Structure And Functional Mechanism Of Two Membrane Proteins:G Protein Coupled Receptor And H~+-coupled Manganese Transporter

There are about30,000encoding genes in human genome, of which30%encode membrane proteins. Membrane proteins play a key role in various basic life processes with important physiological functions. They can act as "carrier" to fulfill the tansmembrane transportation of nutrient molecules, as "receptor" to mediate multiple intracellular signal transduction, and as "channel" to regulate the concentration of small molecules or ions in the intra-/extra-cellular environment. Therefore, study of structure and function of membrane protein may contribute significantly to the major scientific issues of life sciences. Membrane proteins have been found as crucial drug targets and associate directly with a wide range of human diseases, including neurodegenerative disorders, cardiovascular diseases and cancer. On the contrast to its importance and the high proportion in proteins, the structure and function of membrane proteins are poorly understood, which limits the development of basic life sciences. In this thesis, we focused on two membrane proteins:G protein coupled receptor (G2accumulation, G2A) and H+-coupled manganese transporter (MntH). Their functional mechanisms and structures were investigated by several biochemical techniques. The result will benefit to the understanding of the associated diseases and their drug development.G2A (from G2accumulation) receptor is a member of proton-sensing G protein coupled receptor (GPCR) family. It induces signal transduction events that regulate the cell cycle, proliferation, oncogenesis and immunity. The mechanism that G2A-mediated signal transduction is regulated by the extracellular pH remains unknown. Here, we first visualize the pH-dependent G2A distribution in living cells by a sortase A-mediated pulse labeling technique, the short-peptide tag-fused human G2A on human embryo kidney HEK293T cell surfaces was labeled with a small fluorescent dye. The labeled G2A was monitored under acidic and neutral pHs in situ by microscope. G2A internalization was inhibited under acidic condition, while the inhibition was not observed at neutral pH. Additionally, lysophosphatidylcholine (LPC), G2A ligand, antagonized its pH-dependent internalization. However, the internalized G2A redistributed onto cell surfaces when extracellular pH was changed back from neutral to acidic condition. The internalization of proton insensitive mutant (G2A-H174F) showed no difference at acidic and neutral extracellular environment, and the LPC insensitive mutant (G2A-R203A) did not show obvious inhibition on the constitutive internalization either. We thus concluded that the amount of G2A on the cell surface was controlled by suppressing the G2A internalization rate through the extracellular low pH, and this low pH-induced G2A accumulation on cell surface may explain the enhancement of G2A-mediated signal transduction at low pH.Natural resistance associated macrophage proteins (Nramp) have been characterized in mammals as divalent transition metal transporters. The transport mechanism remains unclear. In this thesis, multiple biochemical technologies combined with modern spectroscopic techniques were applied to investigate the transport activity, structure, and transport mechanism of the Nramp bacterial homolog, H+-coupled manganese transporter (MntH) from E.coli. A facile activity assay for an H+-coupled transporter using fluroescent probes was developed with MntH as a model. The transport activity (H+/Mn2+co-transport) of wild type MntH and various conserved mutants can be easily determined and compared via fluorescence intensity changes of the probe,5-(and-6)-carboxyfluorescein (5(6)-FAM). The approach provided a simple and convenient method of the determination of proton-coupled metal ion transporters. In order to investigate the MntH structure and function relationship, as well as its transport mechanism, the third transmembrane fragment, TM3, of MntH (MntH-TM3) containing several crucial, highly conserved residues was synthesized. The secondary-structure of the MntH-TM3in various membrane-mimicking environments were studied by Circular Dichroism (CD) and2D-NMR spectroscopies. The MntH-TM3peptide mainly adopts an a-helical structure in trifluoreoethanol (TFE), SDS and dimyristoyl phosphatidylglycerol liposome. The structure changes of MntH-TM3that induced by the metal ions or protons were observed. The key residues involved in Mn2+transport may contribute to the structure changes of MntH upon its specific interaction with manganese ions or protons, the results lay a base for understanding of MntH structure-function relationship, as well as exploration of other metal ion transporters.