Modulating Transmembrane α-Helix Interactions through pH-Sensitive Boundary Residues.

Changes in pH can alter the structure and activity of proteins and may be used by the cell to control molecular function. This coupling can also be used in non-native applications through the design of pH-sensitive biomolecules. For example, the pH (low) insertion peptide (pHLIP) can spontaneously insert into a lipid bilayer when the pH decreases. We have previously shown that the α-helicity and helix-helix interactions of the TM2 α-helix of the proteolipid protein (PLP) are sensitive to the local hydrophobicity at its C-terminus. Given that there is an ionizable residue (Glu-88) at the C-terminus of this transmembrane (TM) segment, we hypothesized that changing the ionization state of this residue through pH may alter the local hydrophobicity of the peptide enough to affect both its secondary structure and helix-helix interactions. To examine this phenomenon, we synthesized peptide analogues of the PLP TM2 α-helix (wild-type sequence (66)AFQYVIYGTASFFFLYGALLLAEGF(90)). Using circular dichroism and Förster resonance energy transfer in the membrane-mimetic detergent sodium dodecyl sulfate, we found that a decrease in pH increases both peptide α-helicity and the extent of self-association. This pH-dependent effect is due specifically to the presence of Glu-88 at the C-terminus. Additional experiments in which Phe-90 was mutated to residues of varying hydrophobicities indicated that the strength of this effect is dependent on the local hydrophobicity near Glu-88. Our results have implications for the design of TM peptide switches and improve our understanding of how membrane protein structure and activity can be regulated through local molecular environmental changes.

Route of uptake of palmitoylated encephalitogenic peptides of myelin proteolipid protein by antigen-presenting cells: importance of the type of bond between lipid chain and peptide and relevance to autoimmunity.

Previously, we have shown that thiopalmitoylation of peptides of myelin proteolipid protein, as occurs naturally in vivo, increases their ability to induce experimental autoimmune encephalomyelitis, the animal model of multiple sclerosis, and skews the autoimmune response toward a CD4(+)-mediated response. In contrast, the same peptide, when synthesized with a stable amide bond between peptide and lipid, inhibits experimental autoimmune encephalomyelitis and skews the response toward a CD8(+) response. The aim of the current study was to determine the mechanisms responsible for these observations. We show that proteolipid protein lipopeptides, when synthesized with a thioester bond between the lipid and the peptide, are taken up into APCs via an actin-independent endocytic route, the thioester bond is cleaved in the endosome, and the peptide is subsequently displayed on the surface of the APC in the context of MHC class II. The same peptide, when synthesized with the lipid attached via a stable amide bond, rapidly enters into the cytoplasm of the APC and forms micelles; however, the bond between peptide and lipid is not cleaved, and the micelles travel via the endoplasmic reticulum to complex with MHC class I. These findings have implications for vaccine development and for the development of MHC class II-restricted autoimmune diseases, as many human autoantigens thus far identified are thioacylated.

Synthesis and secondary structure of loop 4 of myelin proteolipid protein: effect of a point mutation found in Pelizaeus-Merzbacher disease.

To study the effects of a point mutation found in Pelizaeus-Merzbacher disease (PMD) on the physicochemical and structural properties of the extracellular loop 4 of the myelin proteolipid protein (PLP), we synthesized the peptide PLP(181-230)Pro215 and one mutant PLP(181-230)Ser215 with regioselective formation of the two disulphide bridges Cys200-Cys219 and Cys183-Cys227. As conventional amino acid building blocks failed to give crude peptides of good quality we had to optimize the synthesis by introducing pseudoproline dipeptide building blocks during the peptide elongation. In peptide Pro215 the first bridge Cys200-Cys219 was obtained after air oxidation, but in peptide Ser215 because of aggregation, dimethyl sulfoxide (DMSO) oxidation had to be used. The second bridge Cys183-Cys227 was obtained by iodine oxidation of both Cys (acetamidomethyl, Acm)-protected peptides. The secondary structures of the parent and mutant loops were analysed by circular dichroism (CD) in the presence of trifluoroethanol (TFE) and sodium dodecyl sulphate (SDS) as a membrane mimetic. Analysis of the spectra showed that the content of alpha-helix and beta-sheet varied differently for both peptides in TFE and SDS solutions, demonstrating the sensitivity of their conformation to the environment and the differences in their secondary structure. The ability of both peptides to insert into the SDS micelles was assayed by intrinsic tryptophan fluorescence.