BACKGROUND
in  <dig> the scientific community was gifted with a research tool that profoundly and irreversibly changed the way researchers approach the study of protein function in live cells  <cit> .
the tool was, of course, the gene encoding the aequorea victoria green fluorescent protein   <cit> .
soon after the first demonstrations of functional expression of the gene encoding avgfp in organisms other than jellyfish  <cit> , published reports of the use of fluorescent proteins  for microscopy applications 'took off'  <cit> .
since that time, the impact of fps on the life sciences has continued to increase with each passing year and this growth shows no signs of slowing  <cit> .
one important driving force behind the ever-increasing popularity of fps is the fact that researchers continue to create fps with wavelength-shifted absorbance and/or emission wavelengths and/or improved or novel properties   <cit> .
improved fps facilitate life science research by minimizing technical hurdles that otherwise complicate their use in imaging applications.
for example, fps with improved photostability enable time-lapse imaging over greater durations.
fps with novel properties can inspire the development of entirely new applications that would otherwise be impractical or even impossible.
this has certainly been the case with photoactivatable fps that have enabled cellular imaging at resolutions beyond the diffraction limit  <cit> .
the availability of engineered avgfp variants with altered color, where color refers to the absorbance and/or fluorescence emission spectral profiles, has been a boon to life science research.
access to a wide ranging fp color palette has allowed researchers to simultaneously track multiple proteins or use fluorescence resonance energy transfer  to detect protein-protein interactions in a live cell  <cit> .
fortunately, avgfp has been a fertile source of new colors of fps.
the main classes of color variants derived from avgfp include those that are blue fluorescent  <cit> , cyan fluorescent  <cit> , cyan-excitable green fluorescent  <cit> , uv-excitable green fluorescent  <cit>  and yellow fluorescent  <cit> .
coral is also an abundant source of fps  <cit>  and in recent years this treasure trove, which includes variants with fluorescent hues ranging from cyan to far-red, has yielded a number of exciting new variants  <cit> .
for example, we recently described  <cit>  the engineering of a codon optimized and monomeric version of cfp <dig>  a tetrameric cyan fp  from clavularia coral  <cit> .
the resulting protein, known as monomeric teal fp , has an anionic tyrosine-derived chromophore that is chemically identical to that of enhanced avgfp .
however, the absorbance and fluorescence emission maxima of mtfp <dig>  are blue shifted by about  <dig> nm relative to egfp  owing to numerous amino acid differences in the chromophore-containing cavity  <cit>  .
we have demonstrated that mtfp <dig> is a favorable alternative to avgfp-derived cfps with tryptophan-derived chromophores such as enhanced cfp  or cerulean  <cit> .
the specific advantages of mtfp <dig> include a narrower and single-peaked emission spectrum, improved brightness and improved photostability  <cit> .
in this work we report on our efforts to engineer a series of new colors of mtfp1-derived fps through the use of site-directed mutagenesis and random mutagenesis with library screening.
this work has provided important insight into the amino acid determinants of color in mtfp <dig>  in addition we have undertaken a thorough assessment of mtfp <dig> fusion proteins to determine whether such constructs exhibit their expected pattern of subcellular localization.
together these new results further establish mtfp <dig>  and its suitably optimized hue-shifted variants, as useful new additions to the toolkit of fps for cell biology research.
RESULTS
blue-shifted variants of mtfp1
a series of computational studies have provided support for the idea that there is a partial transfer of charge from the phenolate moiety to the imidazolinone moiety  in the excited state of the avgfp anion  <cit> .
since the phenolate is more electron rich in the ground state than in the excited state, factors that contribute to charge stabilization will tend to increase the energy barrier for charge transfer and shift the excitation and emission peaks to higher-energy wavelengths .
the crystal structures of mtfp <dig>  <cit>  and amfp <dig>  <cit>   revealed that these homologous blue-shifted fps both have structurally analogous histidine imidazoles, his <dig> of mtfp <dig> and his <dig> of amfp <dig>  stacked against the phenolate ring of the chromophore .
owing to the involvement of the imidazole in a quadrupole salt bridge network  it is likely to have significant cationic character.
a simple electrostatic interpretation of the imidazole-chromophore interaction might therefore suggest that this cationic character is helping to stabilize anionic character on the phenolate ring.
other mutagenesis-based studies have provided support for the idea that the side chain of the residue aligning with residue his <dig> of mtfp <dig> , or a buried water molecule that occupies the cavity when the side chain is small , also has an important role in stabilizing anionic character on the phenolate ring  <cit> .
henderson and remington have proposed that the electrostatic interaction with his <dig> is of greater significance than the interaction with the water molecule in the residue  <dig> side-chain cavity for causing the blue-shifted emission of the amfp <dig> chromophore  <cit> .
the relative importance of his <dig> and his <dig> with respect to the blue-shift of the mtfp <dig> chromophore has not been investigated.
we reasoned that if this electrostatic-based mechanism for 'fine tuning' of the emission wavelength is indeed operative in mtfp <dig>  variants with alternative chromophore structures, should also be blue-shifted relative to their avgfp analogs.
