Membrane-inserted conformation of transmembrane domain 4 of divalent-metal transporter.
Divalent-metal transporter 1 (DMT1) is involved in the intestinal iron absorption and in iron transport in the transferrin cycle. It transports metal ions at low pH ( approximately 5.5), but not at high pH (7.4), and the transport is a proton-coupled process. Previously it has been shown that transmembrane domain 4 (TM4) is crucial for the function of this protein. Here we provide the first direct experimental evidence for secondary-structural features and membrane insertions of a 24-residue peptide, corresponding to TM4 of DMT1 (DMTI-TM4), in various membrane-mimicking environments by the combined use of CD and NMR spectroscopies. The peptide mainly adopts an alpha-helical structure in trifluoroethanol, SDS and dodecylphosphocholine micelles, and dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol small unilamellar vesicles. It has been demonstrated from both Halpha secondary shifts and nuclear-Overhauser-enhancement (NOE) connectivities that the peptide is well folded into an alpha-helix from Val(8) to Lys(23) in SDS micelles at pH 4.0, whereas the N-terminus is highly flexible. The alpha-helical content estimated from NMR data is in agreement with that extracted from CD simulations. The highest helicity was observed in the anionic phospholipids [1,2-dimyristoyl- sn -glycero-3-[phospho-rac -(1-glycerol)]], indicating that electrostatic attraction is important for peptide binding and insertion into the membranes. The secondary-structural transition of the peptide occurred at pH 4.3 in the 2,2,2-trifluoroethanol (TFE) water mixed solvent, whereas at a higher pH value (5.6) in SDS micelles, DMT1-TM4 exhibited a more stable structure in SDS micelles than that in TFE in terms of changing the pH and temperature. PAGE did not show high-molecular-mass aggregates in SDS micelles. The position of the peptide relative to SDS micelles was probed by the effects of 5- and 16-doxylstearic acids on the intensities of the peptide proton resonances. The results showed that the majority of the peptide is inserted into the hydrophobic interior of SDS micelles, whereas the C-terminal residues are surface-exposed. The ability of DMT1-TM4 to assume transmembrane features may be crucial for its biological function in vivo.
Li
Hongyan
H
Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Kowloon, Hung Hom, Peoples' Republic of China.
Li
Fei
F
Sun
Hongzhe
H
Qian
Zhong Ming
ZM
D000595:Amino Acid Sequence; D000818:Animals; D027682:Cation Transport Proteins / Q000737:chemistry*; D002942:Circular Dichroism; D003902:Detergents / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D033862:Iron-Binding Proteins / Q000737:chemistry*; D008567:Membranes, Artificial; D008823:Micelles; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D014270:Trifluoroethanol / Q000737:chemistry; D000099236:Solute Carrier Family 11, Member 2
D027682:Cation Transport Proteins; D003902:Detergents; D033862:Iron-Binding Proteins; D008567:Membranes, Artificial; D008823:Micelles; D012967:Sodium Dodecyl Sulfate; D014270:Trifluoroethanol; D000099236:Solute Carrier Family 11, Member 2