Chimeric Autofluorescent Proteins as Photophysical Model System for

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Chimeric Autofluorescent Proteins as Photophysical Model System for Multicolor Bimolecular Fluorescence Complementation (mcBiFC) Sébastien Peter, Sven zur Oven-Krockhaus, Manikandan Veerabagu, Virtudes MiraRodado, Kenneth W. Berendzen, Alfred J. Meixner, Klaus Harter, and Frank E. Schleifenbaum J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b11623 • Publication Date (Web): 27 Feb 2017 Downloaded from http://pubs.acs.org on February 28, 2017

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The Journal of Physical Chemistry

Chimeric Autofluorescent Proteins as Photophysical Model System for Multicolor Bimolecular Fluorescence Complementation (mcBiFC) Sébastien Peter1‡, Sven zur Oven-Krockhaus1‡, Manikandan Veerabagu1, Virtudes Mira Rodado1, Kenneth W. Berendzen1, Alfred J. Meixner2, Klaus Harter1, Frank E. Schleifenbaum1,3* 1

Center for Plant Molecular Biology, Plant Physiology, University of Tübingen, Auf der

Morgenstelle 32, 72076 Tübingen, Germany. 2Institute for Physical and Theoretical Chemistry, Auf der Morgenstelle 18, 72076 Tübingen, Germany. 3Berthold Technologies GmbH & Co KG, Calmbacherstr. 22, 75323 Bad Wildbad, Germany. ‡These authors contributed equally to this work.

The yellow fluorescent protein YFP is frequently used in a protein complementation assay called Bimolecular Fluorescence Complementation (BiFC), employed to visualize protein-protein interactions. In this analysis, two different, nonfluorescent fragments of YFP are genetically attached to proteins of interest. Upon interaction of these proteins, the YFP fragments are brought into proximity close enough to reconstitute their original structure, enabling fluorescence. BiFC allows for a straightforward readout of protein-protein interactions and furthermore facilitates their functional investigation by in vivo imaging. Furthermore, it has been

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observed that the available color range in BiFC can be extended upon complementing fragments of different proteins that are, like YFP, derived from the Aequorea victoria green fluorescent protein, thereby allowing for a multiplexed investigation of protein-protein interactions. Some spectral characteristics of (multicolor) BiFC complexes have been reported before; however, no in-depth analysis has been performed yet. Therefore, little is known about the photophysical characteristics of these multicolor BiFC complexes because a proper characterization essentially relies on in vitro data. This is particularly difficult for fragments of autofluorescent proteins (AFPs) because they show a very strong tendency to form supramolecular aggregates which precipitate ex vivo. In this study, this intrinsic difficulty is overcome by directly fusing the coding DNA of different AFP fragments. Translation of the genetic sequence in E. coli leads to fully functional, well soluble fluorescent proteins with distinct properties. Based on their construction, they are designated chimeric autofluorescent proteins, or BiFC chimeras, here. Comparison of their spectral characteristics with experimental in vivo BiFC data confirmed the utility of the chimeric proteins as BiFC model system. In this study, nine different chimeras were thoroughly analyzed both at the ensemble as well as at the single-molecular level. The data indicates that mutations believed to be photophysically silent significantly alter the properties of AFPs.


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INTRODUCTION One of the most intriguing findings concerning autofluorescent proteins (AFPs) is the fact that they can be successfully employed in so-called protein complementation assays (PCAs). In a PCA, the coding sequence of a reporter protein with known function is split into two parts, each attached to different proteins of interest. Expression of the coding sequence leads to nonfunctional fragments of the reporter. Upon interaction of the two proteins of interest, the fragments of the reporter are brought into close proximity, thereby reconstituting its original function, i.e. fluorescence in case of an AFP. Originally derived from splitting the yellow fluorescent protein1, it has been shown that this concept of bimolecular fluorescence complementation (BiFC) can be generalized for the use of several Aequorea victoria-derived AFPs. Bimolecular fluorescence complementation with fragments of different AFPs, however, may alter the spectral properties. This “multicolor” BiFC (mcBiFC) theoretically enables multiplexed imaging of protein complexes of different compositions. Therefore, AFPs are very interesting PCA-reporters as they allow for functional investigations by microscopy along with a straightforward readout of protein-protein interactions by widely available fluorescence techniques. The employed AFPs are divided into two fragments with non-equal size. In an established scheme, the N-terminal fragment consists of amino acids 1-154, corresponding to the first seven β-sheets of the AFP β-barrel. The complementing C-terminal fraction contains the amino acids 155-238. Although BiFC is widely used in functional imaging in microscopic studies, a precise knowledge of the photophysical parameters, especially when it comes to mcBiFC systems, has been missing so far. This is required in order to choose the appropriate experimental conditions


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in microscopic studies as well as for a robust interpretation of the experimental results. To this end, a photophysical characterization has to be carried out in vitro by investigation of purified BiFC complexes in a well-defined surrounding. The straightforward idea is to heterologously express the fragments and to generate mcBiFC complexes by simply mixing any two AFP fragments of interest, because a weak self-interaction of complementary BiFC fragments has been observed2. However, this is obstructed because the fragments employed in BiFC show a strong tendency to precipitate under the conditions that are typically applied in heterologous protein expression for subsequent protein purification. In fact, very harsh conditions such as using buffers with high amounts of chaotropic agents (e.g. 6M guanidine) were necessary to maintain the AFP fragments in solution3. Obviously, such an environment is not appropriate for forming functional complexes in vitro. As a consequence, all our trials to assemble functional BiFC complexes in vitro by this method failed (data not shown) although Kerppola et al. have reported the observation of targeted complex formation in vitro by quick dilution of denatured samples to a more appropriate regime2, 3. This problem is addressed here by directly fusing the coding sequence of AFP fragments, yielding open reading frames encoding for novel AFPs with the usual length of 238 amino acids, however with the N- and C-terminal sequence originating from different parents. Expression of these chimeric AFPs (called BiFC chimeras here) in E. coli leads to considerable amounts of fully functional, soluble proteins. These BiFC chimeras are therefore used here as model system to access the photophysics of mcBiFC complexes. Three different AFPs were used as parents in this study: the cyan emitting monomeric variant mCerulean, the enhanced green fluorescent protein EGFP and the yellow variant Venus. The chimeras each consisted of an N-terminal fragment, 154 amino acids in length, and a C-terminal


