Action spectroscopy of the isolated red Kaede fluorescent protein chromophore

Abstract

The incorporation of fluorescent proteins into biochemical systems has revolutionized the field of bioimaging. In a bottom-up approach, understanding the photophysics of fluorescent proteins requires detailed investigations on the light-absorbing chromophore, which can be achieved by studying the chromophore in isolation. This paper reports a photodissociation action spectroscopy study on the deprotonated anion of the red Kaede fluorescent protein chomophore, demonstrating that at least three isomers -assigned to deprotomers -are generated in the gas phase. Deprotomer-selected action spectra are recorded over the S 1 ←S 0 band using an instrument with differential mobility spectrometry coupled with photodissociation spectroscopy. The spectrum for the principal phenoxide deprotomer spans the 480-660 nm range with maximum response at ≈610 nm. The imidazolate deprotomer has a blue-shifted action spectrum with maximum response at ≈545 nm. The action spectra are consistent with STEOM-DLPNO-CCSD calculations of excitation wavelengths for the deprotomers. A third gas-phase species with a distinct action spectrum is tentatively assigned to an imidazole tautomer of the principal phenoxide deprotomer. The study highlights the need for isomer-selective methods when studying the photophysics of biochromophores possessing several deprotonation sites.

The hitchhiker's guide to dynamic ion–solvent clustering: applications in differential ion mobility spectrometry

“…These sub-populations can be associated with different conformers or tautomers, 45,48,50,[158][159][160] diastereomers, 44,46,47,139 or with post-DMS fragmentation of an aggregate species (e.g., solvent clusters, proton-bound analyte dimers). 49,161 Although the identity of each feature can be determined by, for example, collision-induced dissociation, 48 hydrogen-deuterium exchange, 162,163 and/or trapped ion spectroscopy, 18,[164][165][166][167][168][169] there is no simple a priori method to determine whether a particular analyte will exhibit multiple features in a DMS ionogram. This complicates the use of DMS in an analytical setting -if an analyte exhibits multiple populations that vary with DMS conditions, but the device is set to select only one sub-population, one runs the risk of underestimating analyte concentration in quantification experiments.…”

“…[11][12][13][14][15] For example, in cases where the gasphase and solution-phase analyte structures differ, the degree of microsolvation can dictate whether the gas-phase or solutionphase favoured isomer will be present in the experimental ensemble. [16][17][18] The number of solvent ligands required to transition between the preferred gas-and solution-phase isomers depend on the analyte. In fact, there is no ''back-of-theenvelope'' method that can estimate this quantity, or the number of solvent ligands required to observe the onset of different condensed-phase properties (e.g., electronic or vibrational spectra, [19][20][21][22][23][24] molecular conformation, [25][26][27] chemical reactivity 28 ).…”

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

“…For these two chromophores, there is a concomitant increase in the calculated vertical detachment energy (VDE, Table ). It is worth noting that gas-phase rKFP – was recently investigated by some of the present authors using isomer-specific photodissociation action spectroscopy, where it was shown that three gas-phase forms may be generated using electrospray ionization (two deprotomers and one tautomer). Following on from that study, judicious choice of solvent and electrospray conditions in the present work allowed generation of a pure gas-phase ensemble of the phenoxide deprotomer shown in Figure .…”

“… a Values in parentheses are solvation-induced spectral shifts, i.e., relative to the bare anion. b Reference . c Reference . …”

The combination of laser photodissociation, action spectroscopy, and mass spectrometry to identify and separate isomers

“…Other examples of experiments that combine ion mobility with PD action spectroscopy are FAIMS separation with cryogenic UV/vis PD spectroscopy of peptides from Rizzo's group, 180,199,200 FAIMS separation with room temperature UV/ vis PD spectroscopy from the Hopkins group, 96,[201][202][203][204] drift mobility separation with cryogenic spectroscopy from Bieske's group, 158,159 and drift mobility separation with room temperature photodissociation and photoisomerization from Bieske's group 72,[205][206][207][208][209][210][211][212][213][214][215][216][217][218] and Dugourd's group. 219,220 Tandem IMS and photoactivation Tandem IMS has been combined with photoactivation to observe photoisomerism.…”