Acceptor Pair in the

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Letter

Non-trivial effect of the color-exchange of a Donor/Acceptor pair in the engineering of Förster resonance energy transfer (FRET)-based indicators Yusaku Ohta, Takanori Kamagata, Asuka Mukai, Shinji Takada, Takeharu Nagai, and kazuki horikawa ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00221 • Publication Date (Web): 27 May 2016 Downloaded from http://pubs.acs.org on May 29, 2016

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ACS Chemical Biology

Non-trivial effect of the color-exchange of a Donor/Acceptor pair in the engineering of Förster resonance energy transfer (FRET)-based indicators Yusaku Ohta†, Takanori Kamagata†‡§, Asuka Mukai†, Shinji Takada‡§, Takeharu Nagai¶ and Kazuki Horikawa*† †

Division of Bioimaging, Institute of Biomedical Sciences, Tokushima University Graduate

School, 3-18-15 Kuramoto-cho, Tokushima City, Tokushima 770-8503, Japan ‡

Okazaki Institute for Integrative Bioscience and National Institute for Basic Biology, National

Institutes of Natural Sciences, Okazaki, Aichi 444-8787, Japan §

SOKENDAI (The Graduate University for Advanced Studies), Okazaki, Aichi

444-8787, Japan ¶

The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki,

Osaka 567-0047, Japan

* To whom correspondence should be addressed. E-mail: [email protected]

Abstract: Genetically encoded indicators driven by the Förster resonance energy transfer (FRET) mechanism are reliable tools for live imaging. While the properties of FRET-based indicators have been improved over the years, they often suffer from a poor dynamic range due to the lack of comprehensive understanding about how to apply an appropriate strategy to optimize the FRET parameters. One of the most successful optimization is the incorporation of circularly permuted fluorescent proteins (cpFPs). To better understand the effects of this strategy, we systematically investigated the properties of the indicators by utilizing a set of FRET backbones consisting of native or one of the most effective cp variants (cp173FPs)

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with considerations of their order. As a result, the ordering of donor and acceptor FPs, which has been ignored in previous studies, was found to significantly affect the dynamic range of indicators. By utilizing these backbones, we succeeded in improving a cGMP indicator with 3.6-fold increased dynamic range and in generating an ultra-sensitive cAMP indicator capable of the environmental imaging, demonstrating the practical importance of the ordering of donor and acceptor in the engineering of FRET-based indicators.

Förster resonance energy transfer (FRET) is a process of non-radiative energy transfer from a donor to an acceptor fluorophore, and FRET-based indicators are ideal tools for the quantitative monitoring of signaling events in living cells and organisms 1. Yellow Cameleon (YC) is one of the first reported FRET-based Ca2+ indicators consisting of a Ca2+ sensing motif sandwiched between color variants of the green fluorescent protein (GFP) as the fluorescent donor and acceptor (D/A) 2. YC’s FRET efficiency reflects the conformational state of the indicator, which is affected by Ca2+ binding, so the emission ratiometry or lifetime measurement of the donor fluorescence allows visualization of the spatiotemporal dynamics of intracellular Ca2+. Encouraged by the success of YC, a variety of FRET-based indicators for the second messengers—phosphorylation—and other enzymatic activities have been developed

3,4

.

However, despite their potential applicability, many FRET-based indicators often suffer from small dynamic range. Among several photo-physical parameters, the FRET efficiency is highly sensitive to changes in the positional parameter, including the distance (r) and relative orientation (κ) between the donor and acceptor fluorophore. More specifically, the FRET efficiency is inversely proportional to the sixth power of the positional parameter “r/R0”, this being the distance between the donor and acceptor normalized by the Förster radius (R0) that gives 50% FRET efficiency

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. Optimizing these parameters improved the indicators’

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performance as has been demonstrated for semi-synthetic FRET indicators by the use of chemical fluorophores

