Secondary structure and position of the cell-penetrating peptide transportan in SDS micelles as determined by NMR.
Transportan is a 27-residue peptide (GWTLN SAGYL LGKIN LKALA ALAKK IL-amide) which has the ability to penetrate into living cells carrying a hydrophilic load. Transportan is a chimeric peptide constructed from the 12 N-terminal residues of galanin in the N-terminus with the 14-residue sequence of mastoparan in the C-terminus and a connecting lysine. Circular dichroism studies of transportan and mastoparan show that both peptides have close to random coil secondary structure in water. Sodium dodecyl sulfate (SDS) micelles induce 60% helix in transportan and 75% helix in mastoparan. The 600 MHz (1)H NMR studies of secondary structure in SDS micelles confirm the helix in mastoparan and show that in transportan the helix is localized to the mastoparan part. The less structured N-terminus of transportan has a secondary structure similar to that of the same sequence in galanin [Ohman, A., et al. (1998) Biochemistry 37, 9169-9178]. The position of mastoparan and transportan relative to the SDS micelle surface was studied by adding spin-labeled 5-doxyl- or 12-doxyl-stearic acid or Mn2+ to the peptide/micelle system. The combined results show that the peptides are for the most part buried in the SDS micelles. Only the C-terminal parts of both peptides and the central segment connecting the two parts of transportan are clearly surface exposed. For mastoparan, the secondary chemical shifts of the amide protons were found to vary periodically and display a pattern almost identical to those reported for mastoparan in phospholipid bicelles [Vold, R., et al. (1997) J. Biomol. NMR 9, 329-335], indicating similar structures and interactions in the two membrane-mimicking environments.
Lindberg
M
M
Department of Biochemistry and Biophysics, The Arrhenius Laboratories, Stockholm University, S-106 91 Stockholm, Sweden.
Jarvet
J
J
Langel
U
U
Gräslund
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001693:Biological Transport, Active; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D003497:Cyclic N-Oxides; D004337:Drug Carriers; D019004:Galanin / Q000737:chemistry; D036341:Intercellular Signaling Peptides and Proteins; D008823:Micelles; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D013113:Spin Labels; D014862:Wasp Venoms / Q000737:chemistry
D003497:Cyclic N-Oxides; D004337:Drug Carriers; D036341:Intercellular Signaling Peptides and Proteins; D008823:Micelles; D010455:Peptides; D011993:Recombinant Fusion Proteins; D013113:Spin Labels; D014862:Wasp Venoms; C110068:transportan; C037404:12-doxylstearic acid; C005072:5-doxylstearic acid; D012967:Sodium Dodecyl Sulfate; C037500:mastoparan; D019004:Galanin