Studies On RNA Chaperone Hfq Interacting With OxyS SRNA And Fabrication Of The Enrichment And Detection Methods For N-phosphorylated Proteins

sRNA(small RNA)involved post-transcription regulatory is an essential mediation in response to the environmental changes in bacteria.This mediation is realized by base-pairing with target mRNA in the sequence of initial code or Shine-Dalgarno(SD),resulting in inhibiting or facilitating gene expression.Protein Hfq(host factor for phage Qβ replication)is a RNA chaperone,which can facilitate the annealing of sRNA-mRNA and result in fast gene regulations.Exploration of how Hfq mediating sRNA-mRNA annealing is beneficial for understanding the function of Hfq as an RNA chaperone and the role in stress response.Besides,protein N-phosphorylation modifications have been found as a kind of important post-translation modification in recent years,involving bacterial stress response.Proteinphosphorylation leads to the structure and the electronic change of the limited domain in protein,leading flexible loop into rigid one.Moreover,the abnormal N-phosphorylated protein have been reported to be related to disease in eukaryotes.Hence,N-phosphorylated proteins are potential biomarkers.Designing sensitive methods to detect N-phosphorylated proteins will be benefit for disease diagnose.Moreover,the study of protein N-phosphorylation is still challenging due to the property of N-P bond including sensitivity to heat,being liable to acid condition.The phosphorylation modifications don’t occur at the same time in cell,leading to a low abundance of phosphorylated proteins,aiding the difficulty for the analysis of N-phosphorylated proteins.Constructing enrichment methods for N-phosphorylated proteins can expand the N-phosphorylated protein data,providing more information for the prediction of the function of N-phosphorylated proteins in both prokaryotes and eukaryotes.Hence,the main work of this thesis is to study how Hfq mediates the structure of sRNA and facilitates the annealing between sRNA with target mRNA.Besides,the methods of enrichment and detection for N-phosphorylated proteins are designed.The specific work is as follow:(1)Real-time monitoring the kinetic process of Hfq opening the stem-loop b of OxyS sRNA based on single-molecule fluorescence resonance energy transfer(smFRET).This process included two steps:the proximal face and distal face of Hfq were wrapped by OxyS sRNA,stopped at this state for about 4.6 s.Hfq then opened the stem-loop b.Also,experiments based on designed OxyS variants(OxySΔSLa、OxySΔSLb)demonstrated that sRNA structure also influenced the mediation of Hfq.The opening of stem-loop b depended on the stem-loop a of OxyS sRNA,illustrating that Hfq mediation was depended on the selfstructure of RNA.Besides,the functional domains of Hfq played different roles in the mediation.Hfq rim was vital for the opening of stem-loop b.while HfqCTD stabilized the Hfq-OxyS complex.Finally,the electrophoretic mobility shift assay(EMSA)showed that Hfq altering the structure of sRNA facilitated the annealing with target mRNA.(2)McsB was the protein kinase for arginine phosphorylation,firstly identified in Bacillus stearothermophilusus in 2009.We constructed a surface-enhanced Raman spectroscopy(SERS)assay to detect McsB.Peptide with positive charges(KRGGGGYIKIIKV,KRV)was selected as a phosphorylation substrate.Basic amino acids in the peptide can induce the aggregation of Au nanoparticles(AuNPs).However,once the arginine in th peptide was phosphoiylated by McsB,it increased electrostatic repulsion between AuNPs,further changing the controllable aggregation of AuNPs.After optimizing the concentration of peptide,incubation time,and pH,a good linear correlation was obtained with the concentration of McsB in the range from 1 nM to 100 nM,the detection limit of 46 pM under optimal conditions.This method demonstrated a high selectivity because the phosphoiylation induced by McsB is sequence-dependence.Besides,the signal of modified 4-MBA was amplified by AuNPs and further amplified once AuNPs were aggregated,promoting the sensitivity of this assay.(3)Construction of Fe3O4@SiO2@Au complex aimed to improve the immobilization of Antiphosphoarginine antibodies(Anti-pArg)on the surface of the nanoparticles,further facilitating the efficiency for the enrichment of N-phosphorylated protein.Compared with commercial protein A/G,the newly prepared Fe3O4@SiO2@Au compound demonstrates a higher load for antibodies(05 vs 0.7).Besides,Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)showed that the functional Fe3O4@SiO2@Au compound can carpture the self-phosphoiylated McsB(pMcsB)from the complicated system.Hence,the above research answers questions of how Hfq mediating the structure of sRNA,illustrating the function of Hfq as an RNA chaperone in stress response.Furthermore,fabricating enrichment and detection methods for N-phosphorylated proteins provide the basis for further protein N-phosphorylation study.