Selection Of Cd²⁺-binding Peptides And The Imulation Of Peptides-Metal Ions

Metal ions play and participate important biological functions in broad life processes. It has become a major reserach area inclduing bioremediation of environmental heavy metal pollution, tumor therapy and bio-mining of precious and heavy metals by using metal-binding proteins/peptides with high affinity to metal ions and surface display technology in recent years. To obtain high-affinity metal binding peptides and the study about the interaction of binding peptides-heavy metal are the focus of current study and hot spots. Many studies has made good progress in this word by using various experimental tchniques and computer simulation techniques, but it is not enough.The purpose and contents of this study were to obtain peptides with high affinity for Cd2+ by using the immobilized metal affinity chromatography(IMAC) and the phage random dodecapeptide library selection; to analyse the characteristics of the obtained peptides by chemical and bioinformatics anlysis; to detect effects of the metal-binding peptides on the detoxification of heavy metal toxicity; to study the interaction of metal-binding proteins/peptides with heavy metal ions by computer simulation using MD and QM in the hope of providing information to obtain high-affinity metal-binding peptides and the study about the interaction of binding peptides-heavy metal.At first, the Cd2+ metal binding peptides were selected and enriched by the glycine(Gly) buffer elution from the filamentous phage dodecapeptide library by using IMAC selection. After amplification of the eluted phages and the preparation of phage single-strand DNA, six phage clones were randomly picked up and sequenced. The homology of the Cd2+-binding peptides were blasted by GeneBank Blast. After the amplification, purification and titering of the monoclonal phages, the relative affinity of the phages for the heavy metal ions(Cd2+) chelated resins were determined. After the amplification, infection of plaque incubation, E.coli paper-ring inhibition experiment was used to determine the detoxification abilities of Cd2+-binding peptides displayed phages for heavy metal ions(Cd2+,Ni2+) treated E.coli. The interaction of binding peptides-heavy metal were studide by computer simulation using DS and Gaussian03 software Package. To analyze the interaction between peptide and metal ions on non-key role as electrostatic and van der Waals forces by using MD model and CDOCKER model in DS; coordinated complex were studied by Gaussian03 software Package.The results of the study were as follows:The titering of the monoclonal phages increased after four rounds glycine (Gly) buffer elution. Six sequences were obtained, and no motif was found by amino acid sequecnce analysis, or no homologization was found by GeneBank Blast. There were rich histidines and many serine and no cysteines in the selected peptides. Metal ions are supposed to tend to interact with O, N and S in peptides. The stability of coordination nickel/cadmium complex is:S> N(imidazolyl)>o(hydrone)> O(carbonyl). The stability of coordination nickel/cadmium complex Ni2+>Cd2+, that was in agreement with Irwing-Williams series. The stability of coordination cadmium complex was as Cd-HH-Cd>HH-Cd.Main conclusion:1. Peptides displayed on the surface of phages with different affinities for Cd2+ were obtained by selection from the phage random dodecapeptide library with IMAC and phage display technology. There were rich histidines and many serine and no cysteines in these selected peptides.2. The selected Cd2+-binding peptides displayed phages also had affinities for Ni2+ by the analysis of the affinities of obtained phage clones for different IMACs, and the affinities for Cd2+ of these peptides were lower than those for Ni2+.3. The two Cd2+-binding peptides had effect on the detoxification of both Cd2+ and Ni2+, and the effect on the detoxification was in the order of Ni2+>Cd2+.4. Ab initio calculation results showed that the stability of coordination cadmium complex was as R2S/RSH/RS>N(imidazolyl).5. The stability of coordination nickel/cadmium cmplex was as Ni2+>Cd2+, that was in agreement with Irwing-Williams series. Metal-binding peptides were rich in histidine or cysteine, not all histidine (or cysteine) also participate in the formation of coordination compounds, that might be due to many factors such as peptide conformation.