Abstract
Previous studies of isolated peptides corresponding to the wild-type signal sequence of the LamB protein of Escherichia coli and to several export-impaired mutants demonstrated that a high tendency to adopt an alpha-helical conformation in low dielectric environments was a property of functional sequences. We have now used nuclear magnetic resonance to establish further characteristics of the helical conformation of these signal peptides in a solvent mixture (50% trifluoroethanol, by volume, in water) which mimics the conformational distribution of these peptides in lipid vesicles. The interactions of signal sequences in vivo may depend on the location of the helix in the sequence, on the length of the helical segment, and on the stability of the helix. We find that the hydrophobic core has the most persistent helix conformation and that the stability of this helix correlates with in vivo function of different mutants of the LamB signal sequence. In the family of signal peptides studied here, the length of the helix required for function appears to be less rigidly restricted since a signal peptide from a functional pseudorevertant with 4 residues deleted from the hydrophobic core takes up helix as stably as wild type but incorporates fewer residues in the helix.
Highlights
Previous studies of isolated peptides corresponding to the wild-type signal sequence of the LamB protein of Escherichia coli and to several export-impaired mutants demonstrated that a high tendency to adopt an ahelical conformation in low dielectric environments was a property of functional sequences
Comparison of the results obtained on the mutants with those obtained on the wild type shows that the functional revertant peptides have approximately 12 residues in a helical conformation, whereas the nonfunctional deletion mutant has only 7 residues in a helix at 25 “C. the wild-type (100% exported) and revertant A78r2 (90% exported) mutant peptides both have stable helices remaining in the hydrophobic core at 50 “C, the nonfunctional deletion mutant A78 (0% exported) has no structure left at 50 “C. The semifunctional revertant A78rl (50% exported) shows some indication of a few turns of a helix, but no stable helical conformation is observed by NMR at 50 “C
The results presented above show that there is a correlation between degree of function of the signal peptide and both the length and stability of the helix formed in the hydrophobic core
Summary
Previous studies of isolated peptides corresponding to the wild-type signal sequence of the LamB protein of Escherichia coli and to several export-impaired mutants demonstrated that a high tendency to adopt an ahelical conformation in low dielectric environments was a property of functional sequences. Most proteins are targeted for export by an N-terminal signal sequence, which is subsequently cleaved, but the roles of the signal sequence are not well understood (for reviews see Rapoport, 1986; Briggs and Gierasch, 1986; Randall et al., 1987; Gierasch, 1989). It is not known what characteristics are required for proper function of signal sequences.
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