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United States Army Medical Research Institute of Infectious Diseases, Department of Cell Biology and Biochemistry, Frederick, Maryland 21702, USA
(RECEIVED May 28, 2004; FINAL REVISION June 29, 2004; ACCEPTED July 2, 2004)
Ricin is a potent toxin presenting a threat as a biological weapon. The holotoxin consists of two disulfide-linked polypeptides: an enzymatically active A chain (RTA) and a galactose/N-acetylgalactosamine-binding B chain. Efforts to develop an inactivated version of the A chain as a vaccine have been hampered by limitations of stability and solubility. Previously, recombinant truncated versions of the 267-amino-acid A chain consisting of residues 1–33/44–198 or 1–198 were designed by protein engineering to overcome these limits and were shown to be effective and nontoxic as vaccines in mice. Herein we used CD, dynamic light scattering, fluorescence, and Fourier-transform infrared spectroscopy to examine the biophysical properties of these proteins. Although others have found that recombinant RTA (rRTA) adopts a partially unfolded, molten globule–like state at 45°C, rRTA 1–33/44–198 and 1–198 are significantly more thermostable, remaining completely folded at temperatures up to 53°C and 51°C, respectively. Deleting both an exposed loop region (amino acids 34–43) and the C-terminal domain (199–267) contributed to increased thermostability. We found that chemically induced denaturation of rRTA, but not the truncated variants, proceeds through at least a three-state mechanism. The intermediate state in rRTA unfolding has a hydrophobic core accessible to ANS and an unfolded C-terminal domain. Removing the C-terminal domain changed the mechanism of rRTA unfolding, eliminating a tendency to adopt a partially unfolded state. Our results support the conclusion that these derivatives are superior candidates for development as vaccines against ricin and suggest an approach of reduction to minimum essential domains for design of more thermostable recombinant antigens.
Keywords: protein folding; stability; ricin; vaccine
Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.04897904.
Reprint requests to: John Carra, United States Army Medical Research Institute of Infectious Diseases, Department of Cell Biology and Biochemistry, 1425 Porter Street, Frederick, MD 21702, USA; e-mail: john. carra{at}det.amedd.army.mil; fax: (301) 619-2348.
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