skip to main content

Title: Ultrafast internal conversion dynamics of bilirubin bound to UnaG and its N57A mutant
Fluorescent proteins (FPs) have become fundamental tools for live cell imaging. Most FPs currently used are members of the green fluorescent protein super-family, but new fluorophores such as bilin-FPs are being developed and optimized. In particular, the UnaG FP incorporates bilirubin (BR) as a chromophore, enhancing its fluorescence quantum yield by three orders of magnitude relative to that in solution. To investigate the mechanism of this dramatic enhancement and provide a basis for further engineering of UnaG and other tetrapyrrole-based fluorophores, we performed picosecond fluorescence and femtosecond transient absorption measurements of BR bound to UnaG and its N57A site-directed mutant. The dynamics of wt-UnaG, which has a fluorescence QY of 0.51, are largely homogeneous, showing an excited state relaxation of ∼200 ps, and a 2.2 ns excited-state lifetime decay with a kinetic isotope effect (KIE) of 1.1 for D 2 O vs. H 2 O buffer. In contrast, for UnaG N57A (fluorescence QY 0.01) the results show a large spectral inhomogeneity with excited state decay timescales of 47 and 200 ps and a KIE of 1.4. The non-radiative deactivation of the excited state is limited by proton transfer. The loss of direct hydrogen bonds to the endo -vinyl dipyrrinone moiety more » of BR leads to high flexibility and structural heterogeneity of UnaG N57A, as seen in the X-ray crystal structure. « less
Authors:
; ; ; ; ; ; ; ;
Award ID(s):
1734006
Publication Date:
NSF-PAR ID:
10092408
Journal Name:
Physical Chemistry Chemical Physics
Volume:
21
Issue:
5
Page Range or eLocation-ID:
2365 to 2371
ISSN:
1463-9076
Sponsoring Org:
National Science Foundation
More Like this
  1. Biliverdin is a bile pigment that has a very low fluorescence quantum yield in solution, but serves as a chromophore in far-red fluorescent proteins being developed for bio-imaging. In this work, excited-state dynamics of biliverdin dimethyl ether (BVE) in solvents were investigated using femtosecond (fs) and picosecond (ps) time-resolved absorption and fluorescence spectroscopy. This study is the first fs timescale investigation of BVE in solvents, and therefore revealed numerous dynamics that were not resolved in previous, 200 ps time resolution measurements. Viscosity- and isotope-dependent experiments were performed to identify the contributions of isomerization and proton transfer to the excited-state dynamics. In aprotic solvents, a ∼2 ps non-radiative decay accounts for 95% of the excited-state population loss. In addition, a minor ∼30 ps emissive decay pathway is likely associated with an incomplete isomerization process around the C15C16 double bond that results in a flip of the D-ring. In protic solvents, the dynamics are more complex due to hydrogen bond interactions between solute and solvent. In this case, the ∼2 ps decay pathway is a minor channel (15%), whereas ∼70% of the excited-state population decays through an 800 fs emissive pathway. The ∼30 ps timescale associated with isomerization is also observed inmore »protic solvents. The most significant difference in protic solvents is the presence of a >300 ps timescale in which BVE can decay through an emissive state, in parallel with excited-state proton transfer to the solvent. Interestingly, a small fraction of a luminous species, which we designate lumin-BVE (LBVE), is present in protic solvents.« less
  2. Natural and laboratory-guided evolution has created a rich diversity of fluorescent protein (FP)-based sensors for chloride (Cl − ). To date, such sensors have been limited to the Aequorea victoria green fluorescent protein (avGFP) family, and fusions with other FPs have unlocked ratiometric imaging applications. Recently, we identified the yellow fluorescent protein from jellyfish Phialidium sp. (phiYFP) as a fluorescent turn-on, self-ratiometric Cl − sensor. To elucidate its working mechanism as a rare example of a single FP with this capability, we tracked the excited-state dynamics of phiYFP using femtosecond transient absorption (fs-TA) spectroscopy and target analysis. The photoexcited neutral chromophore undergoes bifurcated pathways with the twisting-motion-induced nonradiative decay and barrierless excited-state proton transfer. The latter pathway yields a weakly fluorescent anionic intermediate , followed by the formation of a red-shifted fluorescent state that enables the ratiometric response on the tens of picoseconds timescale. The redshift results from the optimized π–π stacking between chromophore Y66 and nearby Y203, an ultrafast molecular event. The anion binding leads to an increase of the chromophore p K a and ESPT population, and the hindrance of conversion. The interplay between these two effects determines the turn-on fluorescence response to halides such as Cl −more »but turn-off response to other anions such as nitrate as governed by different binding affinities. These deep mechanistic insights lay the foundation for guiding the targeted engineering of phiYFP and its derivatives for ratiometric imaging of cellular chloride with high selectivity.« less