6,7

. For fully genetically encoded indicators, indirect tuning of the

positional parameter has been achieved by incorporating distinct sensor motifs and by modifying the peptide linkers connecting the sensor motif with the donor and acceptor, which contributed to increasing the dynamic range 8,9. Modifying the dimerization property intrinsic to Aequorea–derived GFP variants has also contributed to fine tuning of the FRET efficiency 10–12. More direct and effective optimization was demonstrated by replacing native FPs with a series of circularly permuted (cp) ones, which can drastically rearrange the spatial configuration of the D/A. For instance, replacement of the native Venus acceptor of YC with five cp variants (cp49, cp157, cp173, cp195, and cp229) was tested (Supplementary Figure 1), and it was found that cp173Venus yielded YC3.60 with a 5.6-fold increased dynamic range (Supplementary Figure 1C) 13. Replacing the donor with cp173ECFP also expanded the dynamic range of Prickle, an indicator for abl kinase (Supplementary Figure 1D)

14

. In these optimization including an

expanded screening scale (Supplementary Figure 1E) 13,15–17, however, the incorporation of cpFP was solely examined in term of their combination by using backbone libraries having a fixed order of the D/A, and the importance of their ordering that should be another parameter in designing of indicators has not been carefully examined yet (Supplementary Figure 1F). This might be because of the simplified assumption that FRET parameters, including the distance and relative orientation between the donor and acceptor, would not be significantly affected in a color-exchanged pair (e.g., ECFP-Venus vs Venus-ECFP), as these native FPs are almost identical in their overall 3D-structure

18,19

. However, it is not obvious whether the spatial

configuration of the D/A was identical for the pair of color-exchanged indicators when cpFP was incorporated (e.g., ECFP-cp173Venus vs Venus-cp173ECFP), because the 3D-structural information on cpFPs is not yet available.

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In this report, with an aim to systematically evaluate the effect of cpFP incorporation on the dynamic range of indicators, we investigated all the possible combination of native or cp173FPs while still considering the order. Introducing cp173FPs, being the most effective cp variant in rearranging the D/A configuration (Supplementary Figure 1B), to the donor and/or acceptor distinctly affected the dynamic range of the Ca2+ indicator. We further identified that the “color-exchange” of the D/A pair, which has been ignored previously, has an impact on the dynamic range, especially when cpFP was incorporated as the donor or acceptor. We utilized these FRET backbones to improve the existing indicators and successfully developed one for cGMP with a 3.6-fold increased dynamic range and also developed an ultra-sensitive cAMP indicator capable of environmental imaging. Altogether, these results suggest that FRET backbones considering the color-exchanging effect would be promising tools to rapidly develop FRET-based indicators, whose utility would be maximized when other cpFPs than cp173 variants were fully implemented to the backbone library in a future study.

Results and Discussion To evaluate how the incorporation of cpFPs affects the dynamic range of FRET-based indicators, we designed eight FRET backbones harboring native or cp ECFP and Venus, while still considering their order (Figure 1A). For simplicity, we focused on cp173ECFP and cp173Venus among five cp variants (cp49, cp157, cp173, cp195, and cp229) that had been independently reported to improve the dynamic range of YC and Prickle 13,14. We categorized these backbones into two groups, i.e., the N-terminally located donor group (ND-Groups: #1, 2, 3, and 4) and the C-terminally located donor group (CD-Groups: #5, 6, 7, and 8), in which #1 vs #5, #2 vs #6 and so on corresponded to the “color-exchanged” donor/acceptor (D/A) pair. For a proof of concept demonstration, we first investigated the dynamic range of

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indicators carrying CaM-M13 of YC3.60 as a Ca2+ sensing motif (Figure 1B). The recombinant indicators were affinity purified and were then subjected to spectroscopic analysis under freeand saturating-Ca2+ conditions. In all cases, the FRET ratios increased upon Ca2+ addition in distinct patterns, although the indicator with cp173Venus/cp173ECFP (#8) was less efficiently expressed. For the ND-group, replacing the native acceptor with cp173Venus (#2, 203%) increased the dynamic range 5.3-fold compared to ECFP/Venus (#1), as has been demonstrated previously13. Replacing both the donor and acceptor with the cp173 variants increased the FRET ratio change in a smaller amplitude (#4, 48%), while the cp173ECFP/Venus pair displayed a reduced signal change (#3, 8%). The FRET ratio change was similarly affected in the color-exchanged CD-group. Replacing the native donor with cp173ECFP increased the signal change (#5, 33%, #6, 78%), while N-terminal cp173Venus reduced the dynamic range (#7, 22%), confirming that the incorporation of cpFP(s) distinctly affects the signal change, depending on both the combination and the order of the D/A pair. To further characterize the effect of the color-exchange, we compared the spectral pattern of corresponding pairs between the ND-group and the CD-group (Figure 1 and Supplementary Table 1). We found that native D/A pairs (#1 and #5) displayed indistinguishable emission patterns, both under free- and saturating Ca2+ conditions, indicating that the FRET parameters were not affected by exchanging the order of the native donor and acceptor FPs. On the other hand, the color-exchanged pairs displayed different emission patterns when one of the FPs was replaced with its cp variant. For a native to cpFP pair (#2 and #6), #2 displayed a larger FRET emission increase than #6 upon Ca2+ addition, while their baseline emissions in the absence of Ca2+ were comparable. For a cp- to native FP pair (#3 and #7), #7 showed a higher baseline FRET emission with free-Ca2+ and a larger signal change upon Ca2+ addition than #3. These results indicate that the effect of the color-exchange was not neutral when the cpFPs were