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fragment encoding amino acids 155-238. Permutation of all N- and C-terminal fragments led to nine different fluorescent proteins. For clarity, they will be named in this study according to their origin, although the sequences reveal only minor differences. For example, the chimeric fluorescent protein consisting of the first 154 amino acids of mCerulean and the C-terminal fragment of Venus is designated C154-V. Similarly, G154-C is the chimera generated from the N-terminal EGFP fragment and the C-terminal mCerulean fragment. The names of the parent AFPs are of course kept (e.g. G154-G equals EGFP). Care has to be taken not to confound this with unhyphenated designations which describe mutations in the proteins’ amino acid sequences according to common terminology. Essentially, the BiFC chimeras are expected to exhibit spectral properties which are explained by either their chromophore or their surrounding. Generally, the photophysical properties of AFPs are dominated by their chromophore, with the surrounding amino acids exhibiting additional effects. The structure of all AFPs known to date consists of 11 β-sheets forming a socalled β-barrel with a single α-helix spanning its interior. This α-helix contains the chromophore, which is formed from a tripeptide of amino acids 65-67 in an autocatalytic manner. In wild type GFP (wtGFP), the basis for the AFPs of this study, the chromophore is formed from S65, Y66 and G67 in a three-step reaction—by cyclization and dehydration of the protein backbone, an imidazolinone moiety is formed, to which the phenol side group of Tyr66 is covalently bonded. The π-systems are conjugated by the oxidation of Tyr66’s Cα-Cβ-bond. The chromophore of wtGFP exhibits two absorbance maxima at around 400 nm and 480 nm. These transitions are known to originate from two chemically distinct forms of the chromophore differing in the protonation status of the phenolic hydroxyl group4. While the protonated (neutral) form (often referred to as A-form, see Supplemental Figure 1) exhibits its absorbance


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maximum at around 400 nm, deprotonation of this hydroxyl group leads to an anionic chromophore (B-form, Supplemental Figure 1), with its absorbance bathochromically shifted to around 480 nm. wtGFP has been modified by targeted and random mutagenesis in order to extend the available color range of AFPs and to further improve photophysical and biochemical properties such as the fluorescence quantum yield and the chromophore maturation rate. Prominent classes of Aequorea-derived AFPs include the classes of yellow (YFP) and cyan (CFP) fluorescent variants. Yellow fluorescence is achieved by mutation of T203Y. The mechanism involves π-πstacking between Tyr203 and the chromophore, effectively lowering the transition energy. Cyan fluorescence is achieved by substituting the chromophore’s Tyr66 for Trp, effectively shortening the chromophoric π-system, which leads to the characteristic hypsochromic shift of absorbance and emission. Other mutations to the GFP sequence often address biochemical properties, such as the folding efficiency in vivo or the oxidation rate of the chromophore. The BiFC chimeras employed in this work are expected to exhibit spectral properties which are explained by the above-mentioned mutations of their parents. These are listed, relative to wtGFP, in Table 1. A more thorough discussion of the mutations and their effects is also given in the Supporting Information. Table 1 Parent AFPs employed in this study and mutations relative to wtGFP AFP variant

Mutations relative to wtGFP

EGFP

F64L,S65T

mCerulean

F64L,S65T,Y66W,S72A,Y145A,N146I, H148D,M153T,V163A,A206K

Venus

F46L,F64L,S65G,V68L,S72A,M153T, V163A,S175G,T203Y


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As an alternative to resolve the spectral properties of BiFC complexes, the generation of soluble YFP fragments has been previously reported and achieved5 by introducing a set of mutations known from the so-called superfolder GFP6, which has also been explored in the course of this study. Indeed, these modifications led to moderately soluble protein fragments. When mixing two superfolder fragments, very weak absorbance and fluorescence evolved in the course of a couple of days. As the parallel establishment of the chimeric AFPs turned out to be much more efficient in terms of functional protein yield, the superfolder fragments were abandoned in this study. We expected the spectral properties of our chimeras to largely anticipate the photophysical properties of functional BiFC complexes due to the large structural homology of a more recently purified BiFC complex to its uncleaved counterpart7. This hypothesis was probed by comparing the spectra and fluorescence lifetimes of some of our BiFC chimeras to the spectral properties of functional BiFC complexes in living Tobacco (N. benthamiana) leaf cells. Subsequently, the purified BiFC chimeras were thoroughly analyzed in ensemble studies and the molecular dynamics of the AFPs were probed by means of single molecule spectroscopy. MATERIALS AND METHODS Chimeric Autofluorescent Proteins were generated by means of translational fusion by linking the coding DNA of each N-and C-terminal AFP fragment. The final construct was cloned into the bacterial expression vector pET28(b). E.coli BL21(DE3) was used as expression strain. Proteins were purified by metal chelate affinity chromatography. Ensemble absorbance spectra were acquired with a Perkin Elmer Lambda 15 spectrograph (Perkin Elmer, Germany). Fluorescence excitation and emission spectra were recorded using a Varian Cary Eclipse Spectrophotometer (Agilent technologies, Germany). BCA assays were read out by a plate reader (Tecan Safire, Tecan, Germany)


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Fluorescence lifetimes and single-molecule investigations as well as in vivo measurements were performed with a custom-built confocal microscope based on a Zeiss Axiovert 135TV8. A more detailed Materials and Methods description can be found online as Supplemental Information.

RESULTS In vivo Bimolecular Fluorescence Complementation driven by Homo- and Hetero-Dimer formation of bZip63: The fluorescence of a split fluorescent protein whose fragments are attached to different cellular proteins can be reconstituted when the latter interact and bring the fragments into close proximity to each other. The fluorescence spectrum then depends on which fluorescent proteins the respective fragments originated from. To demonstrate this, we fused fragments of the coding DNA of the yellow fluorescent protein Venus, cleaved after amino acid 154, to the Basic Leucine Zipper transcription factor bZip63, which is well-known to form homodimers9. These constructs were cloned into appropriate vectors for transformation of tobacco (N. benthamiana) plant leaves by Agrobacterium tumefaciens. Sections of transformed plant leaves expressing bZip63 fused to the N- and C-terminus of Venus (bZip63-VenN and bZip63-VenC, respectively) were investigated by means of (spectro-) microscopy. Upon illumination at 473 nm (commonly used in our laboratory for fluorescence lifetime imaging), cotransformants of bZip63-VenN and bZip63-VenC gave a strong fluorescence signal located within the nucleus. Fluorescence spectra recorded from different areas in the nucleus continuously exhibited a single maximum at 527 nm, as expected for Venus (Figure 1, orange). Time-resolved measurements revealed a fluorescence lifetime of 3.1 ns, close to reported values of Venus10. A strong fluorescence signal was even observed upon excitation at 438 nm where


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Venus only absorbs weakly, which points to a high concentration of recombined AFPs within the probed volume. In total, the complemented fluorescence signal of Venus exhibited photophysical properties very close to those of uncleaved Venus (Figure 1, gray shaded area).

