ABC  Vol.2 No.2 , May 2012
Two disulfide mutants in domain I of Bacillus thuringiensis Cry3Aa δ-endotoxin increase stability with no effect on toxicity
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.

Cite this paper
Wu, S. , Florez, A. , Homoelle, B. , Dean, D. and Alzate, O. (2012) Two disulfide mutants in domain I of Bacillus thuringiensis Cry3Aa δ-endotoxin increase stability with no effect on toxicity. Advances in Biological Chemistry, 2, 123-131. doi: 10.4236/abc.2012.22015.
[1]   Privalov, P.L. and Gill, S.J. (1988) Stability of protein structure and hydrophobic interaction. Advances in Protein Chemistry, 39, 191-234. doi:10.1016/S0065-3233(08)60377-0

[2]   Horovitz, A., Serrano, L., Avron, B., Bycroft, M. and Fersht, A.R. (1990) Strength and cooperativity of contributions of surface salt bridges to protein stability. Journal Molecular Biology, 21, 1031-1044. doi:10.1016/S0022-2836(99)80018-7

[3]   Burley, S.K. and Petsko, G.A. (1988) Weakly polar interactions in proteins. Advances in Protein Chemistry, 39, 125- 189. doi:10.1016/S0065-3233(08)60376-9

[4]   Stickle, D.F., Presta, L.G., Dill, K.A. and Rose, G.D. (1992) Hydrogen bonding in globular proteins. Journal Molecular Biology, 226, 1143-1159. doi:10.1016/0022-2836(92)91058-W

[5]   Creighton, T.E. (1988) Disulphide bonds and protein stability. Bioessays, 8, 57-63. doi:10.1002/bies.950080204

[6]   Anfinsen, C.B. (1973) Principles that govern the folding of protein chains. Science, 181, 223-230. doi:10.1126/science.181.4096.223

[7]   Alzate, O., You, T., Claybon, M., Osorio, C., Curtiss, A. and Dean, D.H. (2006) Effects of disulfide bridges in domain I of Bacillus thuringiensis Cry1Aa δ-endotoxin on ion-channel formation in biological membranes. Biochemistry, 45, 13597-13605. doi:10.1021/bi061474z

[8]   Schwartz, J.L., Juteau, M. and Grochulski, P., et al. (1997) Restriction of intramolecular movements within the Cry1Aa toxin molecule of Bacillus thuringiensis through disulfide bond engineering. FEBS Letters, 410, 397-402. doi:10.1016/S0014-5793(97)00626-1

[9]   Thornton, J.M. (1981) Disulphide bridges in globular proteins. Journal Molecular Biology, 151, 261-287. doi:10.1016/0022-2836(81)90515-5

[10]   Hodgman, T.C. and Ellar, D.J. (1990) Models for the structure and function of the Bacillus thuringiensis delta- endotoxins determined by compilational analysis. DNA Sequence, 1, 97-106.

[11]   Li, J.D., Carroll, J. and Ellar, D.J. (1991) Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 ? resolution. Nature, 353, 815-821. doi:10.1038/353815a0

[12]   Alzate, O., Hemann, C.F., Osorio, C., Hille, R. and Dean, D.H. (2009) Ser170 of Bacillus thuringiensis Cry1Ab delta-endotoxin becomes anchored in a hydrophobic moiety upon insertion of this protein into Manduca sexta brush border membranes. BMC Biochemistry, 10, 25-34. doi:10.1186/1471-2091-10-25

[13]   Aronson, A. (2000) Incorporation of protease K into larval insect membrane vesicles does not result in disruption of integrity or function of the pore-forming Bacillus thuringiensis δ-endotoxin. Applied Environmental Microbiology, 66, 4568-4570. doi:10.1128/AEM.66.10.4568-4570.2000

[14]   Arnold, S., Curtiss, A., Dean, D. H. and Alzate, O. (2001) The role of a proline-induced broken-helix motif in R- helix 2 of Bacillus thuringiensis δ-endotoxins, FEBS Letters, 490, 70-74. doi:10.1016/S0014-5793(01)02139-1

[15]   Loseva, O.I., Tiktopulo, E.I., Vasiliev, V.D., Nikulin, A.D., Dobritsa, A.P. and Potekhin, S.A. (2001) Structure of Cry3A δ-endotoxin within phospholipid membranes. Biochemistry, 40, 14143-14151. doi:10.1021/bi010171w

[16]   Nair, M.S. and Dean, D.H. (2008) All domains of Cry1A toxins insert into insect brush border membranes. Journal Biological Chemistry, 283, 26324-26331. doi:10.1074/jbc.M802895200

[17]   Potekhin, S.A., Loseva, O.I., Tiktopulo, E.I. and Dobritsa, A.P. (1999) Transition state of the rate-limiting step of heat denaturation of Cry3A δ-endotoxin. Biochemistry, 38, 4121-4127. doi:10.1021/bi982789k

