Delineating Ultrafast Structural Dynamics of a Green-Red Fluorescent Protein for Calcium Sensing.
Biosensors (Basel)
; 13(2)2023 Feb 02.
Article
em En
| MEDLINE
| ID: mdl-36831983
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.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Prótons
/
Cálcio
Idioma:
En
Revista:
Biosensors (Basel)
Ano de publicação:
2023
Tipo de documento:
Article
País de afiliação:
Estados Unidos