Figure 1 Fluorescence spectra acquired from N. benthamiana nuclei transformed with bZip63VenN + bZip63-VenC (orange), ARR18-CerN + bZip63-VenC (cyan) or ARR18-CerN + bZip63-VenC + bZip63-VenC (red). For comparison, the spectrum of Venus purified from E.coli is shown gray-shaded. Similarly, we checked the fluorescence of BiFC complexes reconstituted from an N-terminal fragment of the cyan fluorescent protein mCerulean and the C-terminal fragment of Venus. For this purpose, we transformed N. benthamiana leaves with the aforementioned construct bZip63VenC along with a construct where the N-terminal fragment of mCerulean was fused to the transcription factor ARR18 (construct ARR18-CerN), known to form heterodimers with bZip6311. Fluorescence in co-transformants of bZip63-VenC and ARR18-CerN could be excited efficiently with a 438 nm laser source and was also located in the nucleus. The broad, unstructured fluorescence spectrum peaked at around 500 nm (Figure 1, cyan), whereas mCerulean peaks at around 470 nm. This large bathochromic shift has to be due to the


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interaction of the mCerulean chromophore, located in the N-terminal AFP fragment, with the Cterminal fragment of Venus, carrying Tyr203. This residue is well-known to bathochromically shift absorbance and fluorescence spectra of GFP, yielding the class of yellow fluorescent proteins. Obviously, the photophysical properties of the generated BiFC complex differ largely from both mCerulean and Venus. This is in line with spectral properties described by Hu et al.2 in a similar experimental setting: when complementing an N-terminal fragment, derived from CFP cleaved at amino acid 173, with a C-terminal fragment of YFP (cleaved at the same position), they observed a spectral shift of the CFP chromophore, with the emission peaking at 497 nm. This value is clearly bathochromically shifted compared to a complemented CFP signal observed in the same experimental setting, emitting at 478 nm, and is largely congruent with our observation of fluorescence reconstituted from N-terminal mCerulean and C-terminal Venus. Interestingly, Shyu et al.12 observed spectra closely resembling CFP when combining an Nterminal Cerulean fragment, cleaved at position 173, with a C-terminal Venus fragment, cleaved at position 155. While there can be no doubt that the bathochromic shift observed in the combination of the Cerulean chromophore with a C-terminal Venus fragment is caused by the ππ-stacking between the chromophore and Tyr203 in the C-terminal fragment, the lack of this shift in Shyu et al.s experiments is indicative that the π-systems in the different protein fragments did not properly stack. One cause for this could be that a part of the protein structure, namely amino acids 156 to 173, were duplicated in their experiment, which might distort the protein structure. However, this is not a general rule as Shyu et al. observed yellow fluorescence very close to the native protein when complementing fragments of Venus where the same amino acids were duplicated. It is not clear how these extra amino acids are accommodated in the protein structure but it seems to be intuitive that the structure of a reconstituted BiFC complex will have


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major consequences for its photophysical behaviour. Without structural data, this remains speculative, though. The potential of BiFC to study multiplexed protein-protein interactions at the same time was probed by co-transformation of bZip63-VenC with both bZip63-VenN and ARR18-CerN. Some nuclei exhibited fluorescence spectra that could be assigned to one of the complexes mentioned above. Many nuclei, however, exhibited a combination of spectral characteristics of the aforementioned combination of Cer-N and Ven-C as a pronounced shoulder around 500 nm in spectra otherwise dominated by Venus fluorescence (Figure 1, red). We did, however, not observe any nuclei that were dominated by the spectrum of CerN-VenC. This result could be explained in two different ways: first, the interaction of bZip63 with itself could be much stronger than the interaction with ARR18, leading to an amount of fluorescence complementation that is overall larger in case of Venus than in case of CerN-VenC. Second, one has to take into account that the signal provided by a fluorophore largely depends on its photophysics, mainly its absorbance cross section and its fluorescence quantum yield. While one could infer the photophysical properties of the complemented Venus from its uncleaved parent, this is definitely impossible in case of complemented CerN-VenC because there is no adequate parent to derive the properties from. A quantitative estimation of protein-protein interactions formed in such a multiplexed BiFC assay essentially relies on the precise knowledge of the photophysical properties of the formed BiFC complexes. As mentioned before, such a characterization requires the fluorophores to be available in vitro but is frustrated by the insolubility of the fragments. Therefore all experiments below were performed by translational fusions of the respective AFP fragments, the BiFC chimeras.


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Spectral characterization of the generated BiFC chimeras: The following presentation of the photophysics of the BiFC chimeras often compares values of the photophysical parameters relatively. The exact values determined in this work are summarized in Table 2 in the discussion section. It has to be noted that mCerulean and EGFP differ in their C-terminal fragments (amino acid 155-238) in only two mutations, namely the folding mutation V163A and mutation A206K which is known to suppress dimer formation. Since A206K is not believed to have any effect on either the spectroscopic properties nor the AFP structure13, differences between chimeras containing the C-terminal fragment of either mCerulean or EGFP can directly be assigned to mutation V163A. On the other hand, the C-terminal fragment of Venus differs from the Cterminal EGFP fragment in the folding mutations V163A and S175G along with mutation T203Y, known to cause a lower energy for electronic transitions in the chromophore. Figure 2 shows the ensemble absorbance, excitation and fluorescence spectra of all chimeric AFPs under investigation, recorded at pH 8.0. The absorbance spectra can be easily identified by their strong absorbance band at 280 nm, originating from the aromatic amino acids Trp, Tyr and Phe.


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Figure 2 Solid lines: absorbance and (bathochromically shifted) fluorescence emission spectra of the generated BiFC chimeras; red dashed lines: fluorescence excitation spectra of the BiFC chimeras. The colors of the solid lines roughly reflect the colors of their peak wavelength. Exact values can be found in Table 2.

Regarding the spectra of C154-V (Figure 2c), one finds that they are shifted bathochromically compared to their relatives with the same chromophore. The bathochromic shift of ~20 nm (relative to mCerulean) is most certainly caused by mutation T203Y by π-π stacking of the chromophore with the newly introduced Tyr20314. The observation that the absorbance spectrum


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still exhibits two pronounced absorbance bands supports the hypothesis that the overall band structure of CFP-like chromophores is due to coexisting electronic transitions. This has been proposed to be an intrinsic property of these chromophores15,

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, rather than due to the

simultaneous observation of different three dimensional protein configurations present at least in the enhanced cyan fluorescent protein (Supplemental Figure 2). G154-C (Figure 2d) and EGFP (Figure 2e) both exhibit their absorbance maximum at 490 nm with a shoulder of slightly varying intensity at 403 nm. These bands are known to originate from the deprotonated (~490 nm) and neutral (~400 nm) form of the chromophore, respectively. The corresponding shoulder of G154-C exhibits slightly higher absorbance than in case of EGFP which has to be caused by mutation V163A. Despite the minor difference in the intensity of the protonated band, the spectral properties of these two proteins are almost identical. This picture changes quite dramatically upon incorporation of mutation T203Y in G154-V (Figure 2f). As could be expected, Tyr203 causes a large spectral shift, with the deprotonated form of the chromophore now found to absorb at 515 nm. While the band position of the protonated form stays essentially the same, it is now roughly three times more intense than the deprotonated one. In general, the protonation equilibrium of green fluorescent proteins has been shown to follow complicated pathways. One of the key factors is a hydrogen bond between the amino acid at position 65 (Thr in case of EGFP) and Glu22217. G154-V is closely related to a previously reported variant18 called E2GFP (this corresponds to EGFP_T203Y). This mutant has been investigated with emphasis on the protonation equilibrium, using molecular dynamics simulations19. Following the arguments of Nifosi and Tozzini19, two mechanisms are known to disturb the protonation equilibrium of green fluorescent proteins: suppression of the protonated form can occur either when the aforementioned hydrogen bond is disturbed or when the


