Temperature-Dependent Fluorescence of mPlum Fluorescent Protein from 295 to 20 K

Abstract

The development of bright fluorescent proteins (FPs) emitting beyond 600 nm continues to be of interest both from a fundamental perspective in understanding protein-chromophore interactions and from a practical perspective as these FPs would be valuable for cellular imaging. We previously reported ultrafast spectral observations of the excited-state dynamics in mPlum resulting from interconversion between direct hydrogen bonding and water-mediated hydrogen bonding between the chromophore acylimine carbonyl and the Glu16 side chain. Here, we report temperature-dependent steady-state and time-resolved fluorescence measurements of mPlum and its E16H variant, which does not contain a side-chain permitting hydrogen bonding with the acylimine carbonyl. Lowering the temperature of the system freezes interconversion between the hydrogen-bonding states, thus revealing the spectral signatures of the two states. Analysis of the temperature-dependent spectra assuming Boltzmann populations of the two states yields a 205 cm–1 energy difference. This value agrees with the predictions from a quantum mechanics/molecular mechanics study of mPlum (198 cm–1). This study demonstrates the first use of cryogenic spectroscopy to quantify the energetics and timescales of FP chromophore structural states that were only previously obtained from computational methods and further confirms the importance of acylimine hydrogen-bonding dynamics to the fluorescence spectral shifts of red FPs.

Conformational Dynamics of mCherry Variants: A Link between Side-Chain Motions and Fluorescence Brightness

“…The development of new FPs continues, and new experimentally determined structures are being added to the Protein Data Bank, along with thousands of new AI-predicted structures which recently appeared in the Alpha-Fold database. , Recent advancements on many theoretical and experimental fronts have led to a better understanding of FP photophysics, particularly in the context of the chromophore chemistry. − However, there remains a dearth of information when it comes to linking the success of evolving a dim FP to a brighter variant with the underlying structure–function relationships that impact the chromophore photophysics. We followed our previous evolution efforts for mCherry to provide a local analysis of the conformational space in mCherry, mCherry-XL, mScarlet, C12-3, C12-7, and C12-8 .…”

Elucidating photocycle in large Stokes shift red fluorescent proteins: Focus on mKeima

“…Further, the transition from DHB to WMHB is impeded at temperature below 240 K, during which the fluorescence spectrum reflects the relative populations of the two forms in the ground state, as governed by the Boltzmann distribution. 60…”

“…Thus, these dynamics contribute to a bathochromic shift through a combination of direct excited‐state solvation and regulation of chromophore flexibility. Further, the transition from DHB to WMHB is impeded at temperature below 240 K, during which the fluorescence spectrum reflects the relative populations of the two forms in the ground state, as governed by the Boltzmann distribution 60 …”

Water-soluble red fluorescent protein dimers for hypoxic two-photon photodynamic therapy

“…Moreover, such chromophores possess a large Stokes shift, good water solubility, and outstanding photophysical properties. [14][15][16][17][18] Consequently, they find widespread application in fluorescence imaging and as biological probes. [19][20][21][22][23] Despite their success in these areas, the exploration and reporting of fluorescent protein (FP) chromophore analogues in the field of photodynamic therapy (PDT) remain limited.…”