two qualifications are that formation of the excited state still involves charge transfer to the imidazolinone ring and that significant repacking of the side chains lining the chromophore-containing cavity does not occur with the new chromophore structure.
to investigate whether this mechanism for blue-shifting the fluorescence could be translated to alternative chromophore structures, we created the tyr67trp and tyr67his mutants of mtfp <dig>  the chromophore structures of mtfp1-y67w and mtfp1-y67h are chemically identical to that of avgfp-derived ecfp and ebfp, respectively .
accordingly, we expected that the absorbance and fluorescence emission maxima of mtfp1-y67w and ecfp ) would be similar but not necessarily identical.
if differences between the spectra of the two proteins were observed, they must be attributable to the effect of the protein environment on the chromophore.
measuring the absorbance and emission spectra of purified mtfp1-y67w revealed that this protein is fluorescent, exhibits the typical double-humped peaks associated with a tryptophan-derived chromophore and is blue-shifted relative to ecfp .
considering the mean wavelength at half maximum intensity, the excitation and emission peaks are shifted by  <dig> and  <dig>  nm, respectively.
this result contrasts with a previous report in which it was demonstrated that the analogous substitution in amfp <dig> results in a protein that is not blue-shifted relative to avgfp-derived cfp  <cit> .
the purified mtfp1-y67h variant exhibited no significant fluorescence, but did have a strong absorbance peak that was blue-shifted by  <dig>  nm .
these results demonstrate that the protein-chromophore interactions responsible for blue shifting the absorbance and emission maxima  of mtfp <dig> are not entirely dependent on the presence of a tyrosine-derived chromophore.
in the crystal structure of mtfp <dig>  the imidazole of his <dig> is observed to be making a hydrogen bond with the phenolate oxygen of the chromophore .
an analogous interaction is not possible in the mtfp1-y67h or mtfp1-y67w variants.
in contrast, the close stacking of the his <dig> imidazole against the chromophore phenolate is an interaction that could be preserved in the mtfp1-y67w or mtfp1-y67h variants.
we conclude that the hydrogen bond with his <dig> is not significant with respect to the blue-shift of mtfp <dig> and it is either the close stacking of the his <dig> imidazole and/or a hydrogen bond-independent electrostatic effect of his <dig> that is responsible for the blue-shift.
based on the interaction of his <dig> with the carboxylate of asp <dig> , it is plausible that the imidazole could have significant cationic character.
red shifted variants of mtfp1
a reasonable approach to dissecting the relative importance of his <dig> and his <dig> in blue-shifting mtfp <dig> fluorescence is to examine variants in which the identity of one residue is changed through the use of site-directed mutagenesis.
it has previously been shown that his <dig> of amfp <dig>  which is structurally analogous to his <dig> of mtfp <dig>  is stacked against the chromophore and has multiple critical roles that dictate the spectroscopic properties   <cit> .
we expected that, in the absence of high-resolution crystal structures, interpretation of the effects of mutation at this position would pose a significant challenge.
we opted instead to focus on his <dig> since it is not strictly conserved between the natural cyan-fluorescing proteins and thus less likely to have multiple critical roles.
we performed saturation mutagenesis of mtfp <dig> at position  <dig> and screened the library using a colony-based fluorescence imaging system.
screening revealed that the library contained both brightly cyan-fluorescing and green-fluorescing members.
dna sequencing revealed that the bright cyan-fluorescing members of the library had, as expected, a histidine at position  <dig> and were thus identical to mtfp <dig>  the brightest green-fluorescing member had a methionine at position  <dig> and a fluorescence emission maximum at  <dig> nm .
the fact that the emission maximum of mtfp1-h163m is  <dig> nm red-shifted from that of mtfp <dig> provides strong support for his <dig> contributing to the blue-shift of the mtfp <dig> chromophore by an electrostatic mechanism.
as to why methionine, as opposed to some other amino acid, was identified as the best replacement for his <dig>  we can suggest two possible reasons.
the first is that the methionine side chain could simply be the best steric fit in the cavity formerly filled by the side chain of his <dig>  the second is that the sulfur atom of the methionine side chain may be participating in sulfur-aromatic interactions with the chromophore.
such interactions have previously been used to explain improvements in the extinction coefficient in avgfp variants  <cit> .
aproduct of extinction coefficient and quantum yield.
btime in seconds to bleach from an initial emission rate of  <dig> to  <dig> photons s- <dig> molecule- <dig>
cvalues for both 'humps' are provided.
dnd, not determined.
ethis value is the absorption maximum.
no significant fluorescence was detected for mtfp1-y67h.
fna, not applicable.
gvalues from  <cit> .
hvalues from  <cit> .
so is his <dig> solely responsible for the blue-shift of mtfp <dig> or does his <dig> also play a role?
that is, does the emission maximum of  <dig> nm for mtfp1-h163m represent an appropriate 'reference' state for this particular chromophore structure when located in this particular chromophore cavity and in the complete absence of any blue-shifting effects?
previous work suggests that the answer to the latter question is probably no, and the emission maximum of the reference state is more likely to be approximately  <dig> nm.
one piece of evidence supporting this suggestion is that the emission maximum of amfp486-h199t is  <dig> nm  <cit> .
