PhD Thesis Presentation - Engineering a Near-Infrared Light-Responsive Protein for Bioimaging and New Materials

Biomolecules such as fluorescent proteins (e.g. GFP and its homologs) have long served as versatile tools in a broad range of research areas and, in couple with proper protein engineering, have also generated numerous genetically encoded devices and materials for biomedical applications. However, many of these resulting tools are constrained in their abilities to sense and respond to long wavelength (red or near-infrared; > 600 nm) light, which is important for biomedical applications as far as deep-tissue penetration is concerned. It is therefore necessary to seek for alternative protein molecules or mechanisms to overcome this limitation. The water soluble chlorophyll binding protein (WSCP) derived from plants, which is characterized by its marked stability and responsiveness to >650 nm light, is deemed with such potential but has yet to receive any attention. This thesis aims to develop WSCP into a versatile tool for designing photoresponsive materials, as well as for bioimaging.

Using the WSCP from Lepidium virginicum, genetically encoded click chemistry, and mussel foot proteins (MFP), an entirely protein-based photoresponsive hydrogel was developed. In this material, the WSCP acted as a singlet oxygen generator on exposure to red light, leading to oxidative crosslinking of tyrosine in MFP and strengthening the gel mechanics. Thanks to the photothermal effect of the WSCP, the resulting hydrogel showed the potential for photoacoustic imaging.

The WSCP was also converted to a new class of near infrared fluorescent proteins through the combined use of protein engineering and ligand replacement with bacteriochlorophylls. A key residue that controlled the spectral properties of WSCP was identified and mutated, red-shifting the emission of the protein by >100 nm. These proteins were expressed in mammalian cells and reconstituted with bacteriochlorophylls in vitro.

Together, these studies demonstrated the WSCP as a versatile platform for creating new materials and molecular tools, pointing to great potential in biomedical research.