?

Plant preparation takes several weeks, including germination and growth stages before agroinfiltration can occur.

Is this method applicable to organisms other than plants?

While this study focuses on plants, similar techniques can be adapted for use in other model organisms.

Here we present, a method to visualize ternary complex formation between three protein partners using fluorescent-tagged proteins by BiFC based FRET-FLIM assay. This method is valuable for studying protein-protein interaction complexes in vivo.

The study of protein-protein interactions provides an understanding of the regulation of many biological processes. Here we demonstrate a procedure described initial by Y John shoe and co. Workers in 2007, where the authors have used BiFC in combination with FRET to validate the tripartite interaction between three protein molecules.

However, we have included fluorescence lifetime measurements in this procedure to substantiate FRET measurements. To demonstrate a tripartite interaction, we have chosen a protein, which is known to homodimerize. Furthermore, it has also been validated in-house to interact with a calcium sensor of protein referred to as C protein in this protocol.

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