this variant has essentially an identical chromophore cavity to mtfp <dig>  with the obvious exception of the his199thr replacement.
the second piece of evidence in support of this suggestion is that the avgfp-t203h mutant has a fluorescence emission at  <dig> nm when excited at  <dig> nm .
residue thr <dig> of avgfp  <cit>  is structurally aligned with his <dig> of mtfp <dig> , and thus avgfp-t203h likely has an imidazole-chromophore stacking interaction similar to that of mtfp <dig>  <cit> .
however, unlike the positively charged imidazole of his <dig> in mtfp <dig>  the imidazole of his <dig> in avgfp-t203h is expected to be in the neutral-charge state.
in a later section we describe the discovery of the thr73ala substitution that red-shifted the fluorescence of mtfp1-k139e/h163m from  <dig> to  <dig> nm.
however, it is appropriate to discuss the implications of this fortunate finding in the current context.
in the crystal structure of mtfp <dig>  the hydroxyl group of thr <dig> is hydrogen-bonded to the guanidium group of arg70: a key participant of the quadrupole salt-bridge network responsible for maintaining the imidazole of his <dig> in the positively charged state .
we propose that the loss of the thr73-arg <dig> hydrogen bond in the thr73ala mutant perturbs the salt-bridge network such that the cationic character of his <dig> imidazole is greatly diminished or abolished.
accordingly, the thr73ala mutant effectively separates the electrostatic role of his <dig> from its additional roles in maintaining the chromophore environment and reveals that the electrostatic effect of this residue accounts for a blue-shift of at least  <dig> nm.
our mutagenesis-based study supports the conclusion that his <dig> and his <dig> act in concert to blue-shift the fluorescence emission of the mtfp <dig> chromophore through an electrostatic mechanism.
the contribution of both residues is effectively identical with  <dig> and  <dig> nm of blue-shift attributed to his <dig> and his <dig>  respectively.
this result is essentially consistent with previous studies of amfp <dig> which have suggested complementary roles for his <dig> and the water molecule adjacent to ala <dig> in achieving the blue-shift  <cit> .
the crystallographic and mutational study by henderson and remington supports the conclusion that the water molecule adjacent to ala <dig> has a less significant contribution than his <dig>  <cit> .
in mtfp <dig> the  cationic imidazole group of his <dig> could contribute a significant amount of electrostatic stabilization to electron density on the phenolate ring.
a similar interaction is not possible in amfp <dig> since a lone water molecule sits in the location occupied by the his <dig> imidazole of mtfp <dig>  a caveat is that the electron density modeled as a water in the amfp <dig> structure could actually be a sodium ion or other cation , in which case an electrostatic contribution would not be unreasonable.
directed evolution of the red shifted variant of mtfp1
intrigued by the high apparent fluorescent brightness of mtfp1-h163m , we subjected this template to directed evolution in an effort create a new gfp variant that could potentially be useful for live cell imaging.
error-prone polymerase chain reaction  was used to create libraries of genetic variants, the gene libraries were expressed in escherichia coli, and colonies were screened for bright green fluorescence.
the brightest green fluorescent colony identified in the first round of screening was found to express a g <dig> variant with the additional mutation lys139met .
this variant was used as the template for a second round of library construction and screening.
the brightest variant identified in the second round was found to be mtfp1-t73a/k139m/h163m .
it is interesting to note that the thr73ala mutation present in g <dig> is structurally aligned with the ser72ala mutation that has been reported to improve the brightness of avgfp variants  <cit> .
no further improvements were identified during a third round of screening of randomly mutated variants based on the g <dig> template.
in vitro characterization  of the purified green fluorescing variants revealed that relative fluorescent brightness to be  <dig>   <dig>  and  <dig>  for g <dig>  g <dig> and g <dig>  respectively.
while both g <dig> and g <dig> had fluorescence maxima at  <dig> nm, g <dig> was further red-shifted to  <dig> nm.
the implications of this result have been discussed above.
further investigation of the g <dig> and g <dig> variants revealed that the dimmer g <dig> was  <dig> -fold more photostable than the brighter g <dig> variant.
in our experience mutations that improve fluorescent brightness are much more readily identified than mutants that improve photostability.
for this reason we forsook the brighter g <dig> variant and continued optimization based on the g <dig> template.
saturation mutagenesis at three positions chosen based on their proximity to the chromophore  resulted in the identification of a further improved variant containing the ala66ser substitution.
a subsequent round of random mutagenesis resulted in the identification of the ser216ile substitution.
additional rounds of random mutagenesis yielded no further improvements.
the end product of this process is a brightly fluorescent gfp  that is equivalent to mtfp1-a66s/k139e/h163m/s216i and has been designated mwasabi.
the fluorescence emission maximum of mwasabi is intermediate between that of g <dig> and g <dig>  suggesting that there has been a perturbation of the salt-bridge network.
it has been previously reported that avgfp with a ser at residue  <dig> is  <dig> nm red-shifted from avgfp with an ala at residue  <dig> .
as observed in the avgfp-s65t structure , the hydroxyl group of the ser at residue  <dig> of mwasabi could potentially form a new hydrogen bond with glu <dig> and partially disrupt its ability to contribute to the critical salt-bridge network.
