Both the disulphide bond (Cys(192)-Cys(199)) and the proline-rich motif (pro(193)ProAsnPro(196)) in the long loop connecting the alpha 4-alpha 5 transmembrane hairpin of the Cry4Aa mosquito-larvicidal protein have been found to be unique among the Bacillus thuringiensis Cry delta-endotoxins. In this study, their structural requirements for larvicidal activity of the Cry4Aa toxin were investigated. C192A and C199A Mutant toxins were initially generated and over-expressed in Escherichia coli cells as 130-kDa protoxins at levels comparable to that of the wild-type toxin. When their activities against Aedes acgypti larvae were determined, Eveherichia colicells expressing each Mutant toxin retained the high-level toxicity. Further mutagenic analysis of the PPNP motif revealed that an almost complete loss in larvicidal activity was observed for the C199A/P193A double mutant, whereas a small reduction in toxicity was shown for the C199A/P194A and C199A/P196A Mutants. Increasing the flexibility of the alpha 4-alpha 5 loop through C199A/P193G, C199A/P194G/P196A, C199A/P194A/P196G, and C199A/P194G/P196G mutations significantly decreased the larvicidal activity. Similar to the wild-type protoxin, all Mutant toxins were structurally stable upon solubilisation and trypsin activation in carbonate buffer, pH 9.0. These findings are the first biological evidence for a structural function in larvicidal activity of the unique disulphide bridge as well as the proline-rich motif within the alpha 4-alpha 5 loop of the Cry4Aa toxin. (c) 2005 Elsevier Inc. All rights reserved.