Yersinia pestis YopD 150-287 Fragment is Partially Unfolded in the Native State
Abstract
Yersinia pestis is a human and animal pathogen uses the type III secretion system (TTSS) for delivering virulence factors and effectors into the host cells. The system is conserved in animal pathogens and is hypothesized to deliver the virulence factors directly from bacterial to mammalian cells through a pore composed of YopB and YopD translocation proteins. The YopB and YopD effector proteins must be delivered first to form a functional pore in the mammalian cell. The criteria by which Yersinia selects the two proteins for initial delivery are not known and we hypothesized that the extensive binding by the chaperone and partial unfolding of the unbound region may be the criteria for selection. The YopB and YopD proteins, unlike other effectors, have a common chaperone SycD, which binds through multiple regions. Due to the small size of the pore, we hypothesized that many of the transported virulence factors, YopB and YopD included, are delivered in a partially unfolded state stabilized by binding to specific chaperones. The YopD protein binds the chaperone through amino acid (a.a.) 53-149 and a.a. 278-292 regions but biophysical characterization of YopD has not been possible due to the lack of an expression system for soluble, large fragments of the protein. In our present work, we demonstrated that the YopD 150-287 peptide fragment, almost the full soluble C-terminal part, including the non-interacting peptide fragment YopD150-277, was partially unfolded in its native state by a combination of biophysical methods: circular dichroism, quasi-elastic light scattering, chemical unfolding and 8-anilino-1-naphtalene sulfonate (ANS) binding. The secondary structure of the peptide converted easily between alpha-helical and random coil states at neutral pH, and the alpha-helical state was almost fully recovered by lowering the temperature to 262 K. The current re
Document Details
- Document Type
- Technical Report
- Publication Date
- Nov 17, 2007
- Accession Number
- ADA476407
Entities
People
- Ronald Raab
- Wieslaw Swietnicki!
Organizations
- James Madison University