Identification Of Apocalmodulin-targeting Peptides By Phage Display

Calmodulin （CaM） is widely distributed in nature and is ubiquitous in eukaryoticcells. The overall shape of CaM is often referred to as a “dumbbell”, there are ana-helix in each of the four domains, which consist the “EF hand” Ca²⁺-binding-motif.Calcium-CaM binds to taget proteins and alters their function in such a way as totransducer the Ca²⁺signal. It transmits the Ca²⁺signal by binding to and activatingnumerous enzymes central to cellular regulation.Although much of the research focus has been on the Ca²⁺-bound form, cellularCa²⁺-binding proteins such as CaM can exist primarily in two states, the other beingeffectively Ca²⁺free. Calcuium-CaM binds to target proteins and has other specificeffects. Apocalmoludlin has roles in the cell that apparently do not require the abilityto bind Ca²⁺at all, and these roles appear to be essential for life. Apocalmodulindiffers from Ca²⁺-CaM in its structure. Its way to binds target proteins different,utilizes different binding motifs, such as the noncontiguous binding sites and IQ motif.And there is lots of kinds binding ways potentially await discovery. The ApoCaM canbind to several group proteins, including actin-binding proteins, enzymes, as well ascytoskeletal and membrane proteins which include ion channels and receptors. Muchof the cellular CaM is bound in a Ca²⁺-independent manner to membrane structureswithin the cell, and the proportion bound changes with cell growth and density,suggesting it may be a different form. Although much of the research focus has beenon the roles of Ca²⁺-CaM, the roles of ApoCaM are equally vital but less wellunderstood, especially the home apocalmodulin due to research mostly investigateplant calmodulin.In order to study how the homo ApoCaM the combination interacts with thedownstream proteins, our research constructed homo Calmodulin gene and pET-28（+）recombinant plasmid and expressed in E. coli of BLP strains firstly. Ion chromatography, hydrophobic interaction chromatography, gel chromatography wereused to purify ApoCaM protein. Then, high-throughput screening phage displaypeptide library screening to study protein-protein interactions. After four roundpanning with ApoCaM as the target protein, the selected phage affinity with apoCaMverified with ELISA. Then the apoCaM affinity phage single-stranded DNA wereexacted and sequenced, then four ApoCaM affinity small peptide sequences have beengotten. Using the NCBI website BLAST the sequences, there have been found severalhomo proteins.Among these proteins, some were know to have interactions with CaM,including pleckstrin and neuron plexin, however, the exact affinity isn’t clear. At thesame time, when put the sequence in the Ca²⁺-CaM binding capacity website it showsno force, suggesting that this affinity may be a new mode. Thus, we need to identifythis affinity interaction. Firstly, using solid-phase peptide synthesis methodsynthesized three ApoCaM of small peptides, and purified them by HPLC analysisand preparation. The mass spectrometry molecular weight consists with the theoreticalprediction. Meanwhile, the15N mark ApoCaM has being prepared. NMR is one of thetwo most convincing ways to study of protein structure. Using the NMR, it may find apossible solution of interaction. In this thesis, the experiment divided into two groups,one is short peptides added to ApoCaM, and another is without the peptide as acontrol. The1H-15N HSQC NMR experiments was performed, and we got the2D-NMR spectra of results. By comparing the spectra between the two groups, it canclearly find large displacement of amino acid sites. A significant change in sitesincluding GLY98, ILE¹⁰⁰, ASN¹³⁷, ASP64, LEU¹¹⁶, ILE¹³⁰, ALA⁵⁷, and the extent of siteshift point was measured.In summary, this work using phage display peptide library screening to find thesmall peptides affinity to ApoCaM, and the role and affinity peptide and protein weredetected by NMR methods. Several sites significant shift were found, which proveaffinity small peptides play a role in the apoCaM structural changes. This result couldbe an explanation for the interactions between the ApoCaM and downstream targetproteins, and the affinity pattern. The interesting found describe here show anotherway that the ubiquitous protein CaM especially the apoCaM interact with target peptide.