C Deillon | PubMed Health

PubMed2001

Fusion proteins from artificial and natural structural modules.

The purpose of preparing fusion proteins from designed and natural sequences is mainly twofold; it aims at the stabilization of structure and at the modification of biological activity. Fusion with beta-galactosidase, for example, can increase the intracellular stability and DDT-degrading activity of an artificial DDT-binding peptide, and fusions with a leucine zipper produce mono- and bifunctional single-chain variable domain antibody fragments or homodimeric and heterodimeric DNA-binding proteins like an artificial homodimeric HIV-1 enhancer-binding protein with increased binding specificity and repressor activity. Of importance are also short leader sequences that mediate the translocation of proteins across the cytoplasmic and the nuclear membrane. An interesting by-product of the leucine zipper-mediated dimerization of an HIV-1 enhancer-binding protein was the synthesis and the structural as well as functional characterization of a retro-leucine zipper.

Amino Acid Sequence↗

PubMed2000

The retro-GCN4 leucine zipper sequence forms a stable three-dimensional structure.

The question of whether a protein whose natural sequence is inverted adopts a stable fold is still under debate. We have determined the 2. 1-A crystal structure of the retro-GCN4 leucine zipper. In contrast to the two-stranded helical coiled-coil GCN4 leucine zipper, the retro-leucine zipper formed a very stable, parallel four-helix bundle, which now lends itself to further structural and functional studies.

Amino Acid Sequence↗

PubMed1998

Synthesis, physicochemical characterization, and crystallization of a putative retro-coiled coil.

An artificial HIV enhancer-binding polypeptide has recently been dimerized by covalently linking it to the leucine zipper motif of the yeast transcriptional activator GCN4 (Liu N et al., 1997, Eur Biophys J 25:399-403). Although it seemed that the dimerization of this peptide could be best achieved by the use of the retro sequence of the leucine zipper, this approach was not implemented in the original construct. As the first step toward the synthesis of a basic region-retro leucine zipper HIV enhancer-binding fusion protein, we have now prepared the retro version of the leucine zipper (r-LZ35) and performed initial physicochemical characterization. Circular dichroism and sedimentation equilibrium studies showed that, at concentrations < 100 microM, the retro peptide was an unstructured monomer. At higher concentrations, however, the monomer was in equilibrium with a tetramer and, at 1 mM, the retro peptide was almost fully helical. N-terminal extension of the retro peptide by the tripeptide Cys-Gly-Gly resulted in a 38-residue polypeptide that could be covalently dimerized by forming a disulfide bond between two chains to give the peptide (r-LZ38)2. Even in the low micromolar concentration range peptide (r-LZ38)2 formed a stable, noncovalent, helical dimer as revealed by circular dichroism and sedimentation equilibrium in the presence and absence of guanidinium chloride. (r-LZ38)2 has been crystallized and X-ray structural analysis is under way. The disulfide-crosslinked retro-leucine zipper may lend itself to interesting protein structural studies, including protein design. The present work also highlights the structural and functional potential of retro proteins in general.

Amino Acid Sequence↗