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

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 Ca(2+) 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.

Figure 1. FRET-based method and GFP/His₁₀ tag insertion sites used for structural analysis of RyR1.

(A) Cy3NTA site-specifically binds to a His₁₀ tag inserted within the primary structure of RyR1 (black bar; top) resulting in FRET from a nearby fused GFP fluorescent donor (bottom). The FRET efficiency is indicative of the proximity of the donor and acceptor fluorophores within RyR1. (B) Primary structure of RyR1 (black bar) and the N-terminal functional domain (gray bar) are indicated. Positions of GFP and His₁₀ tag insertions, malignant hyperthermia mutation sites (diamonds), as well as the location of the beta sheet (arrows) and alpha helical (diamond-flanked line) subdomains , are shown.

Figure 2. Functional analysis of His-tagged GFP-RyR1 fusion constructs.

(A) Caffeine-induced Ca²⁺ transients were measured using Fluo-4-based intracellular Ca²⁺ imaging for HEK-293T cells expressing the indicated GFP-RyR1 fusion constructs. A graded series of caffeine concentrations were perfused as indicated (black bars). Individual representative traces indicate changes in Fluo-4 fluorescence normalized to resting fluorescence (F/F₀). Calibration bar = 0.5 F/F₀ ratio units vs. 50 sec. (B-D) Normalized caffeine dose response curves for His-tagged constructs containing GFP fused to position 1 (B), position 291 (C) or position 620 (D) of RyR1. Individual data points represent mean +/− S.E.M.

Figure 3. FRET analysis of His-tagged GFP-RyR1 fusion constructs.

(A–C) FRET efficiencies measured from His-tagged constructs containing GFP fused to position 1 (A), position 291 (B) or position 620 (C) of RyR1 expressed in HEK-293T cells. Data points represent mean FRET efficiency +/− SEM for the indicated constructs determined from recovery of donor fluorescence after acceptor photobleaching as described in Methods.

Figure 4. Calibration of FRET measurements using Cy5NTA.

(A) Predicted structures of Cy3NTA and Cy5NTA. The number of methine groups in each compound is indicated (n). (B) Normalized GFP emission spectrum (green, λ_ex = 476 nm), as well as Cy3NTA (red) and Cy5NTA (blue) absorbance spectra. Shaded regions indicate areas of spectral overlap. (C) FRET efficiencies measured within GFPHis₁₀ using either 2 µM Cy3NTA (red) or 2 µM Cy5NTA (blue) as a FRET acceptor. Values represent mean +/− S.E.M. (D) Donor-acceptor distances within GFPHis₁₀ determined from theoretical FRET curves derived from the R₀ for either GFP/Cy3NTA (R₀ = 62.5 Å; red curve) or GFPCy5NTA (R₀ = 42.9 Å; blue). Black lines indicate observed FRET values from panel (C) and corresponding donor/acceptor distances for each FRET pair.

Figure 5. Triangulation of GFP insertions relative to the crystal structure of an N-terminal RyR1 fragment.

(A) Overall view of the complex indicating the locations of the inserted GFPs and His₁₀ tags. The two individual beta sheet subdomains are indicated in cyan (amino residues 12–204) and dark blue (residues 205–394), respectively. The alpha helical subdomain (residues 395–532) is indicated in red. His₁₀ tag insertion sites are colored white on the ribbon depiction of the crystal structure. The X-ray crystal structure of GFP inserted at each site is indicated in green. (B) The complex rotated 45° relative to the view in (A) along the indicated axis is shown. Scale bars, 20 Å.

Figure 6. Docking of RyR1 N-terminal crystal structure and triangulated GFPs to the RyR1 cryo EM map.

(A) Cryo EM structure of RyR1 (gray) viewed from the “top” (i.e. the cytoplasmic side that would face the T-tubule membrane in situ). Crystal structure of an N-terminal RyR1 fragment is docked to a central location that forms a cytoplasmic vestibule located beneath the area indicated by the dotted circle. The positions of the GFPs (in green) inserted at the indicated positions relative to the crystal structure of the N-terminal domain docked to this position are indicated. Colored dots depict previously published localization sites of GST fused to the N-terminus of RyR3 (orange and red; [9]), as well as CFP fused to position 626 of RyR2 (cyan; [16]). (B) Side view of the cryo EM structure of RyR1 rotated 90° relative to panel A as indicated. (C) Oblique view of the cryo EM structure of RyR1 with the docking to the cytoplasmic vestibule location. (D) Magnified view of this docking from the dotted box in (C). GFP620 and GFP620alt refer to two potential localizations of GFP fused to position 620 discussed in the text. GFP at position 291 is removed to more clearly depict the locations of the X-ray crystal structure of the N-terminal RyR1 fragment as well as the other GFP fusions.