Study On The Molecular Transport Mechanism Of Na⁺(Ca²⁺)/H⁺ Transporters

Membrane transporters exist widely in organisms,which undertake a series of important physiological activities such as material transport and information exchange.Since membrane proteins have low expression levels and are difficult to purify,it is a problem to study their function by obtaining protein crystals.In the early stage,the research team found that some proteins have a very low sequence identity?about 10%?,but have similar core regions.In this paper,the NhaD2 transporter with Na⁺,Li⁺transport function in Halomonas sp.Y2 and the ChaA transporter with Na⁺,K⁺and Ca²⁺transport functions in E.coli were used as research objects,the ion transport method was initially established based on evolutionary conservation analysis,protein purification,and protein conformation analysis,providing a basis for the study of molecular transport mechanisms and the construction of protein models.In this paper,the NhaD2 protein expression system and expression conditions were first optimized,and the protein was purified by Strep-tag II streptavidin-biotin system,qualitative and semi-quantitative analysis by SDS-PAGE and Western Blot.An optimal expression strain p-nhaD2-linker-strep-pEASY-KNabc and the optimal expression conditions were obtained.The purified NhaD2 protein was subjected to trypsin hydrolysis.The results showed that under the condition of pH 8,NhaD2 was hydrolyzed into two short bands,one about 25 kDa and the other about 20 kDa.Cross-linking pre-test showed that the proteins of the three groups of cysteine double mutant strain were expressed normally,providing the basis for the subsequent test.The E.coli ChaA protein belongs to the CaCA superfamily and has a transporter of Na⁺,K⁺,Ca²⁺.Through multi-sequence alignment and evolutionary conservation analysis of ChaA and known structural proteins in CaCA families,we found that they have low sequence identity?about 15%?,but have similar conserved regions,so they are suspected to have similar core regions and molecular transport mechanisms.Sixteen conserved sites in the conserved region of ChaA were subjected to site-directed mutagenesis,and Na⁺and K⁺salt tolerance experiments were performed.The results showed that 62,74,80,87,90,106,109,203,272,282 mutants completely lost Na⁺transport function;62,73,74,85,87,90,282,300 mutants completely lost K⁺transport ability.Based on the above analysis,the 62,74,85,87,90,282 mutants play a crucial role in the Na⁺,K⁺transport process.In order to further study the ChaA molecular transport mechanism,ChaA was subjected to secondary transmembrane structure and three-dimensional structure prediction.Both Swiss-model and Phyre results showed that ChaA has 11 alpha helices;further analysis of three-dimensional structure discovery,TMs 3-4,TMs 8-9 is located in the core region of ChaA protein structure;TMs 3 and TMs 8 are discontinuous transmembrane helices?TMs 3a,3b,3c,TMs 8a,8b?.Most of the sites that have important effects on Na⁺,K⁺transport obtained by mutation analysis are located on the conserved transmembrane region?TMs 3?.The E85 site is located near the"X"structure formed in the core region and plays a crucial role in ion transport.To further study the ChaA conformation,an expression strain p-chaA-linker-strep-pEASY-KNabc was obtained,and the initial purification was performed by the Strep-tag II system.SDS-PAGE and Western Blot were used for qualitative analysis.The target band was subjected to mass spectrometry and the results showed that ChaA protein was present at a position of approximately 30 kDa.Due to poor protein purity,molecular sieve purification experiments will be carried out to obtain high-purity proteins,which will prepare for the study of conformational changes of protein mutants.