Mechanism of Color and Photoacidity Tuning for the Protonated Green Fluorescent Protein Chromophore

Abstract

The neutral or A state of the green fluorescent protein (GFP) chromophore is a remarkable example of a photoacid naturally embedded in the protein environment and accounts for the large Stokes shift of GFP in response to near UV excitation. Its color tuning mechanism has been largely overlooked, as it is less preferable for imaging applications than the redder anionic or B state. Past studies, based on site-directed mutagenesis or solvatochromism of the isolated chromophore, have concluded that its color tuning range is much narrower than its anionic counterpart. However, as we performed extensive investigation on more GFP mutants, we found the color of the neutral chromophore to be much more sensitive to protein electrostatics. Electronic Stark spectroscopy reveals a fundamentally different electrostatic color tuning mechanism for the neutral state of the chromophore that demands a three-form model compared with that of the anionic state, which requires only two forms. Specifically, an underlying zwitterionic charge transfer state is required to explain its sensitivity to electrostatics. As the Stokes shift is tightly linked to the protonated chromophore’s photoacidity and excited-state proton transfer (ESPT), we infer design principles of the GFP chromophore as a photoacid through the color tuning mechanisms of both protonation states. The three-form model could also be applied to similar biological and nonbiological dyes and complements the failure of two-form model for donor–acceptor systems with localized electronic distributions.

Unusual Spectroscopic and Electric Field Sensitivity of Chromophores with Short Hydrogen Bonds: GFP and PYP as Model Systems

“…By extension, we can argue that the S65T/H148D GFP variants likely share the same behavior, but it is concealed by the additional substituent-specific electronic effects from halogenation. Conversely, the SrPYP mutants exhibit the opposite trend upon E46Q mutation, which is different from that expected from the room-temperature SIE study [52], suggesting a nontrivial interplay between the two putative short hydrogen bonds that could be studied through NMR [68].…”

“…In addition, the ΔA(2ω) spectra for both features are dominated by second-derivative lineshapes and no appreciable zeroth derivative component is needed for the fit, suggesting the absence of substantial proton transfer between two wells even when high field strength (~ ƒ • 1.4 MV/cm) is applied. By following the trend from negative to positive ΔpKα cases (i.e., more halogenated to less halogenated chromophores), as the A-like A state grows at the expense of the B-like A state, one can see that the A-like A state has less prominent vibronic feature than the normal A state [68], such that its corresponding Stark signal is greatly reduced and easily overwhelmed by that of the B-like A state, best illustrated by the Stark spectra from monochlorinated and monobrominated variants (the second derivative of a broad vs sharp feature). In contrast, B-like A states still preserve the BLA vibronic structure akin to that of the B state chromophore [3], and this accounts for the dip in the overall absorption spectra at low temperature ( Figure S9 except for the Y66 variant).…”

“…We begin the analysis of the short-hydrogen-bond GFPs with an unsubstituted chromophore (Y66), which is the most well-studied case. As Tonge and coworkers have demonstrated [75], some Raman peak positions strongly correlate with the chromophore's electronic transition energy and therefore reflect the fractions of doubleand single-bond characters for the GFP chromophore, or the relative contributions of the P and I forms (or GS and CT forms for the protonated chromophore [68]) to the groundstate structure from the perspective of Marcus-Hush theory [3]. As a reference, the C=C and C=N stretching bands appear at 1618 (1641) and 1536 (1555) cm -1 for the B (A) state, respectively, for ih:GFP S65T.…”

Quantum mechanical/molecular mechanical studies of photophysical properties of fluorescent proteins

“…The 1PA properties, like VEEs, determined for chromophores in vacuum have been shown to be in reasonable agreement with experimental absorption measurements 52,53 ; comparison has also been facilitated through the availability of experimental measurements on isolated chromophores 52–55 or denatured FPs 8 . On the other hand, the comparison of computationally determined 2PA cross‐sections ( σ 2PA ) for isolated chromophores to experimental measurements, 52,56 almost exclusively on the full FP 56 , has proven more challenging due to both variability in experimental measurements and the strong role played by the environment (see Figure 2 and discussion below).…”

Fluorescence Modulation by Ultrafast Chromophore Twisting Events: Developing a Powerful Toolset for Fluorescent-Protein-Based Imaging

“…Besides the proton, the electronic effect also greatly impacts the FP properties. Strategic mutations of the protein chromophore or local residues to modify their electrostatic interactions 16,17 or additions of a certain electron-withdrawing group (EWG) or electron-donating group (EDG) on a specific site of the chromophore 18,19 have proven to be highly successful in changing the FQY 20 and color. Forster resonance energy transfer (FRET) pairs with two FPs can also modulate the emission color, but the overall protein size is significantly increased.…”