UC San Diego

Understanding the complex knotted topology in protein folding is an important structural and functional indication as increasing families of protein discovered to contain deeply knotted structure. Methyltransferases in SpoU-TrmD (SPOUT) family catalyze the post-translational methylation of RNA assisted by the cofactor, S-Adenosyl methionine (SAM), and have a deeply fold trefoil knot that associated with the dimer interface and ligand binding domain. Misfolding in neuronal ubiquitin C-terminal hydrolases (UCH), where SPOUT family protein associate with, can result in aggregation and cause neurodegenerative disease. “Minimalism” of knotted protein was used to investigate the unfolding/untying process mechanism and its functional relationship of the knot in a more straightforward manner. MTTTM and MTTSA are both SPOUT MTases that contain trefoil knot with similar structure but different stabilities. Thus, studying native and unfolded proteins can provide information about knot forming mechanism and functional approach.

MTTTM purification protocol was modified, and the purification result was analyzed by mass spectroscopy. Fluorescence spectroscopy was utilized to monitor the tertiary structure change of native and unfolded MTases by tracing tryptophane signal for MTTSA and tyrosine signal for MTTTM. Highly refined structural and dynamics information of native and unfolded MTTSA was obtained by NMR spectroscopy. Shifts in unfolded MTTSA emission spectrum maximum as well as tryptophane signal in 1H-15N HSQC spectrum were observed, indicating possible solvent exposure and change in microenvironment of tryptophane. The unusual peak and shoulder appeared in the unfolded MTTTM emission spectrum requires further investigation in tyrosine residues and its relationship with tertiary fold of the protein.