Site-Specific Labeling of the Type 1 Ryanodine Receptor Using Biarsenical Fluorophores Targeted to Engineered Tetracysteine Motifs

James D Fessenden,Mohana Mahalingam,Sabato D'Auria

doi:10.1371/journal.pone.0064686

James D Fessenden, Mohana Mahalingam + Show 1 more

Open Access

https://doi.org/10.1371/journal.pone.0064686

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Journal: PloS one	Publication Date: May 28, 2013
Citations: 8	License type: CC BY 4.0

Affiliation: Brigham and Women's Hospital

Abstract

The type 1 ryanodine receptor (RyR1) is an intracellular Ca2+ release channel that mediates skeletal muscle excitation contraction coupling. While the overall shape of RyR1 has been elucidated using cryo electron microscopic reconstructions, fine structural details remain elusive. To better understand the structure of RyR1, we have previously used a cell-based fluorescence resonance energy transfer (FRET) method using a fused green fluorescent protein (GFP) donor and a fluorescent acceptor, Cy3NTA that binds specifically to short poly-histidine ‘tags’ engineered into RyR1. However, the need to permeabilize cells to allow Cy3NTA entry as well as the noncovalent binding of Cy3NTA to the His tag limits future applications of this technique for studying conformational changes of the RyR. To overcome these problems, we used a dodecapeptide sequence containing a tetracysteine (Tc) motif to target the biarsenical fluorophores, FlAsH and ReAsH to RyR1. These compounds freely cross intact cell membranes where they then bind covalently to the tetracysteine motif. First, we used this system to conduct FRET measurements in intact cells by fusing a yellow fluorescent protein (YFP) FRET donor to the N-terminus of RyR1 and then targeting the FRET acceptor, ReAsH to an adjacent Tc tag. Moderate energy transfer (∼33%) was observed whereas ReAsH incubation of a YFPRyR1 fusion protein lacking the Tc tag resulted in no detectable FRET. We also developed a FRET-based system that did not require RyR fluorescent protein fusions by labeling N-terminal Tc-tagged RyR1 with FlAsH, a FRET donor and then targeting the FRET acceptor Cy3NTA to an adjacent decahistidine (His10) tag. A high degree of energy transfer (∼66%) indicated proper binding of both compounds to these unique recognition sequences in RyR1. Thus, these two systems should provide unprecedented flexibility in future FRET-based structural determinations of RyR1.

Highlights

A complete understanding of a protein’s function in both physiological and pathophysiological states requires an accurate understanding of its structure
A key experimental tool for monitoring changes in protein structure is through measurement of fluorescence energy resonance transfer (FRET) between donor and acceptor fluorophores site- targeted to the protein [1]
This construct exhibited intense yellow fluorescent protein (YFP) fluorescence when expressed in HEK-293T cells (Fig. 1B)

Summary

Introduction

A complete understanding of a protein’s function in both physiological and pathophysiological states requires an accurate understanding of its structure. We found that the RyR can be labeled with either FlAsH or ReAsH when a Tc Tag is fused to the N-terminus of the protein. We created full-length RyR1 constructs that only contained the optimized Tc motif at the N-terminus, with or without a 10 residue histidine tag which we used for FRET measurements between FlAsH and Cy3NTA.

Results

Conclusion