Bombyx mori midgut membrane protein P252, which binds to Bacillus thuringiensis Cry1A, is a chlorophyllide-binding protein, and the resulting complex has antimicrobial activity.
The epithelial cell membrane 252-kDa protein (P252) isolated in our laboratory from Bombyx mori midgut was shown to bind strongly with Cry1Aa, Cry1Ab, and Cry1Ac toxins of Bacillus thuringiensis (15). In the current paper, P252 was shown to bind with chlorophyllide (Chlide) to form red fluorescent protein (RFP) complex, termed Bm252RFP, with absorbance and fluorescence emission peaks at 600 nm and 620 nm, respectively. P252 at a concentration of 1 microM is shown to bind with about 50 microM Chlide in a positively cooperative reaction to form Bm252RFP under aerobic conditions and in the presence of light at 37 degrees C. Various parameters influencing this reaction have been optimized for efficient in vitro chemical synthesis of Bm252RFP. Circular dichroism spectra revealed that P252 is composed of a beta-structure (39.8% +/- 2.2%, based on 5 samples) with negligible contribution of alpha-helix structure. When bound to Chlide, the beta-structure content in the complex is reduced to 21.6% +/- 3.1% (n = 5). Since Chlide had no secondary structure, the observed reduction suggests significant conformational changes of P252 during the formation of Bm252RFP complex. Bm252RFP had antimicrobial activity against Escherichia coli, Serratia marcescens, B. thuringiensis, and Saccharomyces cerevisiae with 50% effective concentrations of 2.82, 2.94, 5.88 microM, and 21.6 microM, respectively. This is the first report ever to show clear, concrete binding characteristics of the midgut protein to form an RFP having significant antimicrobial activity.
Pandian
Ganesh N
GN
Laboratories of Applied Biosciences, Graduate School of Science and Technology, Niigata University, Niigata 950-2181, Japan.
Ishikawa
Toshiki
T
Togashi
Makoto
M
Shitomi
Yasuyuki
Y
Haginoya
Kohsuke
K
Yamamoto
Shuhei
S
Nishiumi
Tadayuki
T
Hori
Hidetaka
H
D000595:Amino Acid Sequence; D000818:Animals; D000890:Anti-Infective Agents / Q000378:metabolism*; D001413:Bacillus thuringiensis / Q000737:chemistry*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D012831:Bombyx / Q000737:chemistry*; D002735:Chlorophyllides / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000378:metabolism*; D005753:Gastric Mucosa / Q000737:chemistry*; D006460:Hemolysin Proteins / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000378:metabolism*; D013696:Temperature; D000097573:Red Fluorescent Protein
D000890:Anti-Infective Agents; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D002735:Chlorophyllides; D004731:Endotoxins; D006460:Hemolysin Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D000097573:Red Fluorescent Protein; C030956:insecticidal crystal protein, Bacillus Thuringiensis