NMR solution structure and position of transportan in neutral phospholipid bicelles

Abstract

Transportan is a chimeric cell-penetrating peptide constructed from the peptides galanin and mastoparan, which has the ability to internalize living cells carrying a hydrophilic load. In this study, we have determined the NMR solution structure and investigated the position of transportan in neutral bicelles. The structure revealed a well-defined -helix in the C-terminal mastoparan part of the peptide and a weaker tendency to form an -helix in the N-terminal domain. The position of the peptide in relation to the membrane, as studied by adding paramagnetic probes, shows that the peptide lies parallel to, and in the head-group region of the membrane surface. This result is supported by amide proton secondary chemical shifts.

Bicelles as model membranes for solid‐ and solution‐state NMR studies of membrane peptides and proteins

“…Andersson and Mäler (112,113) recently investigated the structure and location of several peptides in fast-tumbling bicelles, including the 22 amino acid hormone motilin, two variants of the peptide penetratin (109,114), and transportan (108). More specifically, the structure of motilin has been investigated in zwitterionic and acidic bicelles, in which 30% of DMPC was replaced by DMPG (112).…”

“…Finally, the NMR solution structure of transportan in neutral bicelles has recently been investigated (108). Transportan is a chimeric cell-penetrating peptide constructed from the peptides galanin and mastoparan and it has the ability to translocate through biological membranes and to carry large hydrophilic load through the membranes.…”

“…The size of the peptides investigated varies from five amino acids for methionine-enkephalin (111) to 27 amino acids for the peptide transportan (108). Typically, the peptide structures have been determined by the use of two-dimensional 1 H NMR techniques.…”

“…The first approach has been used to investigate the position of the peptides motilin (113), penetratin (109), and transportan (108), as discussed in the previous section. Several paramagnetic probes have been used to look at different locations in the bicelles, namely Mn 2ϩ , to probe the bicelle surface (headgroup region), and phospholipids with a doxyl group at either the fifth or twelfth position in the fatty acid chain, to explore different depths in the bicelle interior.…”

“…It has been shown that the activity of some proteins and enzymes incorporated into micelles is not readily preserved (8 -10) but that the biological activity of the enzyme diacylglycerolkinase (DAGK) is preserved in bicelles (11,21,82). In addition, it has been shown that the position of a peptide in a micelle can be significantly different from its position in a phospholipid bilayer (108,109). Finally, the lipid composition of bicelles can be varied to better mimic biological membranes.…”