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deprotonated state is additionally stabilized. Mutation S65T is known to shift the protonation equilibrium towards the deprotonated form, arguably due to a steric destabilization of this hydrogen bond. However, mutation T203Y destabilizes the anionic form of the EGFP chromophore20, thereby overcompensating of the effect of S65T, which leads to the protonated form being predominantly present in E2GFP as well as in G154-V. The excitation spectrum of G154-V illustrates that almost no fluorescence is excited upon illumination of the protonated chromophore around 400 nm. In wtGFP, containing the same chromophore as G154-V, fluorescence upon excitation of the protonated chromophore is known to follow a pathway including excited state proton transfer (ESPT) prior to emission from the deprotonated, excited state21. wtGFP is known to exhibit very efficient ESPT and fluorescence of wtGFP fluorescence is therefore excited efficiently in its protonated form as well20. This behaviour is not reflected in G154-V, which is in line with the previously reported loss of fluorescence in the protonated forms of wtGFP_T203Y20 and EGFP_T203Y18. The suppression of fluorescence emission of the excited protonated state in G154-V is even more pronounced than what has been reported for EGFP_T203Y18. As these two proteins only differ in that G154V carries the mutations V163A and S175G, these mutations are therefore identified to additionally suppress fluorescence emission from the excited protonated state of the GFP chromophore. The excitation spectra of G154-C and EGFP underline this statement: in EGFP, the excitation spectrum largely follows the absorbance spectrum in the region around 400 nm. In G154-C, containing mutation V163A but not S175G, the excitation spectrum is lower in this spectral region than the absorbance spectrum. Therefore, V163A seems to influence ESPT. While S175G is apparently too distant to directly affect the ESPT in G154-V, a contribution to the altered protonation equilibrium via different mechanisms still cannot be excluded because


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mutation S175G has large conformational consequences22 that could affect the structure around the chromophore. The spectra of V154-C (Figure 2g), V154-G (Figure 2h) and Venus (Figure 2i) largely follow the trend observed for the BiFC chimeras with an N-terminal EGFP fragment, with V154-C and V154-G being spectrally largely identical. Fluorescence emission seems to be somewhat suppressed upon excitation at ~400 nm in case of V154-C compared to V154-G which is perfectly in line with the observations made for G154-C and EGFP. However, the effect is less obvious for the chimeras with the N-terminal Venus fragment because the protonated form is overall suppressed. The dominant contribution of the deprotonated chromophore has been discussed above: disturbance of the hydrogen bond between the amino acid at position 65 and Glu222 can lead to suppression of the protonated form19 at physiological pH, a condition obviously fulfilled by Gly65. This is also observed for Venus where the protonated form is virtually absent in both the excitation and the absorbance spectrum. pKa determination: Fluorescent proteins are known to be stable over a wide pH range, down to a pH of at least 5.0

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. However, the photophysical properties largely depend on the

protonation state of the chromophore, as could already be seen in the previous section. We therefore investigated how the BiFC chimeras act upon changes in the pH. To this end, the BiFC chimeras were dissolved in buffers differing in pH (ranging from 5.0 to 10.0) and relative fluorescence intensities were measured by exciting the deprotonated forms of the chromophores. The relative fluorescence intensity can be used to determine the chromophores’ pKa values. The structure of the CFP chromophore forbids direct protonation at Trp66. In case of mCerulean, however, the chromophore has been shown to be sensitive to pH changes15 with close-by Asp148 being a candidate for protonation. This residue stabilizes the chromophore via a


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hydrogen bond. It has been observed here that upon protonation, fluorescence is lost. Therefore, protonation of Asp148 probably leads to fluorescence quenching, which is used here for determining the pH stability of mCerulean fluorescence. In Figure 3 (left panel), the relative fluorescence intensity of chimeric AFPs containing the Nterminal mCerulean fragment is shown in dependence of the pH. The relative loss of fluorescence intensity at decreasing pH can be nicely fit by a sigmoidal model function (dashed lines) with the inflection points representing the pKa values of the chromophoric systems. As mentioned before, protonation in these variants likely takes place in the chromophore’s environment rather than at the chromophore itself, probably affecting the hydrogen bonding network within the protein. Whereas the inflection points for mCerulean and C154-G seem to be similar (pKa = 5.2 and 4.9 for mCerulean and C154-G, respectively), C154-V is somewhat more basic (pKa = 5.9). Due to potentially insufficient equilibration of the samples, these results— especially for mCerulean and C154-G—have to be taken cum grano salis, though.


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Figure 3 Evolution of the relative fluorescence intensity depending on pH changes for BiFC chimeras containing an N-terminal mCerulean (left panel), EGFP (middle) or Venus (right panel) fragment. Series were fit by sigmoidal model functions (dashed lines) The titration curves of G154-C and EGFP (Figure 3) are, within experimental errors, identical, with pKa values found at 5.9 (G154-C) and 5.8 (EGFP). In contrast to that, the measured pKa value of 8.5 for G154-V makes this variant the most basic of all AFPs investigated here. The chimeras containing the N-terminal fragment of Venus largely confirm the evaluation made in the previous section with pKa = 6.6 for V154-C and V154-G. For Venus the found pKa value of 5.7. fairly matches the one reported before (5.8022). In the absorbance spectra of G154-C and EGFP (previous section, Figure 2d,e), recorded at pH 8.0, the protonated band was still visible whereas this band was nearly absent for V154-C and V154-G (Figure 2g,h). From the absorbance spectra, one would therefore expect a more acidic nature of the Venus chromophore than for the EGFP chromophore. The titration curves (Figure 3), however, revealed pKa values suggesting a more acidic chromophore for G154-C and EGFP (pKa = 5.8) than for V154- C and V154-G (pKa = 6.6). The pKa values of G154-C and EGFP would suggest only a low population of the protonated state of the chromophore at pH 8.0, but this is not reflected in the bulk spectra. Obviously, the absorbance band at ~400 nm indicates that there is a population of protonated chromophores in EGFP and G154-C that is reluctant to deprotonate. While the postulation of such a “frozen” fraction seems dubious from a thermodynamic point of view, Bizzarri et al. have plausibly proposed a second protonation site in green fluorescent proteins, most likely at Glu22224. They have argued that the protonation sites of the chromophore and at Glu222 exhibit an anticooperative behaviour which explains the presence of a protonated fraction at pH well beyond the pKa of the chromophore. To sum it up,


