Two disulfide mutants in domain I of Bacillus thuringiensis Cry3Aa δ-endotoxin increase stability with no effect on toxicity
| Autor: | Oscar Alzate, Donald H. Dean, Sheng Jiun Wu, Bradley J. Homoelle, Alvaro M. Florez |
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| Rok vydání: | 2012 |
| Předmět: | |
| Zdroj: | Advances in Biological Chemistry. :123-131 |
| ISSN: | 2162-2191 2162-2183 |
| DOI: | 10.4236/abc.2012.22015 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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