Membrane-inserted Structures And Orientation Of The Transmembrane Segments Of Slc11a1

Solute carrier family 11 member 1 is an integral membrane protein which plays an important role in the biological functions. It belongs to Slc11 family which is highly conserved from bacteria to human and is one of a few genes that demonstrated the parasitic resistance at the molecular level. Mammalian Slc11a1 is involved in defense against intracellular pathogens such as Salmonella typhimurium, Mycobacterium bovis and Leishmania donovan. For human, NRAMP1 is associated with the diseases such as tuberculosis, leprosy and rheumatoid arthritis, etc. Understanding the functional mechanism of Slc11a1 may be helpful to study the pathogenesis for these diseases. As a proton-coupled divalent metal ion transporter, Slc11a1 can transport essential metal ions for life including Fe²⁺ and Mn²⁺. People have a certain understanding for biological function of Slc11a1, but are still not clear for its functional mechanism. Some of the transmembrane segments of Slc11a1 may be closely related to the transfer activity of the protein, so the study of the peptides that are crucial for the function of the transporter is helpful to understand the structure and function of the integral protein.Recently, it has become a widely used method in the structural studies of membrane proteins to select function-relating peptides from protein as model peptides and characterize them in the membrane-mimetic environments. The study on the model peptides corresponding to isolated transmembrane domains have been proved to be very useful in providing qualitative information for integral proteins. Both disease-causing mutations in Slc11a1 occurring at glycine 169 (G169D) and Slc11a2 occurring at glycine 185 (G185R) locate within TM4. It has been found by some electrophysiological studies of Slc11 homolog that the replacement of the conservative residues in TM3 severely affected or abrogated the transport function. TM2 is an extremely hydrophobic transmembrane segment and close to TM3. These means that each of the three transmembrane segments may play a specific role in the proton-coupled divalent metal ion transport of the homolog. Therefore, in this thesis, we studied the membrane-inserted structures and orientation of the peptides, including TM2, TM3/TM3-E139A and TM4/TM4-G169D of Slc11a1 in various membrane-mimetic environments, such as SDS micelles, DMPC and DMPG vesicles, using CD spectroscopy, DSC and Fluorescence spectroscopy.First, the second structures of the peptides in model membranes and various pH values were studied using CD. The TM2 and TM4 related peptides adopt predominantlyα-helical conformations in all membrane-mimetic environments used in the study, while TM3 related peptides displayα-helical conformations in DMPG-containing vesicles and SDS micelles but basically are unstructured in DMPC. At all pH values (especially at pH 5.5 and 7) and membrane environments, TM3 is basically less structured than TM2 and TM4. The secondary structures of TM3 in various membranes show evident pH-dependence. The pH sensitivity of TM3 folding is associated to the protonation/deprotonation process of the residue Glu139. The deprotonation of Glu139 is unfavorable for theα-helical folding of TM3. The E139A substitution of TM3 increases theα-helix content and decreases the pH dependence of the helical folding. In comparison with TM3, TM2 and TM4 displays less pH sensitivity of the helical folding. The G169D mutation of TM4 enhances the pH sensitivity of the secondary structure. Moreover, TM4 may be self-associated in the model membranes containing anionic headgroups and more helicity of TM2 in the lipid mixtures at pH 5.5 could be explained for the self-association of the peptide.Secondly, we studied the influences of the peptides on the phase transition behaviors of the model membranes using DSC measurement. We analyzed the peptide/membrane interactions through the changes in the calorimetric parameters of the phase transition of the model membranes in the presence and absence of the peptides. The DSC results showed that TM2, TM3 and TM4 are embedded in the lipid membranes. In pure DMPG and DMPC lipid bilayers, TM4 has more extensive perturbation to the hydrophobic chains of the lipid membranes and is embedded in the lipid bilayers more deeply than TM3. Moreover, the interactions of TM4 with membranes are less affected by pH, whereas the abnormality was observed at pH 5.5. The results studied in DMPG/DMPC lipid mixture also showed that TM3 has less extensive perturbation to the hydrocarbon chains of the lipids than both TM2 and TM4. The phase transition behaviors of the lipid mixture incorporated with peptides are more similar to those of DMPC lipid bilayers in the presence of peptides. The DSC result that the perturbation of TM2 to DMPG/DMPC mixture is less extensive at pH 5.5 than at pH 4 and 7 supports the suggestion that TM2 is self-associated in the mixed membrane at pH 5.5.Finally, we used the quenching constants (Ksv) and the wavelengths of the maximum Trp emission (λmax) obtained by fluorescence measurements to analyze the orientation and location of the peptides in model membranes further. The results showed that TM2 and TM4 have similar topology in membranes and they are inserted more deeply than TM3, especially at pH 5.5 and 7. In addition, the locations of the two peptides are nearly not affected by pH. In contrast, the position of TM3 in membranes is adjusted by pH, more deeply at acidic solution, but less deeply at alkalescent solution. The pH dependence of TM3 is attributed to the protonation/deprotonation of Glu139 residue in the peptide. The pH sensitivity of the topological structure of TM3 in membranes was changed by the E139A substitution, implying that Glu139 plays an important role in the topological structure of TM3. The topological structure of TM4 is hardly affected by the G169D mutation which only increases the pH sensitivity of the Trp microenvironment.We found in the study that the property of lipid headgroups strongly influences the secondary structure, interaction between peptide and membrane and orientation of the peptides. The transmembrane peptides have more helicity in SDS micelles and DMPG-containing vesicles than in zwitterionic DMPC vesicles, implying that the anionic lipid headgroups are favorable to induce anα-helical conformation. The helical stabilization of the TM peptides incorporated with anionic lipid vesicles or SDS micelles could be attributed to the electrostatic interactions between positively charged flanking residues at the N-termini of the peptides and anionic headgroups of lipids. TM3 is embedded in DMPC vesicles less deeply than in DMPG-containing lipid membranes. The stability of the DMPG/DMPC (1:2) mixed vesicles is more similar to that of DMPC vesicles in the presence of peptide, whereas the packing of the mixed vesicles is more similar to that of DMPG vesicles.