서강대학교

초록/요약

The function of a vast majority of biomolecules depends on their specific, noncovalent interactions with other molecules. For example, proteins interact with other proteins, peptides, small molecules, nucleic acids and oligonucleotides, lipids, and polysaccharides. Electrospray ionization mass spectrometry (ESI-MS) is a proper for studying biomolecular noncovalent interactions. This study focused on the noncovalent interactions with a Zincfinger peptide by ESI-MS. Zinc ion is essential as a structural factor of zinc finger proteins which constitute one of the most common DNA binding motif. Zinc finger proteins are known to contain a Cys2His2 domain in common. In our study, an artificial zinc finger peptide which was custom-designed analyzed using an electrospray ionization ion trap mass spectrometer. On the basis of theses results, we determined the binding constants of interested peptides. In this study, the method for the determination of binding constants using reference peptide is presented. The intensity of a reference peptide with a known Kd value is compared with the interested peptide with a unknown Kd. On the basis of the change in the ion intensity of the reference peptide as a function of its concentration, we obtained a calibration curve. In addition, we compared our results with other binding constants which were obtained using the methods developed by Zenobi and De Pauw groups. It is well known that the “zinc-finger” family has a strong tendency to bind to its counterpart deoxyribonucleic acids (DNAs) through sequence-specific interactions of DNA. In therapeutics, there have been extensive efforts to design a new type of zinc-finger proteins and also to develop an analytical method which can facilitate the screening of the specific recognition of the zinc-finger proteins to DNA duplexes. We developed a method for the assaying of an assay that utilizes electrospray ionization mass spectrometry (ESI-MS) to rapidly determine the noncovalent binding of zinc-finger peptides with oligodeoxynucleotides and to assess their relative affinities and stoichiometries. In the present study, the main target of the screening is to find a zinc-finger protein which shows the optimal binding affinity to the target DNAs using ESI-MS. The DNA binding domains of two peptides (Sp11 and CF2-II) and two DNA sequences (one with a preference for Sp11, 5’-GGGGCGGGGC-3’/3’CCCCGCCCCG-5,’ and the other for CF2-II, 5’-GTATATATA-3’/3’-CATATATAT-5’) were selected as a model system to investigate the utility of ESI-MS in detecting sequence-specific peptide-DNA noncovalent complexes. The method uses a set of self-complementary oligodeoxynucleotides that differ in length (9-mer to 10-mer), motif (GC-rich or AT-rich), and sequence, and these were annealed to form duplexes. When a mixture of Sp11 and the DNA duplex of 5’-GGGGCGGGGC-3’/3’CCCCGCCCCG-5’ was electrosprayed, a noncovalent complex of the Sp11-the GC-rich DNA duplex was observed in high abundance. This indicates that a Sp11 peptide has a strong affinity to the DNA duplex of 5’-GGGGCGGGGC-3’/3’CCCCGCCCCG-5.’ The specificity of noncovalent complexes was confirmed by the fact that the noncovalent peak abundance was much higher when a Zn2+ is added. As a negative control, we further examined complexes of Sp11 and DNA duplex of 5’-GTATATATA-3’/3’-CATATATAT-5.’ Indeed, the abundance of Sp11 and the AT-rich DNA duplex was much weaker than that of Sp11 and the GC-rich DNA. Instead, CF2-II showed a strong binding affinity to the 5’-GTATATATA-3’/3’-CATATATAT-5’ duplex. In addition, we examined the Sp11 zinc-finger peptide binding specificity of each middle Sp1 DNA (5’-GGGGCGGGGC-3’) and assessed it before and after the alanine mutation (5’-GGGGAGGGGC-3’) and the ATA mutation (5’-GGGATAGGGC-3’) in the middle while maintaining the complementary sequences. The abundance of Sp11 and the mutated DNA duplex was much weaker than that of Sp11 and the consensus DNA. Electrospray ionization (ESI) mass spectrometry (MS) plays a key role for monitoring protein-ligand interactions. In addition, ESI-MS offers the opportunity to monitor protein-ligand interactions directly. In principle, therefore, ESI-MS represents a simple and novel approach for gaining insights into the binding stoichiometry and affinity of these assemblies. Unfortunately, the formation of nonspecific metal adducts during ESI can be a severe problem, often leading to binding levels that are dramatically higher than those in solution. Focusing on several zinc binding peptide as test systems, this work explores the suitability of different salts to serve as metal source. Despite their widespread use in previous ESI-MS studies, zinc chloride and zinc acetate induce extensive nonspecific adduction. In contrast, much lower levels of artifactual metal binding are observed in the presence of zinc tartrate. In the case of high and intermediate affinity proteins, the resulting ESI-MS data are in excellent agreement with the zinc binding behavior in solution. The situation is more challenging when studying proteins with very low affinities, but in the presence of tartrate qualitative information on protein-metal interactions can still be obtained. The beneficial effects of tartrate also extend to zinc binding experiments. This work does not directly explore the mechanism by which tartrate suppresses nonspecific metalation. The use of tartrate and possibly other weak chelators will greatly enhance the reliability of future ESI-MS studies on the interactions of proteins with divalent metal ions.