ATP Changes the Fluorescence Lifetime of Cyan Fluorescent Protein via an Interaction with His148

Recently, we described that ATP induces changes in YFP/CFP fluorescence intensities of Fluorescence Resonance Energy Transfer (FRET) sensors based on CFP-YFP. To get insight into this phenomenon, we employed fluorescence lifetime spectroscopy to analyze the influence of ATP on these fluorescent proteins in more detail. Using different donor and acceptor pairs we found that ATP only affected the CFP-YFP based versions. Subsequent analysis of purified monomers of the used proteins showed that ATP has a direct effect on the fluorescence lifetime properties of CFP. Since the fluorescence lifetime analysis of CFP is rather complicated by the existence of different lifetimes, we tested a variant of CFP, i.e. Cerulean, as a monomer and in our FRET constructs. Surprisingly, this CFP variant shows no ATP concentration dependent changes in the fluorescence lifetime. The most important difference between CFP and Cerulean is a histidine residue at position 148. Indeed, changing this histidine in CFP into an aspartic acid results in identical fluorescence properties as observed for the Cerulean fluorescent based FRET sensor. We therefore conclude that the changes in fluorescence lifetime of CFP are affected specifically by possible electrostatic interactions of the negative charge of ATP with the positively charged histidine at position 148. Clearly, further physicochemical characterization is needed to explain the sensitivity of CFP fluorescence properties to changes in environmental (i.e. ATP concentrations) conditions

Multi-messenger observations of a binary neutron star merger

On 2017 August 17 a binary neutron star coalescence candidate (later designated GW170817) with merger time 12:41:04 UTC was observed through gravitational waves by the Advanced LIGO and Advanced Virgo detectors. The Fermi Gamma-ray Burst Monitor independently detected a gamma-ray burst (GRB 170817A) with a time delay of ~1.7 s with respect to the merger time. From the gravitational-wave signal, the source was initially localized to a sky region of 31 deg2 at a luminosity distance of 40+8-8 Mpc and with component masses consistent with neutron stars. The component masses were later measured to be in the range 0.86 to 2.26 Mo. An extensive observing campaign was launched across the electromagnetic spectrum leading to the discovery of a bright optical transient (SSS17a, now with the IAU identification of AT 2017gfo) in NGC 4993 (at ~40 Mpc) less than 11 hours after the merger by the One- Meter, Two Hemisphere (1M2H) team using the 1 m Swope Telescope. The optical transient was independently detected by multiple teams within an hour. Subsequent observations targeted the object and its environment. Early ultraviolet observations revealed a blue transient that faded within 48 hours. Optical and infrared observations showed a redward evolution over ~10 days. Following early non-detections, X-ray and radio emission were discovered at the transient’s position ~9 and ~16 days, respectively, after the merger. Both the X-ray and radio emission likely arise from a physical process that is distinct from the one that generates the UV/optical/near-infrared emission. No ultra-high-energy gamma-rays and no neutrino candidates consistent with the source were found in follow-up searches. These observations support the hypothesis that GW170817 was produced by the merger of two neutron stars in NGC4993 followed by a short gamma-ray burst (GRB 170817A) and a kilonova/macronova powered by the radioactive decay of r-process nuclei synthesized in the ejecta

Fluorescence decay curves of CFP – YFP constructs.

<p>Normalized experimental (dotted line) and fitted (solid line) fluorescence decay curves of CFP-xa-YFP (curve 1), CFP-xa-YFP in the presence of 10 mM MgATP (curve 2) or 10 mM ATP (curve 3). The excitation wavelength was 430 nm and the detection wavelength of CFP emission was 480 nm. Weighted residuals are shown in the bottom panel and the recovered parameters (α, τ) are collected in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013862#pone-0013862-t001" target="_blank">Table 1</a>.</p

Fluorescence decay parameters of purified monomeric proteins.

<p>Fluorescence decay parameters of the CFP, CrFP and YFP in absence and presence of ATP or MgATP.</p><p><i>Note.</i> Values in parentheses are the 67% confidence limits. The average fluorescence lifetime (in ns) is calculated as <<i>τ</i>> =  <i>α</i>₁<i>τ</i>₁ + <i>α</i>₂<i>τ</i>₂.</p

Fluorescence decay parameters of CFP – YFP constructs.

<p>Fluorescence decay parameters of the CFP-xa-YFP (CxY), CrFP-xa-YFP (CrxY) and CxY-H148D in absence and presence of ATP or ATP where Mg (MgATP) is added.</p><p><i>Note.</i> Values in parentheses are the 67% confidence limits. The average fluorescence lifetime (in ns) is calculated as <<i>τ></i>  =  <i>α</i>₁<i>τ</i>₁ + <i>α</i>₂<i>τ</i>₂ + <i>α</i>₃<i>τ</i>₃.</p