Dataset Information

Delineating Ultrafast Structural Dynamics of a Green-Red Fluorescent Protein for Calcium Sensing.

ABSTRACT: Fluorescent proteins (FPs) are indispensable tools for noninvasive bioimaging and sensing. Measuring the free cellular calcium (Ca²⁺) concentrations in vivo with genetically encodable FPs can be a relatively direct measure of neuronal activity due to the complex signaling role of these ions. REX-GECO1 is a recently developed red-green emission and excitation ratiometric FP-based biosensor that achieves a high dynamic range due to differences in the chromophore response to light excitation with and without calcium ions. Using steady-state electronic measurements (UV/Visible absorption and emission), along with time-resolved spectroscopic techniques including femtosecond transient absorption (fs-TA) and femtosecond stimulated Raman spectroscopy (FSRS), the potential energy surfaces of these unique biosensors are unveiled with vivid details. The ground-state structural characterization of the Ca²⁺-free biosensor via FSRS reveals a more spacious protein pocket that allows the chromophore to efficiently twist and reach a dark state. In contrast, the more compressed cavity within the Ca²⁺-bound biosensor results in a more heterogeneous distribution of chromophore populations that results in multi-step excited state proton transfer (ESPT) pathways on the sub-140 fs, 600 fs, and 3 ps timescales. These results enable rational design strategies to enlarge the spectral separation between the protonated/deprotonated forms and the Stokes shift leading to a larger dynamic range and potentially higher fluorescence quantum yield, which should be broadly applicable to the calcium imaging and biosensor communities.

SUBMITTER: Krueger TD

PROVIDER: S-EPMC9954042 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Delineating Ultrafast Structural Dynamics of a Green-Red Fluorescent Protein for Calcium Sensing.

Krueger Taylor D TD Tang Longteng L Fang Chong C

Biosensors 20230202 2

Fluorescent proteins (FPs) are indispensable tools for noninvasive bioimaging and sensing. Measuring the free cellular calcium (Ca<sup>2+</sup>) concentrations <i>in vivo</i> with genetically encodable FPs can be a relatively direct measure of neuronal activity due to the complex signaling role of these ions. REX-GECO1 is a recently developed red-green emission and excitation ratiometric FP-based biosensor that achieves a high dynamic range due to differences in the chromophore response to light ...[more]

PMID: 36831983

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Project description:The fluorescence photocycle of the green fluorescent protein is functionally dependent on the specific structural protein environment. A direct relationship between equilibrium protein side-chain conformation of glutamate 222 and reactivity is established, particularly the rate of ultrafast proton transfer reactions in the fluorescence photocycle. We show that parallel transformations in the photocycle have a structural origin, and we report on the vibrational properties of responsive amino acids on an ultrafast timescale. Blue excitation of GFP drives two parallel, excited-state deuteron transfer reactions with 10 ps and 75 ps time constants to the buried carboxylic acid side chain of glutamate 222 via a hydrogen-bonding network. Assignment of 1456 cm(-1) and 1441 cm(-1) modes to nu(sym) and assignment of 1564 cm(-1) and 1570 cm(-1) features to nu(asym) of E222 in the 10 ps and 75 ps components, respectively, was possible from the analysis of the transient absorption data of an E222D mutant and was consistent with photoselection measurements. In contrast to the wild-type, measurements of E222D can be described with only one difference spectrum, with the nu(sym) mode at 1435 cm(-1) and the nu(asym) mode at 1567 cm(-1), also correlating a large Deltanu(asym-sym) with slow excited-state proton transfer kinetics. Density Functional Theory calculations and published model compound and theoretical studies relate differences in Deltanu(asym-sym) to the strength and number of hydrogen-bonding interactions that are detected via equilibrium geometry and COO- stretching frequency differences of the carboxylate. The correlation of photocycle kinetics with side-chain conformation of the acceptor suggests that proton transfer from S205 to E222 controls the rate of the overall excited-state proton transfer process, which is consistent with recent theoretical predictions. Photoselection measurements show agreement for localized C=O vibrations of chromophore, Q69, and E222 with Density Functional Theory and ab initio calculations placed in the x-ray geometry and provide their vibrational response in the intermediates in the photocycle.

Dataset Information

Delineating Ultrafast Structural Dynamics of a Green-Red Fluorescent Protein for Calcium Sensing.

Publications

Delineating Ultrafast Structural Dynamics of a Green-Red Fluorescent Protein for Calcium Sensing.

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