in vitro characterization of mwasabi  revealed that it is  <dig> -fold brighter than egfp, making it one of the brightest and most photostable fps currently available  <cit> .
another notable feature of mwasabi is its very narrow excitation and emission peaks  that are reminiscent of the spectrum of renilla gfp  <cit>  and monomeric azami-green  <cit> .
narrower peaks allow for more efficient excitation and gathering of emission when used in combination with bandpass filters, and reduce the degree of bleed-through in multicolor imaging.
two-color imaging with mwasabi and sapphire
egfp and its descendents have their major absorption peaks at around  <dig> nm .
however, owing to both the breadth of this peak and the fact that in some variants a significant fraction of the protein exists as the uv-excitable neutral chromophore, egfp and related variants are efficiently excited with violet light .
this residual excitation with  <dig> nm light can unnecessarily complicate multicolor imaging in combination with a sapphire-type variant  <cit>  or fluorescence resonance energy transfer  experiments with a blue fp  donor  <cit> .
in terms of fluorescence emission, mwasabi, egfp and emerald have almost identical emission peak shapes .
in contrast, the differences in their excitation spectra are pronounced, with mwasabi showing almost no excitable component below  <dig> nm.
this suggested to us that mwasabi would be superior to egfp for use in two-color imaging with sapphire.
to test this proposal, mwasabi and egfp were fused with a nuclear localization signal  and separately co-expressed with sapphire-β-actin in hela cells.
as shown in figure  <dig>  exciting sapphire with a typical 375– <dig> nm bandpass excitation filter resulted in significant egfp fluorescence as observed in the cell nucleus.
in contrast, no significant fluorescence was observed for mwasabi in the cell nucleus when sapphire was imaged under identical conditions.
this result demonstrates that mwasabi is particularly well suited for multicolor imaging in combination with fluorophores that are excitable with violet light.
imaging of mtfp <dig> and mwasabi fusion proteins
both mtfp <dig> and its green-emitting progeny, mwasabi, are the products of an extensive process of protein engineering and directed evolution.
during the development of these proteins, substantial effort was expended to find variants with the desired color, high fluorescent brightness, high folding and maturation efficiency and high photostability.
our in vitro characterization of these proteins confirms that we were indeed successful in engineering proteins with the desired properties.
however, the ultimate goal of this research is not to simply develop new fps, but rather to develop fps that will be useful tools for fluorescence imaging in living cells.
to be generally useful for live cell imaging, a fp should retain its favorable properties either when fused to a variety of proteins or when targeted to a variety of subcellular compartments.
in addition, the fp should not perturb the normal localization or biological function of the protein to which it is genetically fused.
such a perturbation can be caused by oligomerization of the fp: a problem that should not be relevant to monomeric fps such as mtfp <dig> and mwasabi.
in a previous paper we demonstrated that mtfp <dig> could be successfully fused with β-actin and α-tubulin protein without perturbing the native cytoskeletal structure  <cit> .
in this work we sought to explore the range of proteins that would tolerate fusion to mtfp <dig> and mwasabi.
we created a series of  <dig> different mtfp <dig> fusions  to both the c- and n-terminus of the fp and found that, in all cases, the fusion protein yielded a pattern of localization consistent with that observed for previously validated avgfp fusions.
as shown in figure  <dig>  fusions to histone h2b and annexin a <dig> did not interfere with the normal cellular function of these proteins.
a series of  <dig> similar fusions with mwasabi gave identical results .
it is apparent that mtfp <dig> and mwasabi are robust and versatile fps that tolerate a wide variety of protein fusions and subcellular microenvironments.
CONCLUSIONS
in this manuscript we have described a series of mutagenesis experiments that have provided fundamental insight into the amino acids that dictate the color of the mtfp <dig> chromophore.
our data supports the conclusion that his <dig> and his <dig> act in an independent and additive fashion to increase the energy of the electronic transitions responsible for absorbance and fluorescence.
although determining the precise details of the mechanism are beyond the scope of this paper, our results are consistent with a previous proposal that electrostatic stabilization of charge density on the phenolate ring is a general means of achieving blue-shifted emission in fps  <cit> .
our investigations into the amino acid determinants of the color of mtfp <dig> led us to a series of hue-shifted variants, one of which was subjected to further engineering and directed evolution to eventually produce mwasabi.
while mwasabi is an exceptionally bright and reasonably photostable gfp, we readily acknowledge that for most experiments egfp or emerald should remain the gfp of choice.
however, there are a number of specific applications, such as two-color imaging in conjunction with a sapphire-type variant or as a fret acceptor with a bfp donor, where the negligible excitation of mwasabi at  <dig> nm provides a substantial benefit.
both mtfp <dig> and mwasabi are well behaved in protein chimeras, offering a bright and photostable fluorescent signal with no significant perturbation of the localization or function of the protein of interest.
this combination of desirable features firmly establishes mtfp <dig> and mwasabi as useful members of the fp toolkit.

