Choi Ji Young, Lee Sang Hyoung, Park Chan Young, Heo Won Do, Kim Jong Cheol, Kim Min Chul, Chung Woo Sik, Moon Byeong Cheol, Cheong Yong Hwa, Kim Cha Young, Yoo Jae Hyuk, Koo Ja Choon, Ok Hyun Mi, Chi Seung-Wook, Ryu Seong-Eon, Lee Sang Yeol, Lim Chae Oh, Cho Moo Je
Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Chinju 660-701, Korea.
J Biol Chem. 2002 Jun 14;277(24):21630-8. doi: 10.1074/jbc.M110803200. Epub 2002 Mar 18.
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.
植物表达多种钙调蛋白（CaM）亚型，这些亚型在体外对CaM依赖性酶表现出不同程度的激活或抑制作用；然而，它们对靶酶/蛋白质结合的特异性尚不确定。构建了一个在噬菌体表面展示22肽的随机肽库，以筛选能以Ca2+依赖方式特异性结合植物CaM亚型（本研究使用大豆钙调蛋白（ScaM）-1和SCaM-4）的肽。通过亲和淘选独立分离出的80个噬菌体克隆的推导氨基酸序列分析表明，SCaM亚型需要不同的氨基酸序列才能实现最佳结合。与SCaM-1结合的肽符合1-5-10基序（(FILVW)XXX(FILV)XXXX(FILVW)，其中X表示任意氨基酸），而与SCaM-4结合的肽序列符合1-8-14基序（(FILVW)XXXXXX(FAILVW)XXXXX(FILVW)）。这些基序是根据保守疏水残基的位置分类的。为了进一步研究它们的结合特性，合成了来自每个SCaM亚型结合序列的两个代表性肽，并通过凝胶迁移率变动分析、色氨酸荧光光谱分析和磷酸二酯酶竞争性抑制实验进行分析。这些研究结果表明，SCaM亚型具有不同的结合序列以实现最佳的靶标相互作用，因此这可能为植物中CaM亚型特异性功能提供分子基础。此外，分离出的肽序列不仅可作为有用的CaM结合序列参考，还可作为研究体内CaM亚型特异性功能的潜在试剂。
