Titration of protein transport activity by incremental changes in signal peptide hydrophobicity.
A systematic series of mutants has been generated which provides a means for titrating the dependence of protein transport activity on signal peptide hydrophobicity. These mutants involve replacement of the hydrophobic core segment of the Escherichia coli alkaline phosphatase signal peptide while maintaining the natural amino- and carboxyl-terminal segments and the overall length. The new core regions vary in composition from 10:0 to 0:10 in the ratio of alanine to leucine residues. Thus, a nonfunctional polyalanine-containing signal peptide is titrated with the more hydrophobic residue, leucine. Using precursor processing to quantify transport activity, we observe a clear, nonlinear dependence on hydrophobicity. At ratios of alanine to leucine of less than or equal to 8:2, the signal peptide is essentially nonfunctional; at ratios greater than or equal to 3:7, the signal peptide functions efficiently. The midpoint is between alanine to leucine ratios of 6:4 and 5:5. Signal peptides with hydrophobicity just below the midpoint show substantial, additional precursor processing over time while the others do not. The data are consistent with a simple model involving a two-state equilibrium between the untransported and transported species and a change in the delta G of -0.85 kcal/mol for every alanine to leucine conversion.
Doud
S K
SK
Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269.
Chou
M M
MM
Kendall
D A
DA
D000469:Alkaline Phosphatase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D001483:Base Sequence; D001692:Biological Transport; D002151:Calorimetry; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005798:Genes, Bacterial; D008715:Methionine / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015183:Restriction Mapping
D000596:Amino Acids; D009838:Oligodeoxyribonucleotides; D021382:Protein Sorting Signals; D008715:Methionine; D000469:Alkaline Phosphatase