Analysis Of Streptomyces Coelicolor Membrane Proteome And Characteration Of Several Lipoproteins

Membrane is an important part of prokaryote cells, and its functions involved in energy conversion, material transportation and information transmission. It is significative for us to understand functions of membrane and mechanisms of transmembrane transport.In this study, we optimized BNE and hrCNE for S. coelicolor membrane proteins and then characterized the protein complexes by using a combination of native/native and native/SDS 2D gel electrophoresis. Nucleic acids interference is an main influencing factor for native electrophoresis. In order to remove nucleic acids from membrane protein samples, we have used many methods. The results showed that ultracentrifugation is effective to remove nucleic acids. After removal of the nucleic acids, the membrane proteins separate well on native gels. The maximum sample load of BNE, hrCNE-DDM, and hrCNE-Triton was compared. We found that the optimal protein loads for all three gels were 80-100 μg per lane.A total of 77 proteins were identified by MS/MS analysis. Among these, 61 proteins were detected in 2D gels obtained using Triton X-100, and 48 proteins were detected in 2D gels obtained using DDM, and other proteins were detected in 2D native/native gels. In terms of physiological functions and polymerization, these proteins were classified. 15 protein complexes containing 20 proteins were identified. Several membrane proteins have been identified that are responsible for transmembrane transport. For example, BldKB(SCO5113) and OBP(SCO5477) are responsible for the transportation of oligopeptides, and ABP(SCO2008) is responsible for the transportation of amino acids. OBP and BldKB are the two main proteins observed in the S. coelicolor membrane fractions, and the two proteins are the members of the oligopeptide permease component A(OppA) family. The Opp complexes belong to the group of ATP-binding cassette transporters and play important roles in salvaging extracellular peptides and nutrients, as well as in transporting signaling molecules. OBP and BldKB formed complexes in which the stoichiometry of the subunits differed. There might be three oligomeric states for OBP and BldKB—monomer, dimer, and tetramer. The polymer of OBP and BldKB may be an adjustment mechanism for transportation of oligopeptides.S. coelicolorencodes 223 putative lipoproteins, and 40% ofthese lipoproteins are substrate binding proteins(SBP) for ABCtransporters. These substrate binding proteins are responsible for the transportation ofcarbohydrate, amino acids, oligopeptides, nucleosides and iron. We identified 17 substrate binding proteins in membrane fractions. A Blast search revealed that SCO4884 and SCO4885 have a PBP1_BmpA_PnrA_like conserved domain, and the two proteins might be involved in the uptake of purine nucleosides. In this study, the genes SCO4884 and SCO4885 were cloned into pCOLADuet-1 and overexpressed in Escherichia coli BL21. According to the results of hrCNE, molecular sieve and particle sizedetermination, we suggest that SCO4884 and SCO4885 exist as monomers in solution. The binding of various nucleosides to SCO4884 and SCO4885 was analyzed using isothermal titration calorimetry. The results showed that the two proteins have affinity for adenosine, deoxyadenosine, cytidine, deoxycytidine and uridine. No binding to guanosine, deoxyguanosine and thymidine was detected.Actinorhodin is an type II polyketide. The synthesis of carbon chains was catalyzed by polyketide synthase(PKS). Previous studies have shown that enzymes about polyketide synthesis have interactions, and these enzymes may be located in different locations in the cells. For example, SCO5074 is associatedwith cell wall. SCO5076, SCO5083 and SCO5084 are co-localised with the membrane. SCO5087 and SCO5088 may be also associated with membrane, and the two proteins form a membrane complex(ActKSαβ).The genes SCO5086, SCO5087, SCO5088, SCO5089, SCO5090 were cloned into pSJ7 and overexpressed in E. coliBL21. After purification, antibodies were prepared by immunizing the rabbits. It provides a profitable tool to identify genes function and protein interactions, and cellular localization for polyketide synthase.