Interplay between Locally Excited and Charge Transfer States Governs the Photoswitching Mechanism in the Fluorescent Protein Dreiklang.

We present the results of high-level electronic structure and dynamics simulations of the photoactive protein Dreiklang. With the goal of understanding the details of the Dreiklang photocycle, we carefully characterize the excited states of the ON- and OFF-forms of Dreiklang. The key finding of our study is the existence of a low-lying excited state of a charge-transfer character in the neutral ON form and that population of this state, which is nearly isoenergetic with the locally excited bright state, initiates a series of steps that ultimately lead to the formation of the hydrated dark chromophore (OFF state). These results allow us to refine the mechanistic picture of Dreiklang's photocycle and photoactivation.

π-Stacking Driven Aggregation and Folding of Squaramides.

Competing noncovalent interactions play a pivotal role in the folding and assembly of three-dimensional structures, especially in flexible molecules. Calculations using density functional theory reveal that two squaramide rings aggregate to form a slipped antiparallel π-stacked dimer with high propensity. This π-π stacking interaction is used to design foldamers in which the squaramides are tethered by a simple methylene bridge, and consequently, the structure folds on to itself incorporating a "turn" element. The variation in relative energy with respect to change in dihedral angle for these foldamers show that for all the structures two rings are displaced in space and the folding potential is asymmetric, starting from seemingly symmetric molecules. The addition of successive squaramide rings connected with simple methylene bridges leads to the formation of higher-order structures with a "Turn-Stack-Turn" structural motif. The "Turn-Stack-Turn" motif can be used in designing new synthetic foldamers which could potentially mimic closely related biological systems. Further, it was found that the aggregation of the folded structures was energetically favored over the unfolded structures. The present set of calculations are important in light of the fact that these simple methylene bridged squaramide rings present synthetic challenges.

Influence of the First Chromophore-Forming Residue on Photobleaching and Oxidative Photoconversion of EGFP and EYFP.

Enhanced green fluorescent protein (EGFP)-one of the most widely applied genetically encoded fluorescent probes-carries the threonine-tyrosine-glycine (TYG) chromophore. EGFP efficiently undergoes green-to-red oxidative photoconversion ("redding") with electron acceptors. Enhanced yellow fluorescent protein (EYFP), a close EGFP homologue (five amino acid substitutions), has a glycine-tyrosine-glycine (GYG) chromophore and is much less susceptible to redding, requiring halide ions in addition to the oxidants. In this contribution we aim to clarify the role of the first chromophore-forming amino acid in photoinduced behavior of these fluorescent proteins. To that end, we compared photobleaching and redding kinetics of EGFP, EYFP, and their mutants with reciprocally substituted chromophore residues, EGFP-T65G and EYFP-G65T. Measurements showed that T65G mutation significantly increases EGFP photostability and inhibits its excited-state oxidation efficiency. Remarkably, while EYFP-G65T demonstrated highly increased spectral sensitivity to chloride, it is also able to undergo redding chloride-independently. Atomistic calculations reveal that the GYG chromophore has an increased flexibility, which facilitates radiationless relaxation leading to the reduced fluorescence quantum yield in the T65G mutant. The GYG chromophore also has larger oscillator strength as compared to TYG, which leads to a shorter radiative lifetime (i.e., a faster rate of fluorescence). The faster fluorescence rate partially compensates for the loss of quantum efficiency due to radiationless relaxation. The shorter excited-state lifetime of the GYG chromophore is responsible for its increased photostability and resistance to redding. In EYFP and EYFP-G65T, the chromophore is stabilized by π-stacking with Tyr203, which suppresses its twisting motions relative to EGFP.

Pyridinium Analogues of Green Fluorescent Protein Chromophore: Fluorogenic Dyes with Large Solvent-Dependent Stokes Shift.

Novel fluorogenic dyes based on the GFP chromophore are developed. The compounds contain a pyridinium ring instead of phenolate and feature large Stokes shifts and solvent-dependent variations in the fluorescence quantum yield. Electronic structure calculations explain the trends in solvatochromic behavior in terms of the increase of the dipole moment upon excited-state relaxation in polar solvents associated with the changes in bonding pattern in the excited state. A unique combination of such optical characteristics and lipophilic properties enables using one of the new dyes for imaging the membrane structure of endoplasmic reticulum. An extremely high photostability (due to a dynamic exchange between the free and absorbed states) and selectivity make this compound a promising label for this type of cellular organelles.