FRET-based localization of fluorescent protein insertions within the ryanodine receptor type 1.

Montserrat Samsó,Shweta A Raina,James D Fessenden,Xander H.T Wehrens,Jeffrey Tsai

doi:10.1371/journal.pone.0038594

Montserrat Samsó, Shweta A Raina + Show 3 more

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https://doi.org/10.1371/journal.pone.0038594

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Abstract

Fluorescent protein (FP) insertions have often been used to localize primary structure elements in mid-resolution 3D cryo electron microscopic (EM) maps of large protein complexes. However, little is known as to the precise spatial relationship between the location of the fused FP and its insertion site within a larger protein. To gain insights into these structural considerations, Förster resonance energy transfer (FRET) measurements were used to localize green fluorescent protein (GFP) insertions within the ryanodine receptor type 1 (RyR1), a large intracellular Ca2+ release channel that plays a key role in skeletal muscle excitation contraction coupling. A series of full-length His-tagged GFP-RyR1 fusion constructs were created, expressed in human embryonic kidney (HEK)-293T cells and then complexed with Cy3NTA, a His-tag specific FRET acceptor. FRET efficiency values measured from each GFP donor to Cy3NTA bound to each His tag acceptor site were converted into intermolecular distances and the positions of each inserted GFP were then triangulated relative to a previously published X-ray crystal structure of a 559 amino acid RyR1 fragment. We observed that the chromophoric centers of fluorescent proteins inserted into RyR1 can be located as far as 45 Å from their insertion sites and that the fused proteins can also be located in internal cavities within RyR1. These findings should prove useful in interpreting structural results obtained in cryo EM maps using fusions of small fluorescent proteins. More accurate point-to-point distance information may be obtained using complementary orthogonal labeling systems that rely on fluorescent probes that bind directly to amino acid side chains.

Highlights

In structural studies of proteins using cryo electron microscopy, fusions of fluorescent proteins have been used to localize primary structure elements to cryo electron microscopic (EM) maps of large protein complexes
Forster resonance energy transfer (FRET)-Based Method The Cy3NTA labeling system employed in this study enabled the targeting of small fluorophores to specific locations within ryanodine receptor type 1 (RyR1) via insertion of His10 tags to these locations
Non-specific binding sites for NTA-based fluorophores on RyR1 appeared to be absent since FRET was not observed for green fluorescent protein (GFP)-RyR1 fusion constructs lacking a His10 tag and because these constructs did not bind to an NTA-agarose column

Summary

Introduction

In structural studies of proteins using cryo electron microscopy, fusions of fluorescent proteins have been used to localize primary structure elements to cryo EM maps of large protein complexes In these structural maps, the small fusion protein appears as a ‘‘bulge’’ of density within the larger protein, which is often interpreted as the location of the fusion site. The small fusion protein appears as a ‘‘bulge’’ of density within the larger protein, which is often interpreted as the location of the fusion site This method has been used to localize specific domains in protein complexes such as viral capsids or heteromultimeric GTPases [1,2,3,4]. Both the N-terminus of the type 3 RyR [9] and a regulatory phosphorylation site at position 2808 of RyR2 [10] have been localized to the clamp region

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