An alternative excited‐state proton transfer pathway in green fluorescent protein variant S205V

Abstract

Wild-type green fluorescent protein (wt-GFP) has a prominent absorbance band centered at ;395 nm, attributed to the neutral chromophore form. The green emission arising upon excitation of this band results from excited-state proton transfer (ESPT) from the chromophore hydroxyl, through a hydrogenbond network proposed to consist of a water molecule and Ser205, to Glu222. Although evidence for Glu222 as a terminal proton acceptor has already been obtained, no evidence for the participation of Ser205 in the proton transfer process exists. To examine the role of Ser205 in the proton transfer, we mutated Ser205 to valine. However, the derived GFP variant S205V, upon excitation at 400 nm, still produces green fluorescence. Time-resolved emission spectroscopy suggests that ESPT contributes to the green fluorescence, and that the proton transfer takes place ;30 times more slowly than in wt-GFP. The crystal structure of S205V reveals rearrangement of Glu222 and Thr203, forming a new hydrogenbonding network. We propose this network to be an alternative ESPT pathway with distinctive features that explain the significantly slowed rate of proton transfer. In support of this proposal, the double mutant S205V/T203V is shown to be a novel blue fluorescent protein containing a tyrosine-based chromophore, yet is incapable of ESPT. The results have implications for the detailed mechanism of ESPT and the photocycle of wt-GFP, in particular for the structures of spectroscopically identified intermediates in the cycle.

Excited-state structural dynamics of a dual-emission calmodulin-green fluorescent protein sensor for calcium ion imaging

“…Therein, the Ca 2+ -free protein modes exhibit a time constant of 30-40 ps attributed to ESPT, much longer than the 5-9 ps counterpart for wtGFP in water (22,26). In contrast, the Ca 2+ -bound protein shows characteristic mode decay on the 500-900 ps timescale, revealing that ESPT is essentially blocked and blue emission from A* dominates (17,19).…”

“…In contrast, spectroscopy on wildtype (wt)GFP included infrared pump probe (16), time-resolved fluorescence (17)(18)(19), transient infrared (20,21), and femtosecond Raman spectroscopy (22), as well as computational studies (23)(24)(25), providing a fairly complete picture of the photophysical and photochemical steps leading to green fluorescence (26,27). In the electronic ground state (GS), wtGFP exists as a mixture of neutral chromophore (A, ∼400 nm peak absorbance) and a small population of anionic chromophore (B, ∼475 nm peak Significance Fluorescent proteins (FPs) started their incredible, colorful journey in bioimaging and biomedicine with the extraction and purification of GFP from the Pacific jellyfish Aequorea victoria more than 50 years ago.…”

Optically Modulatable Blue Fluorescent Proteins

“…In addition to the base T203V/S205V, a blue fluorescent protein with additional stabilizing mutations F99S/M153T/V163A was utilized (A gift from J. Remington, here referred to as modBFP). 22 These additional mutations partially increased the fluorescence quantum yield relative to that of mKalama1 (Table 1). Like mKalama1, 405 nm excitation of modBFP yields bright 450 nm emission (Figure 1), but unlike mKalama1, co-excitation of modBFP at 514 nm yields a 4% increase in fluorescence over primary excitation alone.…”

“…Informed by the published S205V crystal structure, 22 modifying key amino acid residues that hydrogen bond to the tyrosine hydroxyl group in either the cis or trans chromophore form should alter modulation depth and frequency. Further, mutations that may inhibit photoisomerization are also likely to affect fluorescent manifold and dark state populations.…”

Wide-dynamic-range kinetic investigations of deep proton tunnelling in proteins

“…However, due to structural rearrangements, an analogous hydroxyl residue (T203) takes the place of S205 in the proton wire of this mutant 45,46 . Thus, deep tunneling that is rate-limited by the hydroxyl proton of T203 can explain the nearly identical room temperature rates found for the S205V mutant and the wild type.…”