Brightness of the fluorescent proteins (FPs) is one of the central properties to their functions in bioimaging applications. The chromophore nonplanarity has long been considered detrimental to fluorescence quantum yield (FQY) that contributes to the brightness (i.e., the product of FQY and extinction coefficient). On the same note, the conformational freedom exerted by the protein matrix has been argued to dictate the chromophore’s nonradiative decay rates which essentially determine the FQY. However, the mechanistic study of how the interplay between nonplanarity and flexibility of a chromophore specifically tunes the protein FQY has been scarce. Herein, we demonstrate a case study wherein the conformational rigidity can overcome the undesirable effect of nonplanarity and generate brighter emission. ChlorON1 and ChlorON3 are two fluorescence “turn-on” chloride (Cl^–) sensors engineered from the bright green-yellow FP, mNeonGreen, by two mutations in the chromophore pocket. Using femtosecond transient absorption (fs-TA) and stimulated Raman spectroscopy (FSRS) with advanced computational methods, we reveal that the chloride binding results in inhomogeneous subpopulations that differ in FQY and H-bonding interactions particularly for ChlorON3. The spectral data unveil two major subpopulations for the Cl^–-bound FP, showing different absorption wavelengths and FQYs. The QM/MM simulations confirm the inhomogeneity and reveal a more twisted chromophore for the fluorescent subpopulation in the presence of bound Cl^– versus the other dim subpopulation (Fig. 1). Our work provides mechanistic insights into the two important factors for FP brightness within the complex and dynamic protein environment on ultrafast timescales, enabling the rational engineering of functional proteins for broader applications.

<b>Fig. 1.</b> The inner-workings of a fluorescent protein (FP)-based chloride (Cl<sup>–</sup>) sensor can be revealed by an integral toolset of femtosecond transient absorption (fs-TA), femtosecond stimulated Raman spectroscopy (FSRS), and advanced QM/MM calculations, focusing on the Cl<sup>–</sup> free (dim, left) and bound (bright, right) states with distinct chromophore structural dynamics and photoinduced spectral features on ultrafast timescales.

Fig. 1. The inner-workings of a fluorescent protein (FP)-based chloride (Cl^–) sensor can be revealed by an integral toolset of femtosecond transient absorption (fs-TA), femtosecond stimulated Raman spectroscopy (FSRS), and advanced QM/MM calculations, focusing on the Cl^– free (dim, left) and bound (bright, right) states with distinct chromophore structural dynamics and photoinduced spectral features on ultrafast timescales.

Vishaka Pathiranage

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