Idealized signal sequences: The role of charge and hydrophobicity in interacting with the secretion pathway

Date of Completion

January 1996

Keywords

Biology, Molecular|Biophysics, General

Degree

Ph.D.

Abstract

Exported prokaryotic proteins typically contain an amino-terminal extension called the signal peptide. Although signal sequences display little primary sequence homology, they share features which promote secretion through the same pathway. These characteristics include a net positive charge in the amino-teminus, a hydrophobic core with $\alpha$-helical propensity, and a cleavage region which contains small side chain residues in the $-$1 and $-$3 positions. The goals of this research are to further analyze the requirement for each property, to determine whether the different features operate at concurrent steps and, ultimately, to correlate the physical features with their functional role(s).^ The alkaline phosphatase signal sequence represents a typical signal peptide and serves as a prototype for designing mutants with simplified sequences that facilitate the delineation of the required physical features. A series of signal sequences varying in amino terminal charge and core region hydrophobicity provided a means of exploring the relative roles of these regions during transport in Escherichia coli. Signal peptides with highly hydrophobic core regions support a rapid rate of transport even in the presence of a negatively charged amino-terminus. These negatively-charged mutants have the same response as the wild-type signal sequence to treatments with SecA and pretonmotive force inhibitors. The negatively-charged mutants demonstrate a lower overall affinity for the transport pathway, evident in the decreased competition with co-expressed $\beta$-lactamase, and indicate that the two regions function in a concerted manner.^ Completely idealized signal sequences, which contain varying ratios of leucine and alanine residues but lack a helix-breaking residue, can be functional if the core region is sufficiently hydrophobic. Structural analyses of the corresponding chemically synthesized peptides by circular dichroism demonstrate solvent dependence and exceptionally stable $\alpha$-helix formation. Although leucines and alanines are predicted to have similar propensities for helix formation, considerably higher $\alpha$-helicity is observed in peptides which contain predominantly polyleucine cores, correlating with the especially efficient transport properties (Rusch et al., 1994). These studies provide the foundation for determining the relative affinities of these isolated signal peptides for purified components of the export pathway. ^

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