Preprint Article Version 1 This version is not peer-reviewed

Structural Domains of the Bacillus thuringiensis Vip3Af Protein Unraveled by Tryptic Digestion of Alanine Mutants

Version 1 : Received: 14 May 2019 / Approved: 16 May 2019 / Online: 16 May 2019 (10:25:26 CEST)

How to cite: Quan, Y.; Ferré, J. Structural Domains of the Bacillus thuringiensis Vip3Af Protein Unraveled by Tryptic Digestion of Alanine Mutants. Preprints 2019, 2019050203 (doi: 10.20944/preprints201905.0203.v1). Quan, Y.; Ferré, J. Structural Domains of the Bacillus thuringiensis Vip3Af Protein Unraveled by Tryptic Digestion of Alanine Mutants. Preprints 2019, 2019050203 (doi: 10.20944/preprints201905.0203.v1).

Abstract

Vip3 proteins are increasingly used in insect control in transgenic crops. To shed light on the structure of these proteins, we used the approach of trypsin fragmentation of mutants altering the conformation of the Vip3Af protein. From an alanine scanning on Vip3Af, we selected mutants with an altered proteolytic pattern. Based on the protease digestion patterns, their effect on oligomer formation, and theoretical cleavage sites, we generated a map of the Vip3Af protein with five domains, which match some of the domains proposed independently by two in silico models. Domain I ranges from aa12-198, domain II from aa199-313, domain III from aa314-526, domain IV from aa527-668 and domain V from aa669-788. The effect of some of the mutations on the ability to form a tetrameric molecule revealed that domains I-III are required for tetramerization, while domain V is not. The involvement of domain IV in the tetramer formation is not clear. Some mutations distributed from near the end of domain I up to the end of domain II affect the stability of the first three domains of the protein and negatively impact oligomerization upon trypsin treatment. Because of the high sequence similarity among Vip3 proteins, we propose that our domain map can be extended to many other members of the Vip3 family of proteins.

Subject Areas

Bt toxins; insecticidal proteins; trypsin cleavage; tetrameric proteins; domain map

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