[18]   Jimenez-Juarez, N., Munoz-Garay, C. and Gomez, I., et al. (2007) Bacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non-toxic to Manduca sexta larvae. Journal Biological Chemistry, 282, 21222-21229. doi:10.1074/jbc.M701314200

[19]   Girard, F., Vachon, V. and Prefontaine, G., et al. (2008) Cysteine scanning mutagenesis of a4, a putative pore-lining helix of the Bacillus thuringiensis insecticidal toxin Cry1Aa. Applied Environmental Microbiology, 74, 2565- 2572. doi:10.1128/AEM.00094-08

[20]   Vachon, V., Prefontaine, G. and Rang, C., et al. (2004) Helix 4 mutants of the Bacillus thuringiensis insecticidal toxin Cry1Aa display altered pore-forming abilities. Applied Environmental Microbiology, 70, 6123-6130. doi:10.1128/AEM.70.10.6123-6130.2004

[21]   Florez, A.M., Osorio, C. and Alzate, O. (2012) Protein Engineering of Bacillus thuringiensis δ-Endotoxins. In: Sansinenea, E., Ed., Bacillus thuringiensis Biotechnology, Springer-Verlag, New York, 350.

[22]   Laflamme, E., Badia, A., Lafleur, M., Schwartz, J.L. and Laprade, R. (2008) Atomic force microscopy imaging of Bacillus thuringiensis Cry1 toxins interacting with insect midgut apical membranes. Journal Membranes Biology, 222, 127-139.

[23]   Groulx, N., McGuire, H., Laprade, R., Schwartz, J.-L. and Blunk, R. (2011) Single molecule fluorescence study of the Bacillus thuringiensis toxin Cry1Aa reveals tetra-merization. Journal Biological Chemistry, 286, 42274- 42282. doi:10.1074/jbc.M111.296103

[24]   Weiner, S.J. and Kollman, P.A. (1986) An all atom force field for simulations of proteins and nucleic acids. Journal Computational Chemistry, 7,230-252. doi:10.1002/jcc.540070216

[25]   Wu, S.J. and Dean, D.H. (1996) Functional significance of loops in the receptor binding domain of Bacillus thuringiensis CryIIIA delta-endotoxin. Journal Molecular Biology, 255, 628-640. doi:10.1006/jmbi.1996.0052

[26]   Kunkel, T.A. (1985) Rapid and efficient site-specific muta- genesis without phenotypic selection. Proceedings National Academic Science USA, 82, 488-492. doi:10.1073/pnas.82.2.488

[27]   Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. doi:10.1038/227680a0

[28]   Berova, N., Nakanishi, K. and Woody, R. (2000) Circular dichroism: Principles and applications, 2nd Edition, Wiley- VCH, New York

[29]   Greenfield, N.J. (2006) Using circular dichroism spectra to estimate protein secondary structure. Nature Protocols, 1, 2876-2890. doi:10.1038/nprot.2006.202

[30]   Cooper, T.M. and Woody, R.W. (1990) The effect of conformation on the CD of interacting helices: A theoretical study of tropomyosin. Biopolymers, 30, 657-676. doi:10.1002/bip.360300703

[31]   Zhou, N.E., Kay, C.M. and Hodges, R.S. (1992) Synthetic model proteins. Positional effects of interchain hydrophobic interactions on stability of two-stranded alpha-helical coiled-coils. Journal Biological Chemistry, 267, 2664-2670.

[32]   Wetlaufer, D.B. (1962) Ultraviolet spectra of proteins and amino acids. Advances in Protein Chemistry, 17, 303-390. doi:10.1016/S0065-3233(08)60056-X

[33]   Monera, O.D., Kay, C.M. and Hodges, R.S. (1994) Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions. Protein Science, 3, 1984-1991. doi:10.1002/pro.5560031110

[34]   Murdock, L.L., Brookhart, G.L. and Dunn, P.E., et al. (1987) Cysteine digestive proteinases in Coleoptera. Computational Biochemistry Physiology, 87, 783-787. doi:10.1016/0305-0491(87)90388-9

[35]   Bravo, A., Gomez, I. and Conde, J. et al. (2004) Oligo- merization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leadingto insertion into membrane microdomains. Biochemistry. Biophysics. Acta, 1667, 38-46. doi:10.1016/j.bbamem.2004.08.013

[36]   Jimenez-Juarez, N., Munoz-Garay, C., Gomez, I., Gill, S.S., Soberon, M. and Bravo, A. (2008) The pre-pore from Bacillus thuringiensis Cry1Ab toxin is necessary to induce insect death in Manduca sexta. Peptides, 29, 318-323. doi:10.1016/j.peptides.2007.09.026