 in the scientific community was gifted with a research tool that profoundly and irreversibly changed the way researchers approach the study of protein function in live cells. the tool was, of course, the gene encoding the aequorea victoria green fluorescent protein. soon after the first demonstrations of functional expression of the gene encoding avgfp in organisms other than jellyfish, published reports of the use of fluorescent proteins for microscopy applications'took off'. since that time, the impact of fps on the life sciences has continued to increase with each passing year and this growth shows no signs of slowing. one important driving force behind the ever-increasing popularity of fps is the fact that researchers continue to create fps with wavelength-shifted absorbance and/or emission wavelengths and/or improved or novel properties. improved fps facilitate life science research by minimizing technical hurdles that otherwise complicate their use in imaging applications. for example, fps with improved photostability enable time-lapse imaging over greater durations. fps with novel properties can inspire the development of entirely new applications that would otherwise be impractical or even impossible. this has certainly been the case with photoactivatable fps that have enabled cellular imaging at resolutions beyond the diffraction limit. the availability of engineered avgfp variants with altered color, where color refers to the absorbance and/or fluorescence emission spectral profiles, has been a boon to life science research. access to a wide ranging fp color palette has allowed researchers to simultaneously track multiple proteins or use fluorescence resonance energy transfer to detect protein-protein interactions in a live cell. fortunately, avgfp has been a fertile source of new colors of fps. the main classes of color variants derived from avgfp include those that are blue fluorescent, cyan fluorescent, cyan-excitable green fluorescent, uv-excitable green fluorescent and yellow fluorescent. coral is also an abundant source of fps and in recent years this treasure trove, which includes variants with fluorescent hues ranging from cyan to far-red, has yielded a number of exciting new variants. for example, we recently described the engineering of a codon optimized and monomeric version of cfp a tetrameric cyan fp from clavularia coral. the resulting protein, known as monomeric teal fp, has an anionic tyrosine-derived chromophore that is chemically identical to that of enhanced avgfp. however, the absorbance and fluorescence emission maxima of mtfp are blue shifted by about nm relative to egfp owing to numerous amino acid differences in the chromophore-containing cavity. we have demonstrated that mtfp is a favorable alternative to avgfp-derived cfps with tryptophan-derived chromophores such as enhanced cfp or cerulean. the specific advantages of mtfp include a narrower and single-peaked emission spectrum, improved brightness and improved photostability. in this work we report on our efforts to engineer a series of new colors of mtfp1-derived fps through the use of site-directed mutagenesis and random mutagenesis with library screening. this work has provided important insight into the amino acid determinants of color in mtfp in addition we have undertaken a thorough assessment of mtfp fusion proteins to determine whether such constructs exhibit their expected pattern of subcellular localization. together these new results further establish mtfp and its suitably optimized hue-shifted variants, as useful new additions to the toolkit of fps for cell biology research.  blue-shifted variants of mtfp1 a series of computational studies have provided support for the idea that there is a partial transfer of charge from the phenolate moiety to the imidazolinone moiety in the excited state of the avgfp anion. since the phenolate is more electron rich in the ground state than in the excited state, factors that contribute to charge stabilization will tend to increase the energy barrier for charge transfer and shift the excitation and emission peaks to higher-energy wavelengths. the crystal structures of mtfp and amfp revealed that these homologous blue-shifted fps both have structurally analogous histidine imidazoles, his of mtfp and his of amfp stacked against the phenolate ring of the chromophore. owing to the involvement of the imidazole in a quadrupole salt bridge network it is likely to have significant cationic character. a simple electrostatic interpretation of the imidazole-chromophore interaction might therefore suggest that this cationic character is helping to stabilize anionic character on the phenolate ring. other mutagenesis-based studies have provided support for the idea that the side chain of the residue aligning with residue his of mtfp, or a buried water molecule that occupies the cavity when the side chain is small, also has an important role in stabilizing anionic character on the phenolate ring. henderson and remington have proposed that the electrostatic interaction with his is of greater significance than the interaction with the water molecule in the residue side-chain cavity for causing the blue-shifted emission of the amfp chromophore. the relative importance of his and his with respect to the blue-shift of the mtfp chromophore has not been investigated. we reasoned that if this electrostatic-based mechanism for'fine tuning' of the emission wavelength is indeed operative in mtfp variants with alternative chromophore structures, should also be blue-shifted relative to their avgfp analogs. two qualifications are that formation of the excited state still involves charge transfer to the imidazolinone ring and that significant repacking of the side chains lining the chromophore-containing cavity does not occur with the new chromophore structure. to investigate whether this mechanism for blue-shifting the fluorescence could be translated to alternative chromophore structures, we created the tyr67trp and tyr67his mutants of mtfp the chromophore structures of mtfp1-y67w and mtfp1-y67h are chemically identical to that of avgfp-derived ecfp and ebfp, respectively. accordingly, we expected that the absorbance and fluorescence emission maxima of mtfp1-y67w and ecfp ) would be similar but not necessarily identical. if differences between the spectra of the two proteins were observed, they must be attributable to the effect of the protein environment on the chromophore. measuring the absorbance and emission spectra of purified mtfp1-y67w revealed that this protein is