2D NMR and structural model for a mitochondrial signal peptide bound to a micelle

Abstract

The 19 amino acid signal peptide of rat liver aldehyde dehydrogenase, possessing a lysine substitution for an arginine and containing 3 extra amino acid residues at the C terminus, was studied by two-dimensional NMR in a dodecylphosphocholine micelle. In this membrane-like environment, the peptide contains two alpha-helical regions, both of which are amphiphilic, separated by a hinge region. The helix located closer to the C terminus is more stable than is the helix located near the N terminus. This suggests that the hydrophobic face of the C-terminal helix is buried within the hydrophobic region of the micelle. On the basis of these results a general model for protein translocation is presented in which the C-terminal amphiphilic helix of the signal region in the preprotein first binds to the mitochondrial membrane and then diffuses to the translocation receptor. The receptor then recognizes the N-terminal helix of the signal region, which is not anchored to the membrane. To explain how this signal peptide was imported into isolated mitochondria in the absence of energy or receptor protein [Pak, Y. K., & Weiner, H. (1990) J. Biol. Chem. 265, 14298-14307], a model for signal peptide translocation across a membrane barrier without the need for auxiliary membrane proteins is proposed. In this model the faces of the two helices fold upon each other, resulting in the mutual shielding of positively charged residues by the complementary hydrophilic face of the other amphiphilic helix.

Cardiolipin modulates the secondary structure of the presequence peptide of cytochrome oxidase subunit IV: a 2D 1H‐NMR study

“…From these data, it appears that the peptide has a helix-breakhelix motive, a motif that has been reported for other presequence peptides as well [9,11,13]. These conclusions extend a previous 2D NMR study on p25 [8], in which it was shown that p25 bound to DPC micelles has one helix at the N-terminus followed by a relatively structureless C-terminal half, of which the authors stated that the structure remained to be defined.…”

“…More detailed information on the conformation of several synthetic mitochondrial presequence peptides in membrane-like environments was obtained using high-resolution 2D NMR. The peptide, corresponding to the presequence of yeast cytochrome oxidase subunit IV, also denoted as p25 [8], as well as the presequence peptide of rat liver aldehyde dehydrogenase [9] and a mutant peptide lacking the flexible linker comprising residues 11-13 [10], were investigated in the presence of DPC micelles. The presequence peptide of Ft-ATPase fl-subunit [11] and the non-cleavable N-terminal targeting extensions of rat rhodanese, rat 3-oxalyl-CoA thiolase [12] and rat chaperonin 10 [13] were analysed in aqueous trifluorethanol solutions.…”

Two‐Dimensional 1H‐NMR of Transmembrane Peptides from Escherichia Coli Phosphatidylglycerophosphate Synthase in Micelles

“…The application of multidimensional NMR to non-water-soluble peptide segments from integral membrane proteins is nevertheless limited. Most investigations have been performed with N-terminal and C-terminal signal peptides (Bruch et al, 1989;Karslake et al, 1990;Rizo et al, 1993;Wang et al, 1993;Chupin et al, 1995;Yamamoto et a]., 1990;Wolff et al, 1994). The structures of gramicidin A, mellitin and alamethicin have been determined in bilayers of phosphatidylcholine (PtdCho) and micelles of dodecylphosphocholine or SDS (Inagaki et al, 1989;Ketchem et al, 1993;Franklin et al, 1994;Okada et al, 1994).…”

“…In addition, the very hydrophobic peptide segments usually will not dissolve in organic solvents or will not adopt the structures that are relevant to a cell membrane. For these reasons, detergent-micellar solutions are used to solubilize peptides where the hydrophobic core of the micelle is assumed to mimic that of a lipid bilayer (Inagaki et al, 1989;Karslake et al, 1990;Rizo et al, 1993;Franklin et al, 1994;Chupin et al, 1995;Gilbert and Baleja, 1995). Even the use of micelles in these studies may introduce non-biological properties, since the lipid packing around the peptides may differ from that in the biological membrane.…”

Studies on Protein Processing for Membrane‐Bound Spinach Leaf Mitochondrial Processing Peptidase Integrated into the Cytochrome bc1, Complex and the Soluble Rat Liver Matrix Mitochondrial Processing Peptidase

“…The mammalian precursors, aldehyde dehydrogenase (pALDH) 1361, rhodanese, and 3-oxoacyl-CoA thiolase (thiolase) [38] contain signal sequences in which the structure has been determined by twodimensional NMR. The presequence of pALDH forms a helixlinker-helix [36]. Linker-deleted ALDH consists of the presequence of ALDH with a deletion of the flexible linker, residues RGP.…”