Signal Sequences: Roles and Interactions by Biophysical Methods

Abstract

Signal sequences are essential for the efficient and selective targeting of nascent protein chains either to the endoplasmic reticulum, in eukaryotes, or to the cytoplasmic membrane, in prokaryotes (for a review, see Gierasch, 1989). Furthermore, signal sequences play a central, although poorly understood, role in the translocation of polypeptide chains across membranes. Despite their ability to perform multiple, common functions, signal sequences lack primary structural homology. Instead, they share several general properties (von Heijne, 1985): (1) an amino-terminal region with a net positive charge; (2) a hydrophobic core of about ten residues; (3) a locus six to eight residues preceding the cleavage site that often contains a prolyl or glycyl residue and has a relatively high predicted turn tendency; and (4) a motif at the cleavage site--AXA in prokaryotes or small-largesmall in eukaryotes. These shared features and the finding that signal sequences are often transportable from one secreted protein to another with retention of function invite the study of isolated signal peptides using physical methods that may reveal the conformational and interactive propensities of these intriguing sequences. This information then can help to elucidate the likely roles of signal sequences in the export pathway. Also, the isolated signal peptides can serve as probes for the interactions of the signal sequence with proteins of the export pathway. By comparing properties of export-competent signal peptides with those of peptides corresponding to export-defective mutants, we have found that all functional signal peptides have both a capacity to form an α helix in membrane environments and an ability to spontaneously insert into the acyl chain region of a membrane (Briggs & Gierasch, 1984; Briggs et al., 1985; McKnight et al.,1989; Hoyt & Gierasch, 1991a, 1991b). As principal methods, we have used circular dichroism (CD) and nuclear magnetic resonance (NMR) to determine the conformational behavior and fluorescence spectroscopy to analyze membrane insertion. Recently, we have developed a signal peptide of enhanced water solubility in order to apply the method of transferred nuclear Overhauser enhancements (trNOEs) to determine directly the conformation of a signal peptide in a lipid bilayer. We find from these studies and our fluorescence work that the hydrophobic core of the signal peptide resides well-embedded in the acyl chain region of the bilayer in a stable α helix, while the N-terminal region associates with the surface. The C-terminal segment adopts a less stable α helix and appears to spend some time at the interface. Fluorescence quenching results on the LamB signal peptide do not support a stable transmembrane arrangement;. work is in progress to establish whether this behavior is general and what the influence is of additional residues past the cleavage site.