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Transmembrane domain mediated self-assembly of major coat protein subunits from Ff bacteriophage
Roman A Melnyk1, Anthony W Partridge, Charles M Deber
1Division of Structural Biology and Biochemistry. Research Institute, University of Toronto, Hospital for Sick Children, Toronto, Ontario M5G 1X8, Canada.
Abstract:
The 50-residue major coat protein (MCP) of Ff bacteriophage exists as a single-spanning membrane protein in the Escherichia coli host inner membrane prior to assembly into lipid-free virions. Here, the molecular bases for the specificity and stoichiometry that govern the protein-protein interactions of MCP in the host membrane are investigated in detergent micelles. To address these structural issues, as well as to circumvent viability requirements in mutants of the intact protein, peptides corresponding to the effective alpha-helical TM segment of wild-type and mutant bacteriophage MCPs were synthesized. Fluorescence resonance energy transfer (FRET) experiments on the dansyl and dabcyl-labeled MCP TM domain peptides in detergent micelles demonstrated that the peptides specifically associate into non-covalent homodimers, as postulated for the biologically relevant membrane-embedded MCP oligomer. MCP peptides labeled with short-range pyrene fluorophores at the N terminus displayed excimer fluorescence consistent with homodimerization occurring in a parallel fashion. Variant peptides synthesized with single substitutions at helix-interactive positions displayed a wide range of dimer/monomer ratios on SDS-PAGE gels, which are interpreted in terms of steric volume, presence or absence of beta-branching, and the effect of polar substituents. The overall results indicate discrete roles for helix-helix interfacial residues as packing recognition elements in the membrane-inserted state, and suggest a possible correlation between phage viability and efficacy of MCP TM-TM interactions.
Insights
Ff bacteriophage major coat protein (MCP) peptides form specific homodimers in membranes, revealing molecular insights into protein interactions critical for phage assembly and viability.
Area of Science:
- Biochemistry
- Structural Biology
- Molecular Biology
Background:
- Ff bacteriophage major coat protein (MCP) is a single-spanning membrane protein in Escherichia coli.
- MCP is essential for assembly into lipid-free virions.
- Understanding MCP's membrane interactions is key to viral assembly.
Purpose of the Study:
- Investigate the molecular basis of MCP's protein-protein interactions in membranes.
- Determine the specificity and stoichiometry of MCP interactions.
- Correlate interaction efficacy with phage viability.
Main Methods:
- Synthesized peptides corresponding to the alpha-helical transmembrane (TM) segment of wild-type and mutant MCPs.
- Utilized Fluorescence Resonance Energy Transfer (FRET) with dansyl and dabcyl labels.
- Employed pyrene fluorophores for N-terminal labeling and analyzed dimerization via excimer fluorescence.
- Assessed dimer/monomer ratios using SDS-PAGE for variant peptides.
Main Results:
- MCP TM domain peptides specifically associate into non-covalent homodimers in detergent micelles.
- Parallel homodimerization was confirmed by pyrene excimer fluorescence.
- Single amino acid substitutions affected dimer/monomer ratios, indicating roles for steric and polar interactions.
- Identified specific helix-helix interfacial residues as key recognition elements.
Conclusions:
- MCP TM domains form specific homodimers in a membrane-like environment.
- Helix-helix interfacial residues play critical roles in packing and recognition.
- Efficacy of TM-TM interactions may correlate with Ff bacteriophage viability.