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the chromophores of G154-C and EGFP are more acidic than those in V154-C and V154-G, however with a fraction of G154-C and EGFP that cannot be deprotonated within the pH regime applied here. The pKa values of the chimeras containing an N-terminal fragment of EGFP or Venus reveal some interesting behaviour regarding the influence of single mutations on the protonation equilibrium. Briefly, mutations S65T (in the EGFP N-terminal fragment) and S65G (in the Venus N-terminal fragment) were both identified to promote deprotonation. From the pKa values (~5.8 for G154-C, EGFP and ~6.6 for V154-C, V154-G) one could assume that the chromophore of EGFP (T65Y66G67) is overall more acidic than the chromophore present in YFP variants (G65Y66G67). When taking into account mutation T203Y, this picture changes. It is observed that G154-V has a higher pKa value (pKa = 8.5) than Venus (pKa = 5.8). Thus, the protonation equilibrium cannot be explained by an isolated investigation of single mutations. We therefore conclude that cooperative effects and the full context, i.e. the structure of a fluorescent protein, have to be respected as well. Determination of ensemble photophysical parameters: A key parameter of autofluorescent proteins is the signal intensity they provide in microscopic studies, determined by the absorbance coefficients and the fluorescence quantum yields. The product of these is generally referred to as “brightness”. These parameters of the BiFC chimeras were recorded at pH 8.0 and are given in Figure 4.


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Figure 4 Ensemble parameters of chimeric AFPs. Red bars: ε at the maximum absorbance of the respective deprotonated forms. Blue bars: fluorescence quantum yields. Green bars: brightness relative to EGFP The brightness of AFPs is largely influenced by the relative number of properly processed protein entities containing a correctly processed chromophore where the observation of nonfunctional protein entities in populations of fluorescent proteins has been reported before17, 25, meaning that AFPs exhibit a variable chromophore formation efficiency (CFE). Accordingly, a change in the relative CFE is reflected by an altered absorbance coefficient ε. For the chimeras containing the N-terminal mCerulean fragment, the absorbance coefficients turned out to be very similar, with a slight decrease of ε in the order mCerulean > C154-G > C154-V. EGFP and G154-C have almost identical absorbance coefficients. For G154-V, the absorbance coefficient of the deprotonated form shown in Figure 4 appears to be very low, rather caused by the low population of the deprotonated state than by an intrinsic low absorbance cross section of this form. Venus is known to have a very high absorbance coefficient26. The Chromophore G65Y66G67, also found in V154-C and V154-G, is found to absorb light very efficiently in the latter variants as well, with V154-C exhibiting a slightly higher extinction coefficient than V154-G.


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Regarding the influence of the C-terminal fragment, mCerulean apparently has a higher absorbance coefficient than C154-G. A similar trend is observed in case of the N-terminal Venus fragment, with V154-C exhibiting a higher extinction coefficient than V154-G. On the other hand, identical absorbance coefficients are found for G154-C and EGFP, but the absorbance spectra have revealed a larger fraction of protonated chromophores in G154-C than in EGFP. Taken together, these findings are overall attributed to a higher CFE in the chimeras with the Cterminal mCerulean fragment than for those with the C-terminal EGFP fragment. This finding is supported by taking into account that the C-terminal fragment of mCerulean contains the “folding mutation” V163A27, 28. Although the exact pathway of how V163A supports the protein folding is unclear, it appears here that overall the mutation leads to a higher fraction of correctly processed protein entities. The presence of dark proteins leads to the consequence that the extinction coefficients found for ensembles of fluorescent proteins do not translate into absorbance cross sections according to the following equation:

= ∙ 10 ∙

10

where σA is the absorbance cross section of the chromophore, ε the molar extinction coefficient and NA represents Avogadro’s number. Rather, deduction of σA of AFPs from their ensemble extinction coefficients leads to an underestimation of their absorbance and fluorescence cross sections. The correct absorbance cross section is by the way found by accounting for the pKa value and the relative number of total chromophores within a population of AFPs. Fluorescence quantum yields and the brightness of BiFC chimeras: One key parameter of fluorescent proteins for their use in vivo is the relative signal they provide in fluorescence


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microscopy. This signal, commonly referred to as brightness, is calculated relative to the EGFP brightness from the fluorescence quantum yield and the extinction coefficient with . ℎ

=

∙ Φ ( ) ∙ Φ ( )

with ε representing the molar extinction coefficients of the chromophores and ΦF being the fluorescence quantum yield. The relative brightness is therefore affected by the CFE, σA and the fluorescence quantum yield ΦF. For fluorescent proteins, εmax is accessible via determination of the extinction coefficient while ΦF has to be determined independently. Fluorescence quantum yields of the chimeric AFPs were thus determined and are shown in Figure 4 along with the brightness, which is given relative to EGFP. Looking at the influence of mutation V163A in the C-terminal fragment, it stands out that the fluorescence quantum yield is considerably higher for mCerulean than for its counterpart carrying the C-terminal fragment of EGFP, whereas V163A does not seem to have any influence in chimeras with other N-terminal fragments (cf. G154-C/EGFP and V154-C/V154-G). The overall higher fluorescence quantum yield of mCerulean, compared to C154-G, could indicate that V163A influences the non-radiative decay rates (see discussion below). This would be plausible because alanine is considerably smaller than valine which could lead to a lower probability of collisional relaxation in the excited state. The fluorescence quantum yields for mCerulean have been somewhat under debate in the past decade. In the original work introducing this protein, Rizzo et al. reported a quantum yield of 0.6229. Using Atto425 as a reference for the determination of ΦF, we found a value of 0.48 which is very close to more recently published values30, 31. The fluorescence quantum yields of C154-G and C154-V were calculated relative to mCerulean which, following Markwardt et al.31, was assumed to exhibit a fluorescence quantum efficiency of 0.48.


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The fluorescence quantum yield of G154-V was determined for its deprotonated form because it contributes most to fluorescence, and was found to be 0.64. Judging from the the absorbance and fluorescence excitation spectra, one can estimate the fluorescence quantum efficiency of the protonated form to be approx. 1.5%. The reasons for the poor emission from the excited protonated state have been discussed above. Evaluation of the brightness reveals that the fusions of each N-terminal fragment to either the C-terminal mCerulean or EGFP fragment does not largely affect the total brightness of the fluorescent proteins. V154-C is found to be slightly brighter than V154-G, presumably due to an enhanced folding efficiency leading to a larger number of intact protein entities. Interestingly, fusion of the C-terminal Venus fragment to the N-terminal fragments of mCerulean and EGFP led to very dim proteins, in case of C154-V because of a poor fluorescence quantum yield, in case of G154-V because the chromophore is mainly present in a nearly non-fluorescent protonated form. This makes G154-V a potential in vivo pH-sensor for compartments with high pH. The reason of the low quantum yield in C154-V is speculative without structural data. However, a plausible explanation would include steric reasons as well as a distortion of the chromophore and/or its surrounding due to the introduction of the bulky Tyr203. Furthermore, a disturbed hydrogen bonding network around the chromophore has to be considered as well since the substitution T203Y is well-known to affect the stabilization of the chromophoric embedment, at least in EGFP19. Fluorescence lifetimes and excited state decay rates: A distinction of fluorescent proteins in vivo is not limited to a discrimination according to their spectra but also possible according to their fluorescence lifetimes (FLT). Therefore, the FLT of the BiFC chimeras have been determined (Figure 5). The radiative and non-radiative decay rates (kF and knr, respectively) can


