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-Å 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. Since the early folding experiments by Anfinsen (1), it has been accepted that structure and function of a protein are determined by its amino acid sequence as read from the N terminus to the C terminus. But how does the structure change if the amino acid sequence of the protein is inverted? Inverted sequences are occasionally found in genomic DNA, but thus far, a native retro-protein has not been detected. Physicochemical properties that are related to the amino acid composition or the hydrophobicity profile should not be affected by the inversion of the sequence, supporting the idea that retro-sequences might fold into a native-like conformation. Modeling experiments suggested that reversal of the backbone direction may result in a topological mirror image of the native structure of the protein (2) or may produce the same topology as that of the parent protein (3, 4). Thus far, no structure elucidation of a retro-Lpeptide at atomic resolution has been reported.Coiled coils, including leucine zippers, consist of two to five intertwined ␣-helices and are frequently found in oligomeric proteins such as transcription factors as well as motility and structural proteins (5). We used the two-stranded coiled-coil domain of the yeast transcription activator GCN4 (6) to dimerize an artificial HIV enhancer-binding peptide, an operation that resulted in increased inhibition of HIