Function Of Ribosomal Proteins In Quantum Dots Biosynthesis And Biomimetic Synthesis

As an important semiconductor nanomaterial,quantum dots（QDs）are widely used in many fields such as medicine,biology,and physics,due to the unique electrical,optical and magnetic properties.QDs used to be synthesized by chemical synthesis.With the advancement of science and technology,the concept of"green chemistry"for the synthesis of QDs has emerged.QDs can be biosynthesized by using the abundant biological resources in the natural world under mild conditions.Biosynthesis provides a practical solution for the green synthesis of QDs.In the previous research,our group proposed a method to synthesize quantum in Saccharomyces cerevisiae cells by using the"temporally-spatially coupling strategy",i.e.,CdSe QDs were synthesized in S.cerevisiae cells by cleverly coupling two unrelated biochemical reaction pathways（selenium metabolism and cadmium detoxification）in time and space.However,the mechanism of quantum dot biosynthesis has not been well figured out,and there are still some difficulties in regulating the biosynthesis of QDs.In addition,the biosynthesis of Ag₂Se QDs has not been reported.Further effort is required to make it clear whether ribosomal protein can regulate the synthesis of Ag₂Se QDs.Based on the previous research on the synthesis of CdSe QDs by S.cerevisiae,in this paper,the protein wrapped on the surface of CdSe QDs is identified to determine whether the ribosomal protein involved in the synthesis of CdSe QDs.The role of ribosomal protein in the synthesis of QDs is explored.The biosynthesis method of Ag₂Se QDs is proposed.This work provides a new perspective for realizing the regulation of the synthesis of QDs.The main research results are as follows:1.The proteins wrapped on the surface of CdSe QDs were identified by liquid chromatography-tandem mass spectrometry（LC-MS/MS）,and 79 ribosomal proteins were detected.By comparing the ability of 23 ribosomal protein-deficient strains to synthesize CdSe QDs with wild-type,it was found that the fluorescence intensity of CdSe QDs synthesized by three types of ribosomal protein-deficient strains（Δrps22a,Δrpl14b,Δrpl20a）was significantly weaker.By increasing the content of S.cerevisiae ribosomal proteins RPS22A,RPL14B and RPL20A,the ability of S.cerevisiae to synthesize CdSe QDs were increased by 2-3 times.It was confirmed that the nanomaterials synthesized by the overexpression strains P_GAL1-RPS22A,P_GAL1-RPL14B and P_GAL1-RPL20A are CdSe QDs by using QDs characterization techniques such as transmission electron microscopy（TEM）、energy dispersive X-ray spectroscopy（EDX）.It showed that these ribosomal proteins are involved in the synthesis of CdSe QDs.2.The expression of RPL14B gene during the selenium enrichment stage and crystal formation stage of the CdSe quantum dot synthesis process was detected by using fluorescence quantitative PCR（q RT-PCR）.The results showed that the expression level did not change significantly during the selenium enrichment stage,while it increased by 2.4 times during the dot crystal formation stage,indicating that the RPL14B protein mainly functions in the quantum dot crystal formation stage.By comparing the ability of the point mutant strain(P_GAL1-RPL14B-C91S)and the strain P_GAL1-RPL14B to synthesize CdSe QDs,it was revealed that the ability of synthesizing CdSe QDs was significantly reduced after the 91st Cys point mutation,indicating that the 91st Cys plays an important role in the synthesis of CdSe QDs by S.cerevisiae.3.The role of ribosomal protein was further explored in two kinds of CdSe QDs biomimetic synthesis systems.Isothermal Titration Calorimetry（ITC）experiments confirmed that the GVCEKW sequence on the ribosomal protein RPL14B can bind to Cd²⁺,with a K_d value of 6.43±0.0749μM,suggesting that RPL14B contains a functional region that binds to Cd²⁺ions.In a system with only Se source,reducing agent and Cd source,the CdSe QDs can be detected after adding RPL14B protein,while CdSe QDs was not detected without adding RPL14B protein,indicating that RPL14B can be used as a template to synthesize CdSe QDs.4.Using P_GAL1-RPL14B as the starting strain,it was discovered that near-infrared QDs can be synthesized in living cells.Fourier transform infrared（FTIR）spectroscopy showed that there are two absorption bands at 1650 cm^-1 and 1544 cm^-1,indicating that the Ag₂Se QDs are coated with proteins on the surface.The results of LC-MS/MS showed that there were 411 kinds of proteins,concluded 67 ribosomal proteins.The ability of P_GAL1-RPL14B and P_GAL1-RPL20A strains to synthesize Ag₂Se QDs was 1-2times higher than that of wild-type strains.This result suggested that it is universal to regulate biosynthetic QDs by regulating ribosomal proteins.In summary,in this work,it was proved that ribosomal protein is involved in the synthesis of CdSe QDs,and the regulatory role of ribosomal proteins in biosynthesis was confirmed by using an in vitro biomimetic synthesis system.In addition,Ag₂Se QDs emitting near infrared second region fluorescence were synthesized by using eukaryotic cells for the first time.It was proved that ribosomal proteins can regulate the synthesis of QDs by regulating S.cerevisiae.The mechanism of biosynthesis of QDs was explained from a new perspective,which provides a new regulatory strategy for the biosynthesis and biomimetic synthesis of QDs.