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

Abstract

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 Siophys 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 <lo0 pM, the refro peptide was an unstructured monomer. At higher concentrations, however, the monomer was in equilibrium with a tetramer and, at 1 mM, the refro peptide was almost fully helical. N-terminal extension of the refro 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)*. 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 refro-leucine zipper may lend itself to interesting protein structural studies, including protein design. The present work also highlights the structural and functional potential of retru proteins in general.Keywords: circular dichroism; coiled coil; retru-peptide; sedimentation equilibrium An artificial 42-residue HN-1 enhancer-binding polypeptide, R42, based on the X-ray structure of the repressor-operator complex of bacteriophage 434 (Anderson et al., 1987;Aggarwal et al., 1988), was able to suppress HIV enhancer-controlled transcription in HeLa cell nuclear extracts (Hehlgans et al., 1993;Stadler et al., 1995;Caderas, 1997;Liu et al., 1997) and in living cells (Caderas, 1997). R42 is monomeric, but most natural regulatory DNA-binding proteins are dimers or tetramers. Many of the dimers recognize short nucleotide sequences that are displayed on both stands of the DNA double-helix in dyad symmetry. This mode of protein-DNA interaction enhances the affinity and specificity of binding and hence the biological activity. Abbreviations: CD, circular dichroism; GCN4-pl, C-terminal 33-residue leucine zipper moiety of yeast transcriptional activator GCN4; GuHCI. guanidine hydrochloride; HIV-I, human immunodeficiency virus type 1 ; r-LZ35 and r-LZ38, 35-and 38-residue rem-leucine zippers based on the sequence of GCN4-p1; (r-LZ38)2, disulfide crosslinked r-LZ38; SMC2 "structural maintainance of chromosomes" protein from yeast; TFA, trifluoroacetic acid; Mw,,,,,,, apparent relative molecular mass.

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

“…1 A) was monomeric and had no appreciable structure in the low micromolar concentration range (10). However, at higher concentrations (1 mM), this peptide is completely ␣-helical (Fig.…”

“…M, r-GCN4-p1 exists as a mixture of monomers and tetramers in solution (10). N-terminal extension of r-GCN4-p1 by the tripeptide Cys-Gly-Gly yielded a 38-residue peptide (r-GCN4-p1Ј) that allowed the formation of a covalently linked dimer.…”

“…The r-GCN4-p1Ј peptide (Fig. 1 A) was synthesized by solid phase chemistry as described (10) and crystallized by hanging drop vapor diffusion. The reservoir solution consisted of 0.2 M sodium chloride͞25% (vol/vol) 2-methyl-2,4-pentanediol͞100 mM sodium acetate, pH 4.8.…”

Chorismate Synthase from the Hyperthermophile Thermotoga maritima Combines Thermostability and Increased Rigidity with Catalytic and Spectral Properties Similar to Mesophilic Counterparts

“…5. The data can be interpreted as resulting from a single ideal species according to the model of Liu et al (18) under all the conditions used. The molecular mass of the enzyme was calculated to be 168,377 Ϯ 1950 Da under each of the conditions used.…”

“…Analytical Ultracentrifugation-Conventional sedimentation equilibrium measurements were made according to the method of Liu et al (18) with a Beckman XL-A analytical ultracentrifuge (Beckman, Palo Alto, CA). The data were collected at 7,000, 9,000, and 12,000 rpm and at 20°C.…”