Targeting Ultrafast Spectroscopic Insights into Red Fluorescent Proteins

Abstract

Red fluorescent proteins (RFPs) represent an increasingly popular class of genetically encodable bioprobes and biomarkers that can advance next‐generation breakthroughs across the imaging and life sciences. Since the rational design of RFPs with improved functions or enhanced versatility requires a mechanistic understanding of their working mechanisms, while fluorescence is intrinsically an ultrafast event, a suitable toolset involving steady‐state and time‐resolved spectroscopic techniques has become powerful in delineating key structural features and dynamic steps which govern irreversible photoconverting or reversible photoswitching RFPs, and large Stokes shift (LSS)RFPs. The pertinent cis‐trans isomerization and protonation state change of RFP chromophores in their local environments, involving key residues in protein matrices, lead to rich and complicated spectral features across multiple timescales. In particular, ultrafast excited‐state proton transfer in various LSSRFPs showcases the resolving power of wavelength‐tunable femtosecond stimulated Raman spectroscopy (FSRS) in mapping a photocycle with crucial knowledge about the red‐emitting species. Moreover, recent progress in noncanonical RFPs with a site‐specifically modified chromophore provides an appealing route for efficient engineering of redder and brighter RFPs, highly desirable for bioimaging. Such an effective feedback loop involving physical chemists, protein engineers, and biomedical microscopists will enable future successes to expand fundamental knowledge and improve human health.

Capturing excited-state structural snapshots of evolutionary green-to-red photochromic fluorescent proteins

“…This is likely influenced by a reduced oscillator strength of the twisted chromophore in LEA-A69T. We note that the correlation between increasing FQY values and decreasing absorption peak wavelengths observed in many other FPs ( Krueger et al, 2023a ; Chen et al, 2023 ), including mCherry variants ( Mukherjee et al, 2022 ), is not observed in the LEA and ALL-FPs. For instance, LEA possesses the highest GB* FQY while maintaining the reddest electronic features among the five FPs studied herein, supporting a photophysical interpretation beyond the energy-gap model ( Englman and Jortner, 1970 ).…”

“…This is likely influenced by a reduced oscillator strength of the twisted chromophore in LEA-A69T. We note that the correlation between increasing FQY values and decreasing absorption peak wavelengths observed in many other FPs (Krueger et al, 2023a;Chen et al, 2023),…”

“…Several photoconversion mechanisms have been proposed for the Kaede-like pcFPs, and most mechanisms involve a β-elimination reaction and backbone breakage to extend the conjugation between Gly64 and His62 that forms the red chromophore, although the complete photocycle remains largely unanswered ( Mizuno et al, 2003 ; Wachter et al, 2010 ; Shcherbakova et al, 2012 ; Subach and Verkhusha, 2012 ; Kim et al, 2015 ; Krueger et al, 2023a ; Krueger et al, 2023b ). In most Kaede-like FPs, the crystal structure, protein chromophore, and local environment display remarkable similarities before and after the photoconversion, which implies that fleeting transient interactions between the chromophore and surrounding residues can induce the necessary conformational changes during photoconversion ( Wachter et al, 2010 ; Kim et al, 2015 ).…”