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

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

YFP/CFP peak ratios of CFP – YFP constructs.

<p>YFP/CFP peak ratios in Cos-1 cells expressing CFP-xa-YFP and CrFP-xa-YFP in cell lysates and in Ni-NTA purified protein from these lysates (see Materials and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013862#s2" target="_blank">Methods</a>) at different ATP concentrations.</p

Fluorescence decay curves of purified monomeric CFP.

<p>Normalized experimental (dotted line) and fitted (solid line) fluorescence decay curves of CFP (curve 1), CFP 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-t002" target="_blank">Table 2</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

Effect of ATP on a GFP based FRET construct.

<p>Steady state fluorescence emission spectrum of a GFP-tdTomato construct (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013862#pone.0013862-vanderKrogt1" target="_blank">[15]</a>) in the presence or absence of 10 mM ATP. Excitation of the GFP was at 480 nm excitation.</p

Effect of ATP on a CFP based FRET construct.

<p>Steady state fluorescence emission spectra of CFP-xa-YFP (A) and CFP alone (B) at different ATP concentrations. Excitation of the CFP was at 420 nm excitation.</p