2025 AIChE Annual Meeting

(182ai) Fluorogenesis - Photoinduced Chromophore Dissociation Enables Aggregation-Driven Red Fluorescence

Authors

Yashwant Kumar, IIT Gandhinagar
Manisha Ojha, IIT Gandhinagar
Reman Kumar Singh, St. Xavier’s College
Karthik S. Pushpavanam, Arizona State University
Fluorescent proteins play a crucial role in bioimaging by enabling the visualization of cellular localization and functionalities, as well as facilitating structural and functional studies of proteins and peptides. PhoCl1, a photocleavable protein, belongs to the class of green-to-red photoconvertible proteins. Upon exposure to 405 nm light, PhoCl1 undergoes cleavage, resulting in two components: an N-terminal empty barrel and a C-terminal nine-amino-acid peptide fragment (CTPF) containing the chromophore. Previous reports have suggested that PhoCl1 becomes nonfluorescent following light-induced dissociation. In this study, we demonstrate that PhoCl1 exhibits red fluorescence upon light irradiation, and we further investigate the underlying mechanism of this fluorogenesis.

We engineered PhoCl1 with a 6xHis tag at its N-terminus to facilitate binding to Ni-NTA resin. Upon irradiation with 405 nm light, the CTPF dissociates and is released into the supernatant, exhibiting red fluorescence. The CTPF present in the supernatant was characterized through MALDI and SDS-PAGE which revealed the absence of impurities. We further performed intensive spectroscopic analyses, including absorbance and fluorescence spectra, lifetime measurements, and quantum yield and fluorescence anisotropy assessment to provide additional insights into the properties of the fluorescent fragment.

To elucidate the mechanism behind the observed red fluorescence, we hypothesized that the CTPF units aggregate which results in the exclusion of water from the chromophore environment, thereby enhancing fluorescence. Dynamic light scattering (DLS) and fluorescence anisotropy measurements supported this aggregation-based mechanism. Quantum mechanics/molecular mechanics (QM/MM) analyses further corroborated these findings. Additionally, we evaluated the fluorescence behavior of the CTPF under various environmental conditions, including changes in pH, NaCl concentration, and temperature.

These findings suggest a reconsideration of the previously proposed model that PhoCl1 loses its fluorescence upon light treatment and suggest an alternative model wherein the cleaved CTPF exhibits red fluorescence due to aggregation.