2023 98 P

Florescent Fusion Proteins for the Study of Styrene Monooxygenase Activity

By: Galilee Samuels, Kyle Cesar

Department: Chemistry & Biochemistry

Faculty Advisor: Dr. George Gassner

Styrene monooxygenase (SMO) serves important roles in both the environmental remediation of contaminated water and soil and as a biocatalyst for the green chemical synthesis of S-styrene oxides. Genetically encoded fluorescent biosensors can be designed by linking light-emitting proteins to a protein under investigation, enabling the measurement of temporal and spatial dynamics involved in protein and small molecule interactions within living systems. Here we report the design of model fusion proteins that can be used in Forster Resonance Energy Transfer (FRET) experiments to evaluate the role of inter-subunit and inter-protein interactions coupled to the FAD-catalyzed oxygen-transfer reaction of NSMOA. In this work structural models were prepared that tether the monomeric green fluorescent protein (mGreenlantern) and red fluorescent protein (mScarlet-1) to the C-terminus of NSMOA with a series of peptide linkers. Structural models prepared by Transform Restrained Rosetta (trRosetta) are found to differ in their interfacial interaction and spatial orientation with respect to the NSMOA subunit to which they are fused. Experimental results from the expression, purification, and spectral characterization of mGreenlantern and mScarlet will be presented and the proposed application of the NSMOA fusion proteins in intra-subunit (FAD --> mScarlet) and inter- subunit (mGreenlantern --> mScarlet) FRET experiments will be discussed. Structural models of the enzymes fused with fluorescent proteins by the proposed linkers will be presented. Considerations involved in the design of peptide linkers and the cloning strategy for expression vector construction will be discussed.