A functional decaisoleucine-containing signal sequence. Construction by cassette mutagenesis

Area of Science:

• Molecular Biology
• Protein Transport
• Biochemistry

Background:

• Signal sequences direct proteins to specific cellular locations.
• Hydrophobic core regions are crucial for signal sequence function.
• Previous studies showed polyleucine enhanced alpha-helix formation and transport.

Purpose of the Study:

• To investigate the role of hydrophobic core structure in signal sequence-mediated protein transport.
• To compare the efficiency of polyisoleucine versus polyleucine core regions.
• To assess the impact of core region length and conformation on protein targeting.

Main Methods:

• Construction of alkaline phosphatase mutants with varying hydrophobic core sequences (polyleucine and polyisoleucine).
• Utilized a new plasmid (CASS3) for efficient mutagenesis and construction of synthetic signal sequences.
• Assessed protein processing and periplasmic targeting in Escherichia coli.

Main Results:

• A decaisoleucine core mutant showed efficient processing and targeting, similar to the polyleucine mutant.
• Truncated polyisoleucine and polyleucine core mutants accumulated as precursors, failing to be processed.
• No significant differences were observed between shortened polyisoleucine and polyleucine segments.

Conclusions:

• Hydrophobic core regions composed of either leucine or isoleucine can efficiently mediate protein transport.
• The conformational preference of isoleucine for beta-sheet formation did not impede signal sequence function.
• Mutants with truncated hydrophobic cores were ineffective, suggesting a critical length requirement for both sequence types.