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be calculated from the fluorescence quantum yield ΦF and the fluorescence lifetime τ according to the following set of equations: " = $ "%& =

Φ # 1 ' Φ #

Figure 5 depicts the radiative and non-radiative decay rates determined for the BiFC chimeras on top of each other. The sum of them corresponds to 1/τ. Figure 5 Radiative (red bars) and non-radiative (gray bars) decay rates of BiFC chimeras. Total

bar height corresponds to 1/τ for each chimera. Analysis of the chimeras with identical N-terminal fragments reveals that the decay rates, both radiative and non-radiative, undergo almost no change when exchanging the C-terminal mCerulean fragment by the C-terminal fragment of EGFP (cf. mCerulean/C154-G, G154C/EGFP and V154-C/V154-G). In line with the spectral characteristics of the BiFC chimeras, this means that mutation V163A only plays a minor role in the photophysical behaviour of these BiFC chimeras. This picture changes dramatically when taking into account the alterations introduced by mutation T203Y. In C154-V, the radiative decay rate slightly decreases with respect to mCerulean and C154-G. Even more obvious, however, is the increase in the non-


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radiative decay rate causing both a low fluorescence quantum yield and a rather low fluorescence lifetime (2 ns for C154-V versus 3.24 ns for mCerulean). This could be attributed to steric clashes between the bulky chromophore of mCerulean, containing Trp66, and the bulky Tyr203. For chimeras containing an N-terminal fragment of mCerulean, it is furthermore interesting to note that the changes in the absorbance coefficients are largely mirrored by their fluorescent decay rates. This could indicate that especially in C154-V the transition dipole moment is rather small, making the spectroscopic transitions under investigation rather unlikely. In case of G154-V and Venus, a different behavior is observed. Surprisingly, introduction of Tyr203 only slightly decreases the radiative decay rates and more considerably lowers the nonradiative decay rates. Apparently, Tyr203 promotes fluorescence, which could already be guessed from the comparably high fluorescence quantum yields of these variants. V154-C and V154-G exhibit amplified non-radiative decay rates compared to G154-C and EGFP. To a lesser extent, this also applies to Venus (compared to G154-V). These comparably high non-radiative decay rates are therefore possibly attributed to an intrinsic property of the chromophore. Single molecule emission spectroscopy and spectral distribution width: Chimeric fluorescent proteins containing an N-terminal fragment of either EGFP or Venus were accessible by means of single molecule spectroscopy. The variants with an N-terminal fragment of mCerulean, however, exhibited a very low photostability under the chosen experimental conditions which precluded them from such an investigation. They are therefore not discussed in this context. Figure 6 gives some examples of single molecule emission spectra of Venus, EGFP and G154V. The maxima positions of the spectra were determined by fitting two Gaussians to every spectrum (gray lines in Figure 6). For comparison, the bulk emission spectra are plotted as well


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(gray shaded areas). Since single molecule experiments probe the individual behavior of emitters32, 33, the spectra do not neccessarily need to exactly overlap with the bulk spectra. This can, for instance, be seen in the EGFP spectrum in Figure 6 which is shifted by ~6 nm with respect to the ensemble where the individual behaviour of single emitters is averaged out.

Figure 6 Representative single molecule emission spectra of distinct BiFC chimeras. The spectra of G154-V exhibit a considerable shift, attributed to emission from different forms of the chromophore. The spectra were fit by two Gaussians (gray lines). The fit (colored line) is shown as a guide to the eye along with the bulk spectrum of the respective BiFC chimeras (shaded areas); the spectrum of the I-form of G154-V is accompanied by the bulk spectrum of EGFP. Generally, the spectra shown here exhibit a tendency of being narrower than the bulk spectra. This is explained by a large number of emitters, each with its individual peak wavelength, contributing to the ensemble spectrum. The single AFP emission spectra were quite homogeneous for every investigated species. In exceptional cases, however, distinct subpopulations of an AFP could be identified. An exemplary case of this behavior is shown in Figure 6, depicting two spectra of single G154-V molecules. Most spectra of this chimera exhibited a single maximum at the position expected from the ensemble, around 525 nm. This spectral form is assigned to emission from the deprotonated form of the chromophore, often referred to as B-form. In a few isolated cases, however, a clearly different form was observed, peaking around 510 nm which is rather typical for (E)GFP.


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Emission from yellow fluorescent protein variants in this spectral regime has been previously described at low temperatures34 and at ambient conditions35. The emission at these wavelengths is attributed to emission from the so-called I-state of the chromophore, which corresponds to a deprotonated chromophore, however in a conformation that is normally engaged by the protonated chromophore. Similarly, yellow emission of green variants has been observed in isolated cases (data not shown). This is in line with previously reported single-molecule data for EGFP35. An interconversion between these forms has not been observed in this study, though. The determination of the spectral distribution width furthermore allows for an evaluation of the rigidity of the AFP shell with narrow distributions translating into a flexible protein shell36. This is attributed to the fact that a flexible system can relax to its ground state in a quite undisturbed manner. For a more rigid system, this relaxation occurs in a stepwise fashion involving transition of several local energetic minima with possible emission from these sub-states, given that they are stable enough to be detected within the experiment36. For this, histograms are calculated from the maximum emission wavelengths observed in single molecule spectra. The spectral distributions are shown in Figure 7 along with Gaussian fits to the histograms. The spectral distribution widths, expressed as 2σ of the Gaussian fits, are indicated. The histograms were calculated from 413, 338 and 167 spectra for G154-C, EGFP and G154-V, respectively and 282, 371 and 300 spectra contributed to the histograms of V154-C, V154-G and Venus.


