Two disulfide mutants in domain I of Bacillus thuringiensis Cry3Aa δ-endotoxin increase stability with no effect on toxicity

Sheng-Jiun Wu, Alvaro M. Florez, Bradley J. Homoelle, Donald H. Dean, Oscar Alzate

Biochemistry Department, Ohio State University, Columbus, USA.
Department of Cell and Developmental Biology, University of North Carolina, Chapel Hill, USA.
Laboratorio de Biología Molecular y Biotecnología, Facultad de Medicina, Universidad de Santander (UDES), Campus Univer- sitario, Bucaramanga, Colombia.

DOI: 10.4236/abc.2012.22015                

Abstract

To increase protein stability and test protein function, three double-cysteine mutations were individually introduced by protein engineering into the cysteine-free Cry3Aa δ-endotoxin from Bacillus thuringiensis. These mutations were designed to create disulfide bonds between α-helices 2 and 5 (positions 110 - 193), and α-helices 5 and 7 (positions 195 - 276 and 198 - 276). Comparison of the CD spectra of the wild-type and the double-cysteine mutant proteins indicates a tighter helical packing consistent with formation of at least two of the disulfide bonds between the central and the outer helices. Thermal stability analysis indi-cates that potential covalent linkages between the central α-helix 5 and the other helices increase resistance to thermal denaturation by 10?C to 14?C com-pared to the thermal stability of the wild-type protein. Spectroscopic analysis of the disulfide-specific absorbance band indicates that the double mutant proteins are more stable to temperature and denaturant (guanidine hydrochloride) than the wild-type protein, as a result of the formation of two of the disulfide bridges. These results indicate that the double muta-tions M110C/F193C and A198C/V276C successfully established disulfide bonds, resulting in a more stable structure of the entire toxin. Despite the increase in stability and structural changes introduced by the disulfide bonds, no effect on toxicity was observed. A possible mechanism involving the insertion of all of domain I of Cry3Aa toxin into the target membrane accounts for these observations.

Keywords

Disulfide Bonds; CD Spectra; Cry3Aa; Site Directed Mutagenesis

Conflicts of Interest

The authors declare no conflicts of interest.

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