fluorescent, exhibits the typical double-humped peaks associated with a tryptophan-derived chromophore and is blue-shifted relative to ecfp. considering the mean wavelength at half maximum intensity, the excitation and emission peaks are shifted by and nm, respectively. this result contrasts with a previous report in which it was demonstrated that the analogous substitution in amfp results in a protein that is not blue-shifted relative to avgfp-derived cfp. the purified mtfp1-y67h variant exhibited no significant fluorescence, but did have a strong absorbance peak that was blue-shifted by nm. these results demonstrate that the protein-chromophore interactions responsible for blue shifting the absorbance and emission maxima of mtfp are not entirely dependent on the presence of a tyrosine-derived chromophore. in the crystal structure of mtfp the imidazole of his is observed to be making a hydrogen bond with the phenolate oxygen of the chromophore. an analogous interaction is not possible in the mtfp1-y67h or mtfp1-y67w variants. in contrast, the close stacking of the his imidazole against the chromophore phenolate is an interaction that could be preserved in the mtfp1-y67w or mtfp1-y67h variants. we conclude that the hydrogen bond with his is not significant with respect to the blue-shift of mtfp and it is either the close stacking of the his imidazole and/or a hydrogen bond-independent electrostatic effect of his that is responsible for the blue-shift. based on the interaction of his with the carboxylate of asp, it is plausible that the imidazole could have significant cationic character. red shifted variants of mtfp1 a reasonable approach to dissecting the relative importance of his and his in blue-shifting mtfp fluorescence is to examine variants in which the identity of one residue is changed through the use of site-directed mutagenesis. it has previously been shown that his of amfp which is structurally analogous to his of mtfp is stacked against the chromophore and has multiple critical roles that dictate the spectroscopic properties. we expected that, in the absence of high-resolution crystal structures, interpretation of the effects of mutation at this position would pose a significant challenge. we opted instead to focus on his since it is not strictly conserved between the natural cyan-fluorescing proteins and thus less likely to have multiple critical roles. we performed saturation mutagenesis of mtfp at position and screened the library using a colony-based fluorescence imaging system. screening revealed that the library contained both brightly cyan-fluorescing and green-fluorescing members. dna sequencing revealed that the bright cyan-fluorescing members of the library had, as expected, a histidine at position and were thus identical to mtfp the brightest green-fluorescing member had a methionine at position and a fluorescence emission maximum at nm. the fact that the emission maximum of mtfp1-h163m is nm red-shifted from that of mtfp provides strong support for his contributing to the blue-shift of the mtfp chromophore by an electrostatic mechanism. as to why methionine, as opposed to some other amino acid, was identified as the best replacement for his we can suggest two possible reasons. the first is that the methionine side chain could simply be the best steric fit in the cavity formerly filled by the side chain of his the second is that the sulfur atom of the methionine side chain may be participating in sulfur-aromatic interactions with the chromophore. such interactions have previously been used to explain improvements in the extinction coefficient in avgfp variants. aproduct of extinction coefficient and quantum yield. btime in seconds to bleach from an initial emission rate of to photons s- molecule- cvalues for both'humps' are provided. dnd, not determined. ethis value is the absorption maximum. no significant fluorescence was detected for mtfp1-y67h. fna, not applicable. gvalues from. hvalues from. so is his solely responsible for the blue-shift of mtfp or does his also play a role? that is, does the emission maximum of nm for mtfp1-h163m represent an appropriate'reference' state for this particular chromophore structure when located in this particular chromophore cavity and in the complete absence of any blue-shifting effects? previous work suggests that the answer to the latter question is probably no, and the emission maximum of the reference state is more likely to be approximately nm. one piece of evidence supporting this suggestion is that the emission maximum of amfp486-h199t is nm. this variant has essentially an identical chromophore cavity to mtfp with the obvious exception of the his199thr replacement. the second piece of evidence in support of this suggestion is that the avgfp-t203h mutant has a fluorescence emission at nm when excited at nm. residue thr of avgfp is structurally aligned with his of mtfp, and thus avgfp-t203h likely has an imidazole-chromophore stacking interaction similar to that of mtfp. however, unlike the positively charged imidazole of his in mtfp the imidazole of his in avgfp-t203h is expected to be in the neutral-charge state. in a later section we describe the discovery of the thr73ala substitution that red-shifted the fluorescence of mtfp1-k139e/h163m from to nm. however, it is appropriate to discuss the implications of this fortunate finding in the current context. in the crystal structure of mtfp the hydroxyl group of thr is hydrogen-bonded to the guanidium group of arg70: a key participant of the quadrupole salt-bridge network responsible for maintaining the imidazole of his in the positively charged state. we propose that the loss of the thr73-arg hydrogen bond in the thr73ala mutant perturbs the salt-bridge network such that the cationic character of his imidazole is greatly diminished or abolished. accordingly, the thr73ala mutant effectively separates the electrostatic role of his from its additional roles in maintaining the chromophore environment and reveals that the electrostatic effect of this residue accounts for a blue-shift of at least nm. our mutagenesis-based study supports the conclusion that his and his act in concert to blue-shift the fluorescence emission of the mtfp chromophore through an electrostatic mechanism. the contribution of both residues is effectively identical with and nm of blue-shift attributed to his and his respectively. this result is essentially consistent with previous studies of amfp which have suggested complementary roles for his and the water molecule adjacent to ala in achieving the