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Figure 7 Histograms of the single molecule emission maxima determined for different chimeric AFPs. The spectral distribution width is indicative for the protein rigidity with broad distributions translating to rigid proteins. From the histograms, Tyr203 is clearly identified as a key factor affecting the spectral distribution width. When comparing the spectral distribution width of EGFP to Venus (Figure 7) it becomes apparent that Venus exhibits a clearly broader spectral distribution width than EGFP, interpreted as a more rigid chromophoric environment. Conceivably, this change is caused by mutation T203Y for steric reasons: the bulkier Tyr203 in Venus allows less vibrational motion of the protein shell than the smaller Thr203 in EGFP. Based on structural data, it has to be noted, however, that Thr203 exhibits a stabilizing effect on the chromophoric environment by participation in the hydrogen bonding network within the protein17,

37

. The fact of a lower

flexibility of Venus therefore indicates that the strain introduced by Tyr203 overcompensates the relaxation in the hydrogen bonds. This effect of Tyr203 is underlined by a comparison of Venus


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with its relatives differing in the C-terminal fragment: V154-C and V154-G clearly exhibit narrower spectral distribution widths than Venus, strongly suggesting an important role of the amino acid at position 203. The most remarkable feature in Figure 7 is the very broad spectral distribution width observed for G154-V, indicating a very rigid protein shell. The N-terminal fragments of EGFP and Venus differ in a number of amino acids, most of which are being attributed to an enhanced folding efficiency. More efficient folding pathways have been discussed to be offered by sterically less demanding amino acid substitutions, which applies at least to mutations S72A and M153T and probably T65G. These mutations are not incorporated into G154-V, but in Venus. Therefore, the internal structure is even more packed in G154-V than in Venus, while the chromophore is additionally stabilized by a hydrogen bond between Thr65 and Glu22219. In total, this leads to the broad spectral distribution width which translates to a very rigid chromophore surrounding. Mutation V163A apparently has an effect especially on the chimeras V154-C (containing Ala163) and V154-G (containing Val163) (2σ = 3.0 nm vs. 2σ = 3.8 nm for V154-C and V154G, respectively). For the chimeras with an N-terminal EGFP fragment, however, there is hardly any difference. This points towards adverse effects of mutation V163A depending on the Nterminal fragment. For chimeras with N-terminal EGFP, mutation V163A only leads to a slightly more rigid protein shell. This observation, however, is at the very detection limit that can be achieved in such an investigation. Contrarily, the chimeras with the N-terminal Venus fragment undergo a considerable structural reinforcement upon replacement of Val163 with Ala163. Thus, the effect of the amino acid at position 163 cannot be regarded in an isolated manner. Rather, cooperative effects by amino acids within the N-terminal fragment of the respective BiFC chimeras have to be considered as well.


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DISCUSSION The purpose of this work has been a spectral characterization of the complexes used in multicolor Bimolecular Fluorescence Complementation (mcBiFC), based on chimeric autofluorescent proteins. The validity of our chimeric autofluorescent proteins as model system for in vivo BiFC complexes can directly be deduced from a comparison of the spectral properties of the chimeras with the respective in vivo data. Comparison of the in vivo BiFC co-transformations bZip63VenN + bZip63-VenC and bZip63-VenC + ARR18-CerN with their respective in vitro chimeric counterparts Venus and C154-V shows excellent agreement in both spectral (Figure 8, a and b) and fluorescence lifetime measurements. The in vivo BiFC spectra are almost indistinguishable from their in vitro analogues, which allows for linear spectral unmixing of in vivo spectra of multiplexed BiFC complexes using the in vitro spectra of the relevant chimeric autofluorescent proteins (Figure 8c).

Figure 8 Validation of BiFC chimeras as model system for in vivo BiFC complexes. a) and b): spectra of BiFC complexes in vivo (squares) and the respective BiFC chimeras in vitro (lines). c): the in vivo spectrum derived from complexes involving three BiFC fragments (circles) can be assigned to the formation of two distinct BiFC complexes by linear spectral unmixing using the spectra of their chimeric counterparts.


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It has to be furthermore noted that the set of quantitative parameters listed in Table 2 even allows for determination of the relative amount of each BiFC complex in a mixed sample (the details of the calculus can be found in the Supplemental Information). To demonstrate this, we calculated the relative fluorescence contributions of the single BiFC complexes to the spectrum in Figure 8c to be 75% (bZip63-VenN + bZip63-VenC) and 25% (ARR18-CerN + bZip63VenC). By division of these relative contributions with the luminosity that can be expected upon illumination at the chosen wavelength ((() ∙ Φ ), we find that 91% of the observed BiFC complexes are formed from bZip63 homodimers and 9% are assigned to ARR18-bZip63 heterodimers. Taking furthermore into account that bZip63 could also be bound in bZip63-VenN or bZip63-VenC homodimers that do not result in functional BiFC complexes, we can even calculate that ~45% of bound bZip63 is to be expected in non-fluorescent dimers and the relative amount of bZip63 bound in heterodimers involving ARR18 drops to 5%. With the caveat of small deviations between our in vivo and in vitro data, this is, to the best of our knowledge, the first quantitative estimation of the relative amounts of protein complexes in living cells. Table 2 Photophysical and photochemical parameters of the BiFC chimeras mCerulean

C154-G

C154-V

G154-C

EGFP

G154-Va

V154-C

V154-G

Venus

λabs [nm]

434

434

469

490

490

515

503

504

516

λexc [nm]

453

453

471

491

488

515

504

505

516

λem [nm]

477

477

508

511

510

525

514

515

529

pKa

5.2

4.9

5.9

5.9

5.8

8.5

6.6

6.6

5.7

ε [M-1 cm-1]

43000

38000

35000

55000

56000

7600

78000

71000

92200

ΦF

0.48

0.42

0.16

0.60

0.60

0.64

0.43

0.43

0.58

Brightnessb

0.61

0.48

0.16

0.98

1

0.15

1.01

0.91

1.58

τ [ns]

3.24

3.15

2.02

2.69

2.73

3.36

2.14

2.16

3.15

kFl [108 s-1]

1.36

1.23

0.71

2.23

2.20

1.92

2.03

2.00

1.83


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Σknr [108 s-1]

1.73

1.94

4.24

1.48

1.47

1.06

2.64

2.64

1.34

2σ (SMS) [nm]

n.d.

n.d.

n.d.

3.4

3.3

10.1

3.0

3.8

4.5

a

Values given for the fluorescent (deprotonated) form; b Brightness relative to EGFP

Similarly, the fluorescence lifetimes of bZip63-VenN + bZip63-VenC (3.05 ns) and Venus (3.15 ns) are nearly identical, whereas the fluorescence lifetimes of ARR18-CerN + bZip63VenC (1.77 ns) and C154-V (2.02 ns) are similar, but with a larger deviation. This is not surprising, as the local environment (e.g. pH38, cellular localization39) in (living) cells can alter the fluorescence lifetime of fluorophores. The small deviations in fluorescence lifetimes are therefore to be expected and we are very confident that our approach to probing otherwise unattainable photophysical properties of BiFC complexes is robust and sound. Like frequencyresolved spectra, time-correlated single photon counting data acquired for determining fluorescence lifetimes can be used for the calculation of the relative amounts of fluorescent species within a sample (see Supplement). Based on time-resolved measurements, we found that, for the nucleus discussed above, ~11.5% of the fluorescence originates from ARR18-bZip63 heterodimers. Taking into account a probable dark fraction of bZip63 homodimers (~42%), we find that ~6% of bZip63 is bound in bZip63-ARR18 heterodimers, which fairly matches the numbers estimated from the frequency-resolved spectrum. This congruence underlines the overall robustness of our in vitro data as well as their applicability to in vivo investigations. The chemical, photophysical and molecular parameters that have been probed in this study are summarized in Table 2 and certainly offer a somewhat more general understanding of the effect of some amino acid substitutions in AFPs. Looking for fragment combinations that can be used for observing (multiplexed) interactions in vivo, some beneficial pairings with clearly different properties can be identified. For instance, combination of an N-terminal AFP fragment with


