Identification of calmodulin isoform-specific binding peptides from a phage-displayed random 22-mer peptide library.
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.
Choi
Ji Young
JY
Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju 660-701, Korea.
Lee
Sang Hyoung
SH
Park
Chan Young
CY
Heo
Won Do
WD
Kim
Jong Cheol
JC
Kim
Min Chul
MC
Chung
Woo Sik
WS
Moon
Byeong Cheol
BC
Cheong
Yong Hwa
YH
Kim
Cha Young
CY
Yoo
Jae Hyuk
JH
Koo
Ja Choon
JC
Ok
Hyun Mi
HM
Chi
Seung-Wook
SW
Ryu
Seong-Eon
SE
Lee
Sang Yeol
SY
Lim
Chae Oh
CO
Cho
Moo Je
MJ
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
D000596:Amino Acids; D002147:Calmodulin; D010455:Peptides; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D010727:Phosphoric Diester Hydrolases