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incorporated, and therefore systematic screening by using a set of FRET backbones covering the previously ignored D/A configuration could help expand the dynamic range. To demonstrate the utility of the FRET backbones in the engineering of FRET indicators, we tried to improve the dynamic range of the existing cGMP indicator

20–22

cGES-GKIB is one of cGMP specific FRET-based indicators comprised of the cGMP binding domain B from the cGMP-dependent protein kinase IB (GKIB) sandwiched between EYFP and ECFP 20. We inserted the cGMP binding domain B from GKIB into our eight FRET backbones (named as PKG #1-8), and examined the fluorescence spectra of the recombinant indicators in the absence or presence of cGMP (Figure 2 and Supplementary Table 2). In all cases, the FRET ratios decreased upon cGMP addition, as reported for cGES-GKIB, and the dynamic range was differently affected depending on both the combination and the order of the D/A pair. Among our FRET backbones, PKG #7 was identified to display a 3.6-fold higher dynamic range (136%) compared to #5 (38%, corresponding to cGES-GKIB), while the others showed a reduced dynamic range. Comparison of the emission spectra between the color-exchanged pair confirmed its non-neutral effect, as the dynamic range of PKG #7 was 5.7 times larger than its counterpart (#3, 24%). We then performed tests to characterize the in vitro property of the developed PKG #7. Titration with cGMP revealed the apparent Kd for cGMP of PKG #7 as 2.64 µM (Supplementary Figure 2 and Supplementary Table 3). The affinity to cAMP was 3 orders of magnitude lower than that to cGMP, indicating its high specificity to cGMP (2,536 µM, Supplementary Figure 2). To examine the in-cell performance of PKG #7, we introduced the developed indicators into N1E-115 cells, which display a transient increase of intracellular cGMP when NO signaling is activated. Upon stimulation by 100 µM sodium nitroprusside (SNP), a transient decrease of the emission ratio of Venus/ECFP was observed for cells expressing PKG #7 with a twice larger

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amplitude than PKG #5 (Supplementary Figure 3). The decrease of the FRET ratio was eliminated in cells expressing PKG #7_T317A—a cGMP-insensitive mutant—confirming that the observed signal change was free from environmental artifacts, including cellular pH change 23

. To further explore the applicability of the developed indicator in detecting more

physiological cGMP dynamics but not the cellular response to artificially applied stimuli, we observed a population of Dictyostelium discoideum (D. discoideum) cells that are known to display the oscillation of the intracellular cGMP in association with spontaneously established chemotactic locomotion (Figure 2 and Supplementary Movie 1)

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. An approximately 6 min

interval of FRET ratio change was observed, with a twice larger amplitude than PKG #5 (Supplementary Figure 4), demonstrating the increased detection sensitivity of PKG #7 with good reversibility. We finally tried to develop a new indicator for cAMP by using our backbone set. In the population of developing D. discoideum cells, oscillatory dynamics of the extracellular cAMP ([cAMP]ex) that is synthesized intracellularly and acts as a chemo-attractant in the extracellular space, has been known to control the collective chemotaxis. Although biochemical analysis suggested the peak concentration of effective [cAMP]ex was 5 to 30 nM

