Effects of Threonine 203 Replacements on Excited-State Dynamics and Fluorescence Properties of the Green Fluorescent Protein (GFP)

Abstract

We report a comparative study of wild-type green fluorescent protein (GFP) and single-site mutants in which threonine at position 203 has been replaced by aliphatic and aromatic residues, i.e., by valine (V), isoleucine (I), phenylalanine (F), tyrosine (Y), and histidine (H). Steady-state absorption spectra reveal changes that reflect different charge distributions in the mutants as compared to wild-type GFP. While the absorption peak of the protonated fluorophore, RH, undergoes only a small red shift in all T203 mutants, a pronounced red shift is observed for the deprotonated form R-, ca. 1000 cm-1 for the aliphatic mutants T203V and T203I, ca. 1200 cm-1 for T203F, and 1360 cm-1 for T203Y. Thus, we conclude that a ground-state conformation higher in energy than the wild-type R- state is the predominant origin of the red shift in all the T203 mutants investigated. Furthermore, mutant-dependent changes in the ground-state equilibria of RH and R- result from at least two modes of electrostatic stabilization, one resting on hydrogen bonding as in T203 and the other one on π−π-stacking as in T203F and T203Y. Surprisingly, the deprotonation dynamics of RH* is only weakly affected by the mutations at position 203. Only in the most red-shifted mutant T203Y an additional ultrafast (1.7 ps) excited-state decay channel of RH* has been observed. The identical kinetics of both processes, decay of RH* and ground-state recovery of RH in T203Y, is discussed in terms of two mechanisms: (i) rate-determining electron transfer from the protonated (or deprotonated) tyrosyl 203 residue to RH* followed by considerably faster recombination processes, which cannot occur in T203F for energetic reasons, and (ii) internal conversion in RH* favored by rotational motion around the exocyclic double bond.

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

“…S4) diminish the functional relevance of a phenolic ring wag in the Ca 2+ -free GEM-GECO1, which instead adopts a 170 cm −1 in-plane rocking motion to drive ESPT (SI Text) but less efficiently. This is in accord with previous reports of longer ESPT time in wtGFP upon T203V mutation (17,18).…”

“…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.…”

Crystal Structures of the GCaMP Calcium Sensor Reveal the Mechanism of Fluorescence Signal Change and Aid Rational Design

“…As GCaMP2 is largely monomeric under typical imaging conditions, the Ca 2ϩ -monomer structures served as the basis for mutagenesis studies to address function. All mutations were made in the context of GCaMP2-T116V (gfp T203V), a mutation previously described to increase the wild type EGFP excited-state proton transfer (32)(33)(34). 3 We created groups of GCaMP2 mutants to test three hypotheses about GCaMP function: (a) that mutating CaM or the cpEGFP-CaM linker to block solvent access to the EGFP chromophore would improve brightness ("solvent access" mutants), (b) disrupting the cpEGFP-CaM interfaces seen in the crystal structure would decrease sensor performance ("interface" mutants), and (c) mutating EGFP ␤-barrel positions observed to be solvent-exposed in the GCaMP2 structure (but not in EGFP itself) due to the circular permutation would alter sensor function ("inner barrel" mutants).…”

Concerted Asynchronous Proton Transfer in H-Bonding Relay Model: An Implication of Green Fluorescent Protein

“…23 For instance, the effect of Threonine 203 was reported. 24 It was found that T203F and T203Y could stabilize the deprotonated form, while T203V, T203I and …”