title: Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library.
abstract: Plants express numerous calmodulin (CaM) isoforms that exhibit differential activation or inhibition of CaM-dependent enzymes in vitro; however, their specificities toward target enzyme/protein binding are uncertain. A random peptide library displaying a 22-mer peptide on a bacteriophage surface was constructed to screen peptides that specifically bind to plant CaM isoforms (soybean calmodulin (ScaM)-1 and SCaM-4 were used in this study) in a Ca2+-dependent manner. The deduced amino acid sequence analyses of the respective 80 phage clones that were independently isolated via affinity panning revealed that SCaM isoforms require distinct amino acid sequences for optimal binding. SCaM-1-binding peptides conform to a 1-5-10 ((FILVW)XXX(FILV) XXXX(FILVW)) motif (where X denotes any amino acid), whereas SCaM-4-binding peptide sequences conform to a 1-8-14 ((FILVW)XXXXXX(FAILVW)XXXXX(FILVW)) motif. These motifs are classified based on the positions of conserved hydrophobic residues. To examine their binding properties further, two representative peptides from each of the SCaM isoform-binding sequences were synthesized and analyzed via gel mobility shift assays, Trp fluorescent spectra analyses, and phosphodiesterase competitive inhibition experiments. The results of these studies suggest that SCaM isoforms possess different binding sequences for optimal target interaction, which therefore may provide a molecular basis for CaM isoform-specific function in plants. Furthermore, the isolated peptide sequences may serve not only as useful CaM-binding sequence references but also as potential reagents for studying CaM isoform-specific function in vivo.
authors.affiliation: Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju 660-701, Korea.
authors.forename: Ji Young
authors.initials: JY
authors.lastname: Choi
authors.forename: Sang Hyoung
authors.initials: SH
authors.lastname: Lee
authors.forename: Chan Young
authors.initials: CY
authors.lastname: Park
authors.forename: Won Do
authors.initials: WD
authors.lastname: Heo
authors.forename: Jong Cheol
authors.initials: JC
authors.lastname: Kim
authors.forename: Min Chul
authors.initials: MC
authors.lastname: Kim
authors.forename: Woo Sik
authors.initials: WS
authors.lastname: Chung
authors.forename: Byeong Cheol
authors.initials: BC
authors.lastname: Moon
authors.forename: Yong Hwa
authors.initials: YH
authors.lastname: Cheong
authors.forename: Cha Young
authors.initials: CY
authors.lastname: Kim
authors.forename: Jae Hyuk
authors.initials: JH
authors.lastname: Yoo
authors.forename: Ja Choon
authors.initials: JC
authors.lastname: Koo
authors.forename: Hyun Mi
authors.initials: HM
authors.lastname: Ok
authors.forename: Seung-Wook
authors.initials: SW
authors.lastname: Chi
authors.forename: Seong-Eon
authors.initials: SE
authors.lastname: Ryu
authors.forename: Sang Yeol
authors.initials: SY
authors.lastname: Lee
authors.forename: Chae Oh
authors.initials: CO
authors.lastname: Lim
authors.forename: Moo Je
authors.initials: MJ
authors.lastname: Cho
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001483:Base Sequence; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D002942:Circular Dichroism; D015723:Gene Library; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D011485:Protein Binding; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence
chemical_list: D000596:Amino Acids; D002147:Calmodulin; D010455:Peptides; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D010727:Phosphoric Diester Hydrolases