Photoinduced chromophore dissociation resulting in aggregation-induced red fluorescence

Abstract

Fluorescent molecules are essential for bioimaging and visualizing cellular localization, functionalities, including biosensing, ion sensing, and photochromism. The photocleavable fluorescent protein PhoCl1 belongs to a sub-class of green-to-red photoconvertible β-barrel fluorescent protein and has a characteristic green fluorescence conferred by the chromophore p-HBI. In contrast to other photoconvertible proteins, that shift their fluorescence from green-to-red upon photoexposure, PhoCl1 has been reported to render itself non-fluorescent by releasing the 9 amino-acid C-terminal peptide fragment (CTPF) bearing the photo-transformed red chromophore from the β-barrel. Here we show the fate of photoreleased chromophore which shows an unexpected dim red fluorescence. We attribute this dim red fluorescence to the aggregation of CTPF molecules which is validated through dynamic light scattering measurements. We further characterize the aggregated CTPF through various optical techniques to determine the excitation/emission maxima, fluorescence lifetime, quantum yield and rotational correlation time through fluorescence anisotropy. We assessed the red fluorescence behavior under diverse environmental conditions including variations in pH, NaCl, and temperature. Molecular dynamics simulations support our experimentally observed aggregation of CTPF molecules. We supplemented these studies with quantum mechanics/molecular mechanics study which indicated the role of the chromophore in the photodissociated peptide fragment in the generation of dim red fluorescence. These findings not only provide insight into the behavior of fluorescent chromophore-peptide conjugate but also potentially lay the groundwork for developing light-activated fluorescence systems, AIE-based biosensors, and tunable biomaterials for protein tagging and responsive material design.