25,26

, the live

imaging has not been possible due to the lack of sensitive cAMP indicators with a low nM sensitivity. We here focused on a high affinity cAMP binding motif of the regulatory subunit of human PKA (RIα, cAMP binding site B)

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and incorporated it into our FRET-backbones

(Supplementary Figure 5A). The cell lysates of HEK293 cells expressing these indicators were subjected to spectroscopic analysis and only RIα #7 was found to display a clear FRET ratio change upon cAMP addition (Figure 3A). Characterization of purified PKA RIα #7 revealed that the dynamic range and Kd for cAMP was 38 % and 37.2 nM, respectively (Figure 3B and Supplementary Figure 5B). To test the applicability of the developed indicator for [cAMP]ex

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imaging, purified PKA RIα #7 was supplemented into the culture medium containing a population of chemotacting D.discoideum cells. As a result, 7 min interval of FRET ratio change in phase with the periodic chemotaxis was clearly observed (Figures 3C, 3D and Supplementary Movie 2). Titration analysis under the microscopic observation revealed that [cAMP]ex oscillates at the concentration range of approx. 5-100 nM that is in a good agreement with previous biochemical reports (Figure 3D)

25,26

. This is the first demonstration of live [cAMP]ex imaging

benefited from a rapid development of new indicator with a large dynamic range and nM-sensitivity. In summary, we identified how the color-exchange of the D/A pair has an impact on the performance of FRET-based indicators. The utility of our developed FRET backbones considering both the combination and ordering of D/A was demonstrated by the successful improvement of the cGMP indicator, whose dynamic range increased 3.6-fold and developing an ultra-sensitive cAMP indicator capable of the environmental imaging. The color-exchange of the D/A pair was originally assumed to have a neutral effect on indicators’ performance because it was assumed that the structural similarity of ECFP and Venus with just 8 a.a. differences would not affect any parameters controlling the FRET efficiency. Although this was valid for native pairs such as YC #1/5 and PKG #1/5, a maximum 5.7 times difference in the dynamic range of the color-exchanged pairs harboring cpFP (e.g., PKG #3/7, Supplementary Table 1 and Supplementary Table 2) clearly indicated some FRET parameters are affected, although the question is which? In a formulation, the FRET efficiency is explicitly controlled by r and R0, the latter is a function of other opto-physical properties of D/A including κ, the quantum yield of the donor (ΦD), the extinction coefficient of the acceptor (εA), and the spectral overlap integral of the donor emission and acceptor absorption (J) 5. It is also non-explicitly affected by the additional properties of D/A, including the stability and the

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efficiency of folding and maturation. As the information on these parameters is not available, especially for cpFPs, it is difficult to obtain information on the underlying mechanisms. However, a focused comparison of the emission patterns of YC #2/6 and PKG #3/7 raises an important notion about the involvement of the dimerization interface of FPs that could account for part of the mechanisms. In these pairs, i.e., the best performing one and its color-exchanged counterparts, most predominantly affected was the emission ratio at the FRET-ON state, while that of the FRET-OFF state was left almost unchanged (e.g., the ON-FRET ratio of YC #2 and #6 was 5.8 and 3.4, and that of FRET-OFF state for both YCs was 1.9, Supplementary Table 1 and Supplementary Table 2). This was similarly observed for the previously reported YC3.60s, in which the monomeric mutation (A206K) was introduced at the dimerization interface of FPs that plays a crucial role in achieving the high FRET efficiency 10. It thus would be plausible to speculate that the high FRET efficiency at the FRET-ON state of YC #2 (corresponding to YC3.60) and PKG #7 was brought about by the closer interaction of D/A through their dimerization interface, while it was sub-optimal for their color-exchanged counterparts (YC #6 and PKG #3). To support this idea, the monomeric mutation on PKG #7 (A206K) decreased the emission ratio just at the FRET-ON state, while the effect of the monomeric mutation on PKG #3 was minimal (Supplementary Figure 6 and Supplementary Table 2), suggesting that the local structure at the dimerization interface might be differently affected by the circular permutation for ECFP and Venus, respectively. Alternatively, the spatial configuration of D/A could be drastically altered by the color-exchange. Contrary to the above discussed pairs, YC #3/7 displayed a distinct emission pattern both at the FRET-ON and FRET-OFF state, indicating the involvement of other FRET parameters, such as r, R0, and associated properties of D/A. The distinct folding efficiency of cp173FPs is another point of action that could affect the overall structure of the color-exchanged indicators. Future analysis on the ultra-structures and on the