blue-shift. the crystallographic and mutational study by henderson and remington supports the conclusion that the water molecule adjacent to ala has a less significant contribution than his. in mtfp the cationic imidazole group of his could contribute a significant amount of electrostatic stabilization to electron density on the phenolate ring. a similar interaction is not possible in amfp since a lone water molecule sits in the location occupied by the his imidazole of mtfp a caveat is that the electron density modeled as a water in the amfp structure could actually be a sodium ion or other cation, in which case an electrostatic contribution would not be unreasonable. directed evolution of the red shifted variant of mtfp1 intrigued by the high apparent fluorescent brightness of mtfp1-h163m, we subjected this template to directed evolution in an effort create a new gfp variant that could potentially be useful for live cell imaging. error-prone polymerase chain reaction was used to create libraries of genetic variants, the gene libraries were expressed in escherichia coli, and colonies were screened for bright green fluorescence. the brightest green fluorescent colony identified in the first round of screening was found to express a g variant with the additional mutation lys139met. this variant was used as the template for a second round of library construction and screening. the brightest variant identified in the second round was found to be mtfp1-t73a/k139m/h163m. it is interesting to note that the thr73ala mutation present in g is structurally aligned with the ser72ala mutation that has been reported to improve the brightness of avgfp variants. no further improvements were identified during a third round of screening of randomly mutated variants based on the g template. in vitro characterization of the purified green fluorescing variants revealed that relative fluorescent brightness to be and for g g and g respectively. while both g and g had fluorescence maxima at nm, g was further red-shifted to nm. the implications of this result have been discussed above. further investigation of the g and g variants revealed that the dimmer g was -fold more photostable than the brighter g variant. in our experience mutations that improve fluorescent brightness are much more readily identified than mutants that improve photostability. for this reason we forsook the brighter g variant and continued optimization based on the g template. saturation mutagenesis at three positions chosen based on their proximity to the chromophore resulted in the identification of a further improved variant containing the ala66ser substitution. a subsequent round of random mutagenesis resulted in the identification of the ser216ile substitution. additional rounds of random mutagenesis yielded no further improvements. the end product of this process is a brightly fluorescent gfp that is equivalent to mtfp1-a66s/k139e/h163m/s216i and has been designated mwasabi. the fluorescence emission maximum of mwasabi is intermediate between that of g and g suggesting that there has been a perturbation of the salt-bridge network. it has been previously reported that avgfp with a ser at residue is nm red-shifted from avgfp with an ala at residue. as observed in the avgfp-s65t structure, the hydroxyl group of the ser at residue of mwasabi could potentially form a new hydrogen bond with glu and partially disrupt its ability to contribute to the critical salt-bridge network. in vitro characterization of mwasabi revealed that it is -fold brighter than egfp, making it one of the brightest and most photostable fps currently available. another notable feature of mwasabi is its very narrow excitation and emission peaks that are reminiscent of the spectrum of renilla gfp and monomeric azami-green. narrower peaks allow for more efficient excitation and gathering of emission when used in combination with bandpass filters, and reduce the degree of bleed-through in multicolor imaging. two-color imaging with mwasabi and sapphire egfp and its descendents have their major absorption peaks at around nm. however, owing to both the breadth of this peak and the fact that in some variants a significant fraction of the protein exists as the uv-excitable neutral chromophore, egfp and related variants are efficiently excited with violet light. this residual excitation with nm light can unnecessarily complicate multicolor imaging in combination with a sapphire-type variant or fluorescence resonance energy transfer experiments with a blue fp donor. in terms of fluorescence emission, mwasabi, egfp and emerald have almost identical emission peak shapes. in contrast, the differences in their excitation spectra are pronounced, with mwasabi showing almost no excitable component below nm. this suggested to us that mwasabi would be superior to egfp for use in two-color imaging with sapphire. to test this proposal, mwasabi and egfp were fused with a nuclear localization signal and separately co-expressed with sapphire-β-actin in hela cells. as shown in figure exciting sapphire with a typical 375– nm bandpass excitation filter resulted in significant egfp fluorescence as observed in the cell nucleus. in contrast, no significant fluorescence was observed for mwasabi in the cell nucleus when sapphire was imaged under identical conditions. this result demonstrates that mwasabi is particularly well suited for multicolor imaging in combination with fluorophores that are excitable with violet light. imaging of mtfp and mwasabi fusion proteins both mtfp and its green-emitting progeny, mwasabi, are the products of an extensive process of protein engineering and directed evolution. during the development of these proteins, substantial effort was expended to find variants with the desired color, high fluorescent brightness, high folding and maturation efficiency and high photostability. our in vitro characterization of these proteins confirms that we were indeed successful in engineering proteins with the desired properties. however, the ultimate goal of this research is not to simply develop new fps, but rather to develop fps that will be useful tools for fluorescence imaging in living cells. to be generally useful for live cell imaging, a fp should retain its favorable properties either when fused to a variety of proteins or when targeted to a variety of subcellular compartments. in addition, the fp should not perturb the normal localization or biological function of the protein to which it is genetically fused. such a perturbation can be caused by oligomerization of the fp: a problem that should not be relevant to monomeric fps such as mtfp and mwasabi. in a previous paper we demonstrated that mtfp could be successfully fused with β-actin and α-tubulin protein without perturbing the native cytoskeletal structure. in this work we sought to explore the range of proteins that would tolerate fusion to mtfp and mwasabi. we created a series of different mtfp fusions to both the c- and n-terminus of the fp and found that, in all cases, the fusion protein yielded a pattern of localization consistent with that observed for previously validated avgfp fusions. as shown in figure fusions to histone h2b and annexin a did not interfere with the normal cellular function of these proteins. a series of similar fusions with mwasabi gave identical results. it is apparent that mtfp and mwasabi are robust and versatile fps that tolerate a wide variety of protein fusions and subcellular microenvironments.  