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Updated: Feb 16, 2026

Monitoring the Assembly of a Secreted Bacterial Virulence Factor Using Site-specific Crosslinking
Published on: December 17, 2013
Genetically encoded photochemical covalent crosslinking within the Hcp-1 self-assembling bacterial secretion
Alicja K Antonczak1, Kedric Milholland1, Eric M Tippmann2
1Department of Chemistry, Indiana-Purdue University, Fort Wayne, IN, 46805, USA.
Abstract:
The target protein, Hcp1, was first described as part of the bacterial Type VI secretion system from Pseudomonas aeruginosa. The protein first self-assembles into a hexamer and then the hexamers further stack into a nanotubular structure. Hcp1 monomers were targeted for mutagenesis with two widely used photoactivatable amino acids: para-benzoyl phenylalanine or para-azidophenylalanine. The ability of these amino acids to form covalent adducts within the Hcp1 self-assembled system was investigated. Multiple residues, putatively of equal distance between the monomer-monomer interface were targeted. The efficiency of each amino acid to covalently link self-assembled hexamers was determined. The results demonstrate the choice and role of genetically encoded tools applied to complicated biological processes such as self-assembly and also suggested some structural dynamics of the Hcp-1 protein not obvious from crystallographic structures.
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