title: Effect of specific mutations in helix alpha7 of domain I on the stability and crystallization of Cry3A in Bacillus thuringiensis.
abstract: Insecticidal crystal (Cry) proteins of Bacillus thuringiensis crystallize after synthesis forming large inclusions that stabilize these toxins in the environment after cell lysis until eaten by an insect. Despite the biological importance of crystallization, little is known about the structural elements of Cry molecules that facilitate this process. We identified subdomains that affect Cry3A structure possibly through improper folding by chimeric-scanning mutagenesis, substituting short peptides of a truncated 70-kDa Cry1C molecule that does not crystallize into Cry3A, a wild-type 70-kDa molecule that crystallizes readily. Cry3A consists of three domains that contain five different blocks of conserved amino acids. Domain substitution and mutagenesis within these blocks suggested that the specific structure of block 2, which spans the junction between domains I and II, was important to the relative stability of Cry3A and subsequent crystallization. Amino acid sequences of particular importance to stability in Cry3A block 2 were identified using three substitution mutants, each spanning about a third of this block. One that consisted of Cry1C helix alpha7 yielded no detectable protein, whereas the other two produced characteristic Cry3A crystals. Specific mutations in this region showed tyrosine 268 was critical to normal stability of Cry3A and subsequent crystallization in that a mutant, Y268L, was less stable than wild-type Cry3A and failed to form a characteristic Cry3A crystal. Circular dichroism analysis showed a decrease in this mutant's alpha-helicity, indicating the importance of tyrosine 268 to the specific conformation of helix alpha7 that facilitates stability and normal crystallization.
authors.affiliation: Department of Entomology, University of California Riverside, CA 92521, USA.
authors.forename: Hyun-Woo
authors.initials: HW
authors.lastname: Park
authors.forename: Brian A
authors.initials: BA
authors.lastname: Federici
mesh_terms: D000595:Amino Acid Sequence; D001413:Bacillus thuringiensis / Q000235:genetics* / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics*; D001427:Bacterial Toxins / Q000737:chemistry* / Q000235:genetics*; D001709:Biotechnology / Q000379:methods*; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics*; D006460:Hemolysin Proteins; D008855:Microscopy, Electron, Scanning; D008858:Microscopy, Phase-Contrast; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D014357:Trypsin / Q000494:pharmacology; D014443:Tyrosine / Q000737:chemistry
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D011506:Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D014443:Tyrosine; D014357:Trypsin