FRET-Based Localization of Fluorescent Protein Insertions Within the Ryanodine Receptor Type 1

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

Detection and characterization of nitric oxide synthase in the mammalian cochlea

The messenger molecule nitric oxide (NO) is involved in blood flow regulation, cytotoxicity, and neural signalling, processes that are important in the physiology and pathophysiology of the mammalian cochlea. However, neither the presence of NO nor its synthetic enzyme, NO synthase, has been established in the peripheral auditory system. NO synthase activity, measured as the enzymatic conversion of radioactive arginine to citrulline, was predominantly soluble in the auditory nerve, lateral wall, vetibule and cochlear neuroepithelium. and trifluoperazine inhibited NO synthase activity in the lateral wall and auditory nerve. Histochemical staining by NADPH-diaphorase localized NOS activity to the lateral wall and the neuronal elements of the organ of Corti. Based on these results, the predominant NO synthase isoform in the cochlea is the neuronal type-I isoform.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/31105/1/0000001.pd

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His-tagged GFP-RyR1 fusion proteins bind to NTA-agarose.

<p>NTA-agarose-based fractionation of crude lysates from HEK-293T cells expressing GFP-RyR1 fusion proteins. Columns were washed as indicated (dotted lines). Datum points indicate relative levels of RyR immunoreactivity in consecutive 120 µl fractions quantified by an RyR-specific ELISA assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007338#s2" target="_blank">Methods</a>.</p

TLC analysis and structure of Cy3NTA.

<p>(A) Products from reactions consisting of Cy3-bis-succinimidyl ester alone (lane 1), Cy3-mono-succinimidyl ester and AB-NTA (lane 2), and Cy3-bis-succinimidyl ester and AB-NTA (lane 3) separated by TLC as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007338#s2" target="_blank">Methods</a>. Arrow indicates Cy3NTA used for FRET studies. R_f = relative index of mobility. (B) Predicted structure of Cy3NTA. Locations of Cy3 and the two NTA/Ni²⁺ moieties are indicated.</p

Cy3NTA can be used to measure FRET between internal sites on RyR1.

<p>(A) GFP-RyR1 fusion constructs containing a His₁₀ tag in divergent region 3 at amino acid residue 1861. Bars represent amino acid sequence of GFP (green) and RyR1 (black). For construct GFP(618)DR3His, GFP inserted at amino acid residue 618 was flanked by poly-glycine segments as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007338#s2" target="_blank">Methods</a>. Scale indicates RyR1 amino acid number. (B) Cy3NTA concentration-dependence of quenching of GFP(1)DR3His (green trace) and GFP(618)DR3His (purple) expressed in HEK-293T cells. Data was fit to determine EC₅₀ and the level of energy transfer (E) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007338#s2" target="_blank">Methods</a>. (C) Degree of quenching of the indicated GFP-RyR1 fusion proteins by 1 µM Cy3NTA. Asterisk indicates significant level of quenching (p<0.01) relative to GFP-RyR1 (-His) as determined using 1-way ANOVA followed by a Dunnett's post test.</p

Cy3NTA quenches His-tagged GFP-RyR1 fluorescence in a concentration-dependent manner.

<p>(A) Concentration dependence of Cy3NTA quenching of GFP-RyR1 (N-term His) fluorescence. The indicated Cy3NTA concentrations were added to HEK-293T cells expressing GFP-RyR1 (N-term His) at the time point indicated (arrowhead) and the change in GFP fluorescence normalized to initial fluorescence (F/F₀) was measured. Pre-incubation with 5 mM EDTA (top trace) abolished fluorescence quenching by Cy3NTA. (B) Cy3NTA concentration dependence of quenching of the indicated GFP-RyR1 fusion proteins expressed in HEK-293T cells. Values represent mean +/− S.E.M. for 7–14 cells. EC₅₀ and FRET efficiency values were determined from these curves as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0007338#s2" target="_blank">Methods</a>.</p