Conformational and Structural Characterization of Knotted Proteins.

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Knotted proteins possess unique topological features but their folding, stability, and function remain poorly understood.
• Understanding knotted protein structures is crucial for deciphering their biological roles.

Purpose of the Study:

• To characterize the structural transitions of two model knotted proteins, ubiquitin C-terminal hydrolase (UCH) and α-haloacid dehalogenase (DehI).
• To investigate the impact of solution composition and temperature on knotted protein stability and unfolding pathways.

Main Methods:

• Native trapped ion mobility spectrometry-mass spectrometry (TIMS-MS) was employed for structural characterization.
• Enzymatic digestion and molecular dynamics simulations were used to identify key residues within the knot core.

Main Results:

• Both UCH and DehI exhibited structural transitions across varying methanol concentrations and temperatures, suggesting a three-step unfolding pathway.
• The UCH knot core demonstrated greater resistance to thermal unfolding than DehI, though both were susceptible to disruption by organic solvent.
• Specific residue ranges (UCH: Glu20-Glu188; DehI: Arg89-His304) were identified as crucial for maintaining knot core integrity.

Conclusions:

• Solution organic content and temperature are critical factors influencing knotted protein structural transitions.
• Native TIMS-MS is a powerful tool for comprehensive characterization of knotted proteins, aiding in understanding their stability and unfolding mechanisms.