@article{12740,
  author = {Taecheon Lyu and So Sohn and Ralph Jimenez and Taiha Joo},
  title = {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.},
  year = {2022},
  journal = {The Journal of Physical Chemistry B},
  month = {2022-03},
  publisher = {American Chemical Society},
  isbn = {1520-6106},
  url = {https://doi.org/10.1021/acs.jpcb.1c10516},
  doi = {10.1021/acs.jpcb.1c10516},
  note = {doi: 10.1021/acs.jpcb.1c10516},
}