Complexation of Green and Red Kaede Fluorescent Protein Chromophores by a Zwitterion to Probe Electrostatic and Induction Field Effects

Abstract

The photophysics of green fluorescent protein (GFP) and red Kaede fluorescent protein (rKFP) are defined by the intrinsic properties of the light-absorbing chromophore and its interaction with the protein binding pocket. This work deploys photodissociation action spectroscopy to probe the absorption profiles for a series of synthetic GFP and rKFP chromophores as the bare anions and as complexes with the betaine zwitterion, which is assumed as a model for dipole microsolvation. Electronic structure calculations and energy decomposition analysis using Symmetry-Adapted Perturbation Theory are used to characterize gas-phase structures and complex cohesion forces. The calculations reveal a preponderance for coordination of betaine to the phenoxide deprotonation site predominantly through electrostatic forces. Calculations using the STEOM-DLPNO-CCSD method are able to reproduce absolute and relative vertical excitation energies for the bare anions and anion–betaine complexes. On the other hand, treatment of the betaine molecule with a point-charge model, in which the charges are computed from some common electron density population analysis schemes, show that just electrostatic and point-charge induction interactions are unable to account for the betaine-induced spectral shift. The present methodology could be applied to investigate cluster forces and optical properties in other gas-phase ion–zwitterion complexes.

Protomers of the green and cyan fluorescent protein chromophores investigated using action spectroscopy

“…Earlier benchmarking calculations on p HBDI-based deprotonated anions with the same level of theory determined that vertical excitation energies, on average, are within ≈0.05 eV (≈15 nm in the present spectral range) of experiment. 22 The calculated vertical excitation wavelength for protomer I is within 10 nm of the action spectrum peak. On the other hand, the calculated value for protomer II is red-shifted by ≈20 nm compared with the longest-wavelength shoulder feature in the cryogenic action spectrum.…”

“…48 and 49, while the DLPNO-STEOM-CCSD method has previously reproduced vertical transition energies for a series of p HBDI-based anions and other biochromophores to within ≈0.1 eV. 22,50…”

“…The pHBDI molecule used in this work was available from an earlier study. 22 The Cyan molecule was synthesised in a two-step reaction according to an established procedure involving the preliminary synthesis of an azalactone followed by its aminolysis with methylamine. 27 The structures and purity of the compounds were confirmed by 1 H spectroscopy -see ESI.…”

“…While the gas-phase deprotonated anions of pHBDI chromophores have been studied extensively (see ref. 22 for a recent summary of relevant literature) and deprotonated Cyan has been studied with photoelectron spectroscopy, 23 the protonated cations have been studied at room-temperature only, [24][25][26] with limited discussion of protomers. In this work, photodissociation action spectra of pHBDI + and Cyan + were recorded using two instruments, the first offering the capabilities for measurements at T E 300 K and T = 40 AE 10 K, and the second offering isomer (protomer) selectivity, although the ions are heated to T = 400-500 K during the isomer selection step.…”

Photophysics of the red-form Kaede chromophore

“…We and others investigated the fundamental spectroscopy of the anion rK1 − in the gas phase. 48,52,53 Here, we present a detailed study of the condensed phase photophysics of both rK1H and rK1 − , including ultrafast time-resolved uorescence (TRF), transient absorption (TA), and femtosecond stimulated Raman spectroscopy (FSRS). These experiments are applied to investigate effects of solvent polarity and viscosity on excited state spectroscopy and dynamics.…”