Conformational requirement of signal sequences functioning in yeast: circular dichroism and 1H nuclear magnetic resonance studies of synthetic peptides.
Recently, we have designed a series of simplified artificial signal sequences and have shown that a proline residue in the signal sequence plays an important role in the secretion of human lysozyme in yeast, presumably by altering the conformation of the signal sequence [Yamamoto, Y., Taniyama, Y., & Kikuchi, M. (1989) Biochemistry 28, 2728-2732]. To elucidate the conformational requirement of the signal sequence in more detail, functional and nonfunctional signal sequences connected to the N-terminal five residues of mature human lysozyme were chemically synthesized and their conformations in a lipophilic environment [aqueous trifluoroethanol (TFE) or sodium dodecyl sulfate micelles] analyzed by circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectroscopy. The helix content of the peptides, including functional (L8, CL10) and nonfunctional (L8PL, L8PG, L8PL2) signal sequences, was estimated from CD spectra to be 40-50% and 60-70%, respectively, indicating that the helical structure is more abundant in the nonfunctional signal sequences. Two-dimensional NMR analyses in 50% TFE/H2O revealed that each peptide adopted a helical conformation throughout the sequence except for a few residues at the N- and C-termini. Furthermore, H-D exchange experiments indicated that the helical structure of the C-terminal region of the functional signal sequences (L8 and CL10) was less stable than that of the nonfunctional signal sequences (L8PL and L8PL2). On the basis of these results, a model was developed in which the functional signal sequence is inserted in the membrane with a helical conformation and the C-terminal helix unraveled in an extended conformational form through an interaction with the signal peptidase.
Yamamoto
Y
Y
Protein Engineering Research Institute, Osaka, Japan.
Ohkubo
T
T
Kohara
A
A
Tanaka
T
T
Tanaka
T
T
Kikuchi
M
M
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002942:Circular Dichroism; D006801:Humans; D006859:Hydrogen; D009682:Magnetic Resonance Spectroscopy; D008823:Micelles; D008969:Molecular Sequence Data; D009113:Muramidase; D011487:Protein Conformation; D021382:Protein Sorting Signals / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate; D012995:Solubility; D014270:Trifluoroethanol; D015003:Yeasts / Q000032:analysis*
D008823:Micelles; D021382:Protein Sorting Signals; D012967:Sodium Dodecyl Sulfate; D014270:Trifluoroethanol; D006859:Hydrogen; D009113:Muramidase