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photochemical and biochemical properties of cpFPs is needed to better understand how the minor difference in their primary sequences (as few as 8 a.a. substitutions between the two cp173FPs) causes rather drastic effects on the indicator’s performance. So far, the engineering of high-performance FRET indicators has been routinely achieved by screening the backbone libraries with a variety of D/A configurations, whose coverage has been expanded by up to a hundred candidates considering the distinct size of the inter-domain linkers, sensor motifs, and the incorporation of a series of cpFPs as either a donor or acceptor or both

9,16,17,28,29.

Although these have enabled the successful identification of

indicators for a variety of biological activities, the libraries utilized in these studies missed half the parameter space by just examining the fixed order of the D/A pair (Supplementary Fig1). The identified effects of the color-exchange fully compatible with the above-mentioned strategies would be beneficial for increasing the chance of success in generating indicators with a much greater dynamic range, which in turn would facilitate the live imaging of physiological events at the cell and organism level.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We thank A. Miyawaki (RIKEN BSI) for YC3.60, T. Izumi (Kyoto University) for their helpful discussions, and A. Ichiraku for assisting the molecular biology and cell culture. This work was

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supported in part by the Nano-Macro Materials, Devices and System Research Alliance, by a Grant-in-Aid for Young Scientists (B) (25840050) and by a Grant-in-Aid for Scientific Research on Innovative Areas "Spying minority in biological phenomena (No. 3306)" from MEXT (23115003) to KH.

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Abbreviations The abbreviations used are: cpFP, circularly permuted fluorescent proteins; GKIB, cGMP-dependent protein kinase IB; NO, nitric oxide; PKA RIα, protein kinase cAMP-dependent type I regulatory subunit α; ΦD, the quantum yield of the donor; εA, extinction coefficient of the acceptor; J, the spectral overlap integral of the donor emission and acceptor absorption

Figure legends Figure 1. Development of a backbone set of a FRET-based indicator.

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(a) Schematic representation of a backbone set of a FRET-based indicator. Dashed boxes are the sensor motifs. ND- and CD-Group, N- and C-terminally located donor group, respectively. (b) Fluorescence spectra of Ca2+ indicators under free (dashed line) and saturated Ca2+ (solid line) conditions. DR, The dynamic range of the indicator.

Figure 2. Improvement of the dynamic range of cGMP indicator. (a) Fluorescence spectra of PKG-based cGMP indicators under free (dashed line) and saturated cGMP (solid line) conditions. DR, The dynamic range of the indicator. (b, c) Physiological cGMP oscillation observed in the population of D. discoideum cells. (b) FRET ratio images of cells expressing PKG #7, representing the spatial propagation of intracellular cGMP transients (from the right upper to left lower corner). Scale bar, 10 µm. (c) Time courses of the intracellular [cGMP] change in ROIs indicated in a. See also Supplementary Movie 1.

Figure 3. Extracellular cAMP imaging by an ultra-sensitive cAMP indicator. (a) The dynamic range of PKA RIα #1-8 assayed by using lysates of HEK293 cells. (b) Fluorescence spectra of purified PKA RIα #7 under free (dashed line) and saturated cAMP (solid line) conditions. (c-d) The spatio-temporal dynamics of the environmental cAMP released from chemotacting D. discoideum cells. (c) Bright-field images show repeated chemotaxis of wild-type cells. The wave propagation of [cAMP]ex revealed by FRET ratio images of the culture medium containing PKA RIα #7. Scale bar, 200 µm. (d) Time courses of the [cAMP]ex change and the velocity of representative chemotacting cells (N = 20 cells). See also Supplementary Movie 2.

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Graphical ToC 79x59mm (300 x 300 DPI)



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Figure 1. Development of a backbone set of a FRET-based indicator 140x201mm (300 x 300 DPI)


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Figure 2. Improvement of the dynamic range of cGMP indicator 140x233mm (300 x 300 DPI)



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Figure 3. Extracellular cAMP imaging by an ultra-sensitive cAMP indicator 140x226mm (300 x 300 DPI)


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Acceptor Pair in the

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