in this manuscript we have described a series of mutagenesis experiments that have provided fundamental insight into the amino acids that dictate the color of the mtfp chromophore. our data supports the conclusion that his and his act in an independent and additive fashion to increase the energy of the electronic transitions responsible for absorbance and fluorescence. although determining the precise details of the mechanism are beyond the scope of this paper, our results are consistent with a previous proposal that electrostatic stabilization of charge density on the phenolate ring is a general means of achieving blue-shifted emission in fps. our investigations into the amino acid determinants of the color of mtfp led us to a series of hue-shifted variants, one of which was subjected to further engineering and directed evolution to eventually produce mwasabi. while mwasabi is an exceptionally bright and reasonably photostable gfp, we readily acknowledge that for most experiments egfp or emerald should remain the gfp of choice. however, there are a number of specific applications, such as two-color imaging in conjunction with a sapphire-type variant or as a fret acceptor with a bfp donor, where the negligible excitation of mwasabi at nm provides a substantial benefit. both mtfp and mwasabi are well behaved in protein chimeras, offering a bright and photostable fluorescent signal with no significant perturbation of the localization or function of the protein of interest. this combination of desirable features firmly establishes mtfp and mwasabi as useful members of the fp toolkit. 
we also employed site-directed and random mutagenesis in combination with library screening to create new hues of mtfp1-derived variants with wavelength-shifted excitation and emission spectra. our results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mtfp operate independently of the chromophore structure. based on the results of our mutagenesis study, we conclude that the two histidine residues in close proximity to the chromophore are approximately equal determinants of the blue-shifted fluorescence emission of mtfp with respect to live cell imaging applications, the mtfp1-derived mwasabi should be particularly useful in two-color imaging in conjunction with a sapphire-type variant or as a fluorescence resonance energy transfer acceptor with a blue fp donor. one of the latest additions to the toolkit is monomeric teal fp, a bright and photostable fp derived from clavularia cyan fp. to gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. this conclusion is supported by the observation that the tyr67trp and tyr67his mutants of mtfp retain a blue-shifted fluorescence emission relative to their avgfp counterparts. based on previous work with close homologs, his and his are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. to assess the usefulness of mtfp and mwasabi in live cell imaging applications, we constructed and imaged more than different fusion proteins. in the years that have passed since the cloning of aequorea victoria green fluorescent protein, the expanding set of fluorescent protein variants has become entrenched as an indispensable toolkit for cell biology research.
 in the years that have passed since the cloning of aequorea victoria green fluorescent protein, the expanding set of fluorescent protein variants has become entrenched as an indispensable toolkit for cell biology research. one of the latest additions to the toolkit is monomeric teal fp, a bright and photostable fp derived from clavularia cyan fp. to gain insight into the molecular basis for the blue-shifted fluorescence emission we undertook a mutagenesis-based study of residues in the immediate environment of the chromophore. we also employed site-directed and random mutagenesis in combination with library screening to create new hues of mtfp1-derived variants with wavelength-shifted excitation and emission spectra.  our results demonstrate that the protein-chromophore interactions responsible for blue-shifting the absorbance and emission maxima of mtfp operate independently of the chromophore structure. this conclusion is supported by the observation that the tyr67trp and tyr67his mutants of mtfp retain a blue-shifted fluorescence emission relative to their avgfp counterparts. based on previous work with close homologs, his and his are likely to be the residues with the greatest contribution towards blue-shifting the fluorescence emission. indeed we have identified the substitutions his163met and thr73ala that abolish or disrupt the interactions of these residues with the chromophore. the mtfp1-thr73ala/his163met double mutant has an emission peak that is nm red-shifted from that of mtfp itself. directed evolution of this double mutant resulted in the development of mwasabi, a new green fluorescing protein that offers certain advantages over enhanced avgfp. to assess the usefulness of mtfp and mwasabi in live cell imaging applications, we constructed and imaged more than different fusion proteins.  based on the results of our mutagenesis study, we conclude that the two histidine residues in close proximity to the chromophore are approximately equal determinants of the blue-shifted fluorescence emission of mtfp with respect to live cell imaging applications, the mtfp1-derived mwasabi should be particularly useful in two-color imaging in conjunction with a sapphire-type variant or as a fluorescence resonance energy transfer acceptor with a blue fp donor. in all fusions attempted, both mtfp and mwasabi give patterns of fluorescent localization indistinguishable from that of well-established avgfp variants. 