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either C-terminal mCerulean or EGFP was expected to lead to largely identical spectral properties, which could be confirmed with the BiFC chimeras employed here. On the other hand, fusions of any N-terminal fragment exhibit considerably different properties when being fused to the C-terminal fragment of Venus. However, C154-V and G154-V turned out to be very dim proteins. The former exhibits very unfavorable photophysical properties such as fast nonradiative decay rates that lead to a very poor fluorescence quantum yield. The latter is altered in its chemical properties, shifting G154-V predominantly to its protonated form which turned out to be nearly non-fluorescent. The N-terminal Venus fragment is comparably bright irrespective of the C-terminal fragment. Furthermore, V154-C and V154-G exhibit fairly different photophysics than Venus. This makes these AFP fragments excellent candidates for imaging a protein of interest with different putative interaction partners in an mcBiFC assay. According to the parameters found in the BiFC chimeras, a differentiation can also be done based on the fluorescence lifetimes, allowing even more flexibility. Again, N-terminal fragments being fused to the C-terminal fragment of either mCerulean or EGFP are hardly distinguishable. However, the BiFC chimeras G154-C and EGFP (τ≈2.69 ns) show considerably different fluorescence lifetimes than V154-C and V154-G (τ≈2.14 ns) while exhibiting a close spectral similarity. Moreover, both pairs exhibit decidedly different fluorescence lifetimes compared to mCerulean and C154-G (τ≈3.24 ns). Based on this, one could establish multiple protein-protein interactions by fusing the C-terminal fragment of EGFP or mCerulean to a protein of interest and the N-terminal fragments of EGFP, mCerulean and Venus to its putative interaction partners. The influence of different, single mutations reveal partly complex patterns. The most obvious changes are observed when substituting Thr203 with Tyr. While the bathochromic shift of the spectra could be expected, a number of other parameters change as well. First, mCerulean


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exhibits a somewhat higher sensitivity to acidification upon introduction of mutation T203Y, expressed in a higher pKa value of C154-V. This is considerably more pronounced in case of EGFP, which was almost exclusively present in its protonated form in the chimera G154-V. This changes in case of Venus, which is more acidic than the chimeras V154-C and V154-G. Furthermore, mutation T203Y leads to longer fluorescence lifetimes and higher fluorescence quantum yields in G154-V (compared to G154-C, EGFP) and Venus (compared to V154-C, V154-G). A closer look at the radiative and non-radiative decay rates reveals that these phenomena are closely related. More precisely, T203Y induces large changes in the nonradiative decay rates and only moderately affects the radiative decay rates. This is also different when the N-terminal mCerulean fragment is fused to the C-terminal Venus fragment, yielding C154-V. In this case, the changes are more pronounced. Overall, this chimera exhibits very unfavorable photophysical properties making it inappropriate for in vivo studies when high signal intensities are needed. Finally, Tyr203 is observed to lead to a considerable reinforcement of the chromophoric environment, as found by investigation of the spectral distribution width in single molecule studies. This could, however, be expected from the fact that tyrosine is a much bulkier amino acid than threonine. In the course of this study, mutation V163A again and again appeared to influence many photophysical properties in the BiFC chimeras, yet with little effect on the bulk spectra. However, chimeras containing Ala163 exhibit higher bulk extinction coefficients than their counterparts with Val163. Mutation V163A is therefore interpreted to result in a slightly higher fraction of AFPs containing a chromophore. This is in line with its presumed ability to increase the folding efficiency of AFPs. The determination of the pKa values has revealed that mutation V163A marginally increases the pKa of the chromophores in the N-terminal EGFP and Venus


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fragments. The observation that V163A inhibits fluorescence from the excited protonated state in the chromophores of EGFP and Venus via ESPT is more prominent, though. This is visible mainly upon comparison of EGFP (containing Val163) to G154-C (containing Ala163). In EGFP, the fluorescence excitation spectrum largely follows the absorbance spectrum. In G154C, fluorescence excitation is suppressed upon excitation of the protonated form of the chromophore. This is attributed to frustrated excitated state proton transfer, thereby suppressing the excited, deprotonated state. In addition to that, V163A affects the molecular dynamics of single BiFC chimeras, however not in a canonical way. Rather, cooperative effects originating from the specific BiFC chimeras play a role as well. In more detail, G154-C is observed to exhibit a spectral distribution width very similar to EGFP. More pronounced is the observation that V154-G, carrying Val163, exhibits a much lower spectral distribution width than V154-C which contains Ala163. This translates into a higher flexibility of the chromophoric environment in V154-C than in V154-G. Therefore, mutation V163A may exhibit more influences on the intramolecular forces than known at present. A deeper analysis of the underlying mechanisms might be provided by structural and computational studies. CONCLUSIONS In this work, complexes of fluorescent proteins formed upon Bimolecular Fluorescence Complementation (BiFC) were under investigation in vitro. The intrinsic solubility problem with fragments of fluorescent proteins was circumvented by fusing the coding DNA of AFP fragments, yielding soluble, fully functional chimeric AFPs. A thorough analysis of these BiFC chimeras revealed superior combinations of different fragments that can be employed for multiplexed analysis of protein-protein interactions. Furthermore, mutations were identified that influence not only the spectral properties of autofluorescent proteins, but also their protonation


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equilibrium in the ground and excited states, their photophysics and molecular dynamics. Along with the previously described effects of these mutations on biochemical properties, a very complex pattern of effects of single amino acid substitutions is identified in this study. Overall, it was demonstrated that a thorough analysis of autofluorescent proteins, including their photophysics, is mandatory for their reasonable use in in vivo studies.

ASSOCIATED CONTENT Supplemental discussion on previously reported effects of single amino acid substitutions, supplementary materials and methods and calculation of fractions of different protein complexes within a sample. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Author to whom correspondence should be addressed: E-mail: [email protected]; Tel.: +49-7071-29-76222; Fax: +49-7071-29-5490

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. (match statement to author names with a symbol)


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39. Treanor, B.; Lanigan, P.; Suhling, K.; Schreiber, T.; Munro, I.; Neil, M.; Phillips, D.; Davis, D.; French, P., Imaging fluorescence lifetime heterogeneity applied to gfp‐tagged mhc protein at an immunological synapse*. Journal of microscopy 2005, 217 (1), 36-43.


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Chimeric Autofluorescent Proteins as Photophysical Model System for

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