  3. Fluorescent proteins (FPs) are indispensable tools for noninvasive bioimaging and sensing. Measuring the free cellular calcium (Ca2+) concentrations in vivo with genetically encodable FPs can be a relatively direct measure of neuronal activity due to the complex signaling role of these ions. REX-GECO1 is a recently developed red-green emission and excitation ratiometric FP-based biosensor that achieves a high dynamic range due to differences in the chromophore response to light excitation with and without calcium ions. Using steady-state electronic measurements (UV/Visible absorption and emission), along with time-resolved spectroscopic techniques including femtosecond transient absorption (fs-TA) and femtosecond stimulated Raman spectroscopy (FSRS), the potential energy surfaces of these unique biosensors are unveiled with vivid details. The ground-state structural characterization of the Ca2+-free biosensor via FSRS reveals a more spacious protein pocket that allows the chromophore to efficiently twist and reach a dark state. In contrast, the more compressed cavity within the Ca2+-bound biosensor results in a more heterogeneous distribution of chromophore populations that results in multi-step excited state proton transfer (ESPT) pathways on the sub-140 fs, 600 fs, and 3 ps timescales. These results enable rational design strategies to enlarge the spectral separation between the protonated/deprotonated forms and the Stokes shift leading tomore »a larger dynamic range and potentially higher fluorescence quantum yield, which should be broadly applicable to the calcium imaging and biosensor communities.« less
  4. The electronically excited singlet states of nitroaromatic compounds are often presumed to be essentially non-fluorescent. Nonetheless, a growing number of reports in the literature have demonstrated that certain structural types of nitroaromatics can indeed fluoresce, and often quite efficiently. Consideration of the mechanisms responsible for the typical fast or ultrafast non-radiative deactivation of the excited singlet states of nitroaromatics points to several general principles for their design that combine the strong electron-withdrawing properties of the nitro group with reasonable fluorescence quantum yields. An overview of published examples of fluorescent nitroaromatics emphasizes these concepts in the context of the importance of chromophore architecture and conformation and the defining roles of excited state charge transfer and solvent polarity in modulating the non-radiative decay channels that compete with fluorescence. Overcoming the stigma that nitroaromatics are intrinsically destined to be non-fluorescent thus paves the way for incorporating the strongly electron-withdrawing nitro group into the existing toolbox for the development of new nitro-substituted fluorophores and chromophores tuned to specific applications.
  5. Coumarins are bioactive molecules that often serve as defenses in plant and animal systems, and understanding their fundamental behavior is essential for understanding their bioactivity. Aesculetin (6,7-dihydroxycoumarin) has recently attracted attention due to its ability to act as an antioxidant, but little is known about its photophysical properties. The fluorescence lifetimes of its neutral and anion form in water are 19 ± 2 ps and 2.3 ± 0.1 ns, respectively. Assuming the short lifetime of the neutral is determined by ESPT, we estimate k(PT )~ 5 × 10^10 s^–1. Using steady-state and time-resolved fluorescence spectroscopy, we determine its ground and excited-state p𝐾𝑎1 to be 7.3 and –1, respectively, making it one of the strongest photoacids of the natural coumarins. Aesculetin exhibits a strong pH dependence of the relative fluorescence quantum yield becoming much more fluorescent above p𝐾𝑎1. The aesculetin anion has slightly photobasic character. We also report that aesculetin forms a fluorescent catechol-like complex with boric acid, and this complex has a p𝐾𝑐 of 5.6.