Designed coiled-coil proteins: synthesis and spectroscopy of two 78-residue alpha-helical dimers.
Receptor-adhesive modular proteins are nongenetic proteins designed to contain ligand, spacer, coil, and linker modules and to interact strongly with integrins or other types of cell-surface receptors. We have designed, chemically synthesized, and characterized a 39-residue peptide chain having a 6-residue ligand module (Gly-Arg-Gly-Asp-Ser-Pro-) for adherence to Arg-Gly-Asp-binding integrin receptors, a 3-residue spacer module (-Gly-Tyr-Gly-) for flexibility, and a 30-residue coil module [-(Arg-Ile-Glu-Ala-Ile-Glu-Ala) 4-Arg-Cys-NH2] containing four 7-residue repeats for dimerization. This chain was designed to form a 78-residue noncovalent dimer (P39) by folding the coils of two chains into an alpha-helical coiled coil through hydrophobic interaction of eight pairs of Ile residues. Air oxidation of P39 gave P78, a 78-residue covalent dimer having a disulfide bridge linking its C termini. Raman spectroscopy indicated that both synthetic proteins have high alpha-helical content. Ultraviolet circular dichroic spectroscopy indicated that both dimers contain stable alpha-helical coiled coils. Its C-terminal disulfide bridge renders P78 significantly more stable than P39 to thermal denaturation or denaturation by urea. The coiled coil of P39 was 30% unfolded near 55 degrees C and half-unfolded in 8 M urea, while that of P78 was 30% unfolded only near 85 degrees C. These studies have demonstrated the feasibility of using these ligand, spacer, and coil modules to construct the designed coiled-coil proteins P39 and P78, a stage in the nanometric engineering of receptor-adhesive modular proteins.
Engel
M
M
Department of Chemistry, University of North Carolina, Chapel Hill 27599-3290.
Williams
R W
RW
Erickson
B W
BW
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011489:Protein Denaturation; D011506:Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D011956:Receptors, Cell Surface / Q000378:metabolism*; D013059:Spectrum Analysis, Raman; D013816:Thermodynamics
D007202:Indicators and Reagents; D046911:Macromolecular Substances; D011506:Proteins; D011956:Receptors, Cell Surface