ChemRxiv
These are preliminary reports that have not been peer-reviewed. They should not be regarded as conclusive, guide clinical practice/health-related behavior, or be reported in news media as established information. For more information, please see our FAQs.
1/1
2 files
0/0

Structural and Spectroscopic Characterization of Photoactive Yellow Protein and Photoswitchable Fluorescent Protein Constructs Containing Heavy Atoms

preprint
submitted on 21.03.2020 and posted on 23.03.2020 by Matthew Romei, Chi-Yun Lin, Steven Boxer
Photo-induced structural rearrangements of chromophore-containing proteins are essential for various light-dependent signaling pathways and optogenetic applications. Ultrafast structural and spectroscopic methods have offered insights into these structural rearrangements across many timescales. However, questions still remain about exact mechanistic details, especially regarding photoisomerization of the chromophore within these proteins femtoseconds to picoseconds after photoexcitation. Instrumentation advancements for time-resolved crystallography and ultrafast electron diffraction provide a promising opportunity to study these reactions, but achieving enough signal-to-noise is a constant challenge. Here we present four new photoactive yellow protein constructs and one new fluorescent protein construct that contain heavy atoms either within or around the chromophore and can be expressed with high yields. Structural characterization of these constructs, most at atomic resolution, show minimal perturbation caused by the heavy atoms compared to wild-type structures. Spectroscopic studies report the effects of the heavy atom identity and location on the chromophore’s photophysical properties. None of the substitutions prevent photoisomerization, although certain rates within the photocycle may be affected. Overall, these new proteins containing heavy atoms are ideal samples for state-of-the-art time-resolved crystallography and electron diffraction experiments to elucidate crucial mechanistic information of photoisomerization.

Funding

National Institutes of Health GM118044

National Science Foundation CHE-1740645

History

Email Address of Submitting Author

mromei@stanford.edu

Institution

Stanford University

Country

United States

ORCID For Submitting Author

0000-0001-6798-7493

Declaration of Conflict of Interest

No conflict of interest

Exports