Selection of a carbohydrate-binding domain with a helix-loop-helix structure.
We obtained a novel carbohydrate-binding peptide having a helix-loop-helix scaffold from a random peptide library. The helix-loop-helix peptide library randomized at five amino acid residues was displayed on the major coat protein of a filamentous phage. Affinity selection with a ganglioside, Galbeta1-3GalNAcbeta1-4(Neu5Acalpha2-3)Galbeta1-4Glcbeta1-1'Cer (GM1), gave positive phage clones. Surface plasmon resonance spectroscopy showed that a corresponding 35-mer synthetic peptide had high affinity for GM1 with a dissociation constant of 0.24 microM. This peptide preferentially binds to GM1 rather than asialo GM1 and GM2, suggesting that a terminal galactose and sialic acid are required for the binding as for cholera toxin. Circular dichroism spectroscopic studies indicated that a helical structure is important for the affinity and specificity. Furthermore, alanine scanning at randomized positions showed that arginine and phenylalanine play an especially important role in the recognition of carbohydrates. Such a de novo helix-loop-helix peptide would be available for the design of carbohydrate-binding proteins.
sato@bio.keio.ac.jp.
Teruhiko
T
Matsubara
Mie
M
Iida
Takeshi
T
Tsumuraya
Ikuo
I
Fujii
Toshinori
T
Sato
D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D030562:Databases, Protein; D005732:Gangliosides / Q000737:chemistry* / Q000378:metabolism*; D018257:Helix-Loop-Helix Motifs; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020349:Surface Plasmon Resonance
D005732:Gangliosides; D010455:Peptides