Dissecting Optical Response and Molecular Structure of Fluorescent Proteins With Non-canonical Chromophores.

Tracking the structural dynamics of fluorescent protein chromophores holds the key to unlocking the fluorescence mechanisms in real time and enabling rational design principles of these powerful and versatile bioimaging probes. By combining recent chemical biology and ultrafast spectroscopy advances, we prepared the superfolder green fluorescent protein (sfGFP) and its non-canonical amino acid (ncAA) derivatives with a single chlorine, bromine, and nitro substituent at the ortho site to the phenolate oxygen of the embedded chromophore, and characterized them using an integrated toolset of femtosecond transient absorption and tunable femtosecond stimulated Raman spectroscopy (FSRS), aided by quantum calculations of the vibrational normal modes. A dominant vibrational cooling time constant of ~4 and 11 ps is revealed in Cl-GFP and Br-GFP, respectively, facilitating a ~30 and 12% increase of the fluorescent quantum yield vs. the parent sfGFP. Similar time constants were also retrieved from the transient absorption spectra, substantiating the correlated electronic and vibrational motions on the intrinsic molecular timescales. Key carbon-halogen stretching motions coupled with phenolate ring motions of the deprotonated chromophores at ca. 908 and 890 cm-1 in Cl-GFP and Br-GFP exhibit enhanced activities in the electronic excited state and blue-shift during a distinct vibrational cooling process on the ps timescale. The retrieved structural dynamics change due to targeted site-specific halogenation of the chromophore thus provides an effective means to design new GFP derivatives and enrich the bioimaging probe toolset for life and medical sciences.

Molecular Structure, Equilibrium Conformation, and Ring-Puckering Motion in 1,1,3,3-Tetramethylcyclobutane. An Electron-Diffraction Investigation Augmented by Molecular Orbital and Normal Coordinate Calculations.

The molecule cyclobutane (CB) has a nonplanar carbon skeleton folded around a line connecting diagonally opposite atoms. The puckering angle (the change from planarity) of ∼30° is generally attributed to steric repulsion between the four sets of adjacent methylene groups that would be opposed in a planar ring and is relieved by the puckering. According to this criterion, a similar molecule, 1,1,3,3-tetramethylcyclobutene (TMCB), in which adjacent methylene groups do not exist, would be expected to have a planar ring in the equilibrium form. We have investigated the structure of TMCB to test this expectation. Two models were designed for the tests: one having D2h symmetry (planar ring) and one of C2v symmetry (nonplanar ring). Each model incorporated the dynamics of large-amplitude bending around a line joining the methylene groups. Our results suggest the D2h model is to be preferred. Dynamic averages (rg/Å; ∠g/deg) of the more important distances and angles in the D2h model with estimated 2σ uncertainties, are as follows. = 1.105 (5), C1-C5 = 1.524 (10), C1-C2 = 1.559 (11), C2-C1-C4 = 87.4 (8), C1-C2-C3 = 92.0 (7), C5-C1-C6 = 109.0 (13), and C2-C1-C5 = 115.8 (8). The large-amplitude bending of the ring leads to a thermal average value of the folding angle equal to 177.1°. The results, including the differences between TMCB and CB, are discussed.

Combined Electron-Diffraction and Spectroscopic Determination of the Structure of Spiropentane, C5H8.

Gas-phase electron-diffraction (GED) data have been combined with recent spectroscopic rotational constants to determine the rα0 structural parameters for spiropentane, C5H8. The structure has D2d symmetry, and the results yield values of 1.105(2) Å for the CH bond length, 1.557(3) Å for the distal CC bond length, and a smaller value of 1.482(1) Å for the four lateral CC bonds that connect to the central carbon atom. The HCH angle is 113.7(13)°, and the HCH flap angle, defined as the angle of the HCH bisector and the distal CC bond, is 150.2(16)°. Corresponding rg values are 1.122(2) Å, 1.560(3) Å, 1.485(1) Å, 115.1(13)°, and 148.9 (16)°. The results are in good accord with values from density functional calculations (B3LYP/cc-pVTZ) and resolve some questions about the structure reported in an earlier GED study, in particular about the HCH angle and anomalous rotational constants calculated for the structure.