Sequence determinants of C-terminal substrate recognition by the Tsp protease

Area of Science:

• Biochemistry
• Molecular Biology
• Proteolysis

Background:

• Cytochrome b562 is a bacterial hemoprotein.
• Proteolytic cleavage regulates protein levels and function.
• The tail-specific protease Tsp (TSP) in Escherichia coli targets specific protein substrates.

Purpose of the Study:

• To investigate the role of the C-terminal sequence of cytochrome b562 in its susceptibility to Tsp-mediated cleavage.
• To identify specific amino acid requirements at the C-terminus for efficient Tsp substrate recognition and cleavage.
• To correlate in vitro Tsp cleavage rates with in vivo degradation patterns.

Main Methods:

• Site-directed mutagenesis of the cytochrome b562 C-terminus.
• Expression analysis of cytochrome b562 variants in wild-type and tsp-deficient E. coli strains.
• Purification of cytochrome b562 variants.
• In vitro proteolytic assays using purified Tsp.
• Pulse-chase experiments to determine in vivo cleavage rates.
• Thermal denaturation assays (Tm) to assess protein stability.

Main Results:

• Cytochrome b562 requires a specific C-terminal sequence (WVAAA) to be efficiently cleaved by Tsp.
• Randomization of the C-terminal three residues yielded 54 mutants with varying Tsp susceptibility.
• Cleavage rates in vitro correlated well with in vivo degradation rates.
• The identity of the C-terminal residue is most critical, with small, uncharged residues being preferred.
• Non-polar residues are favored at the second and third positions from the C-terminus.

Conclusions:

• The C-terminal sequence of cytochrome b562 is a major determinant of its proteolytic fate, primarily mediated by Tsp.
• Tsp exhibits distinct selectivity rules for each of the three C-terminal positions.
• Protein stability (Tm) is not directly correlated with Tsp cleavage rates, indicating sequence-specific recognition is dominant.