Synthesis of alpha-helix-forming peptides by gene engineering methods and their characterization by circular dichroism spectra measurements.
Two kinds of peptides which were considered to form alpha-helices were designed and characterized. One was "alpha(3)-peptide' with 21 residues comprising three repeats of the seven-residue sequence Leu-Glu-Thr-Leu-Ala-Lys-Ala. This peptide appeared to be amphipathic due to a hydrophobic surface of Leu residues and a hydrophilic surface of Lys and Glu residues, thus forming a bundle structure. The other was "alpha(3)-GPRRG-alpha(3) peptide' with 47 residues in which two alpha(3)-peptides were connected by the five-residue sequence Gly-Pro-Arg-Arg-Gly. The genes encoding these peptides were fused to the adenylate kinase gene via a methionine codon. The resulting fused protein was expressed as an inclusion body, and the peptides were purified after cleavage with BrCN. The stability of the peptides in various buffers was then examined by measuring their circular dichroism spectra. The alpha(3)-peptide showed concentration-dependent stabilization of the alpha-helix. Sedimentation equilibrium ultracentrifugation indicated that it formed a bundle structure composed of four polypeptide chains, and a dimer intermediate during oligomerization was also detected by analytical gel-filtration. The stability of the alpha(3)-peptide was decreased by shifting the pH to 2 or 12, due to electrostatic repulsion of charged residues. Thus, the alpha(3)-peptide was stabilized by increasing the ionic strength, particularly in acidic or alkaline buffer, through the masking of the repulsion by high salt concentration. In buffer of neutral pH and a high salt concentration, the alpha(3)-peptide at high concentration formed visible aggregates, due possibly to the exposed hydrophobic surfaces of the alpha-helical bundles. On the other hand, alpha(3)-GPRRG-alpha(3) peptide did not show concentration-dependent reversible dissociation and association. It was shown to exist as a trimer even at low concentration, indicating very tight association of the alpha(3)-GPRRG-alpha(3) peptide. In contrast to the alpha(3)-peptide, the alpha(3)-GPRRG-alpha(3) peptide was very stable at various pH values and salt concentrations. This seemed to be due to increased hydrophobic interactions resulting from the increase in the number of seven-residue repeats from three to six, even though each group of three repeats was separated by a five-residue sequence.
Kojima
S
S
Institute for Biomolecular Science, Gakushuin University, Tokyo, Japan.
Kuriki
Y
Y
Sato
Y
Y
Arisaka
F
F
Kumagai
I
I
Takahashi
S
S
Miura
K
K
D000595:Amino Acid Sequence; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D005818:Genetic Engineering; D005822:Genetic Vectors; D006863:Hydrogen-Ion Concentration; D008961:Models, Structural; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010452:Peptide Biosynthesis; D010455:Peptides / Q000737:chemistry*; D017433:Protein Structure, Secondary
D009838:Oligodeoxyribonucleotides; D010455:Peptides