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. 2015 Feb;104(2):424-32.
doi: 10.1002/jps.24047. Epub 2014 Jun 10.

Biophysical characterization of the type III secretion tip proteins and the tip proteins attached to bacterium-like particles

Shyamal P Choudhari  1 , Xiaotong Chen, Jae Hyun Kim, Maarten L Van Roosmalen, Jamie C Greenwood 2nd, Sangeeta B Joshi, William D Picking, Kees Leenhouts, C Russell Middaugh, Wendy L Picking
Affiliations

Biophysical characterization of the type III secretion tip proteins and the tip proteins attached to bacterium-like particles

Shyamal P Choudhari et al. J Pharm Sci. 2015 Feb.

Abstract

Bacterium-like particles (BLPs), derived from Lactococcus lactis, offer a self-adjuvanting delivery vehicle for subunit protein vaccines. Proteins can be specifically loaded onto the BLPs via a peptidoglycan anchoring (PA) domain. In this study, the tip proteins IpaD, SipD, and LcrV belonging to type III secretion systems of Shigella flexneri, Salmonella enterica, and Yersinia enterocolitica, respectively, were fused to the PA and loaded onto the BLPs. Herein, we biophysically characterized these nine samples and condensed the spectroscopic results into three-index empirical phase diagrams (EPDs). The EPDs show distinctions between the IpaD/SipD and LcrV subfamilies of tip proteins, based on their physical stability, even upon addition of the PA. Upon attachment to the BLPs, the BLPs become defining moiety in the spectroscopic measurements, leaving the tip proteins to have a subtle yet modulating effect on the structural integrity of the tip proteins-BLPs binding. In summary, this work provides a comprehensive view of physical stability of the tip proteins and tip protein-BLPs and serves as a baseline for screening of excipients to increase the stability of the tip protein-BLPs for future vaccine formulation.

Keywords: circular dichroism; fluorescence spectroscopy; light scattering (static); pH; physical characterization; physical stability.

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Figures

Figure 1
Figure 1
Three-index empirical phase diagrams (EPDs) for IpaD (A), SipD (B) and LcrV (C), representing the conformational stability of the tip proteins as a function of pH and temperature. The red, green and blue panels at the right define individual component indices for secondary structure (CD), tertiary structure (fluorescence peak position) and aggregation behavior (LS), respectively.
Figure 2
Figure 2
Three-index empirical phase diagrams (EPDs) for IpaD-PA (A), SipD-PA (B) and LcrV-PA (C), representing the conformational stability of the proteins as a function of pH and temperature. The red, green and blue panels at the right define individual component indices for secondary structure (CD), tertiary structure (fluorescence peak position) and aggregation behavior (LS), respectively.
Figure 3
Figure 3
Three-index empirical phase diagrams (EPDs) for IpaD-BLPs (A), SipD-BLPs (B) and LcrV-BLPs (C), representing the conformational stability of the tip proteins attached to the BLPs as a function of pH and temperature. The red, green and blue panels at the right define individual component indices for secondary structure (CD), tertiary structure (fluorescence peak position) and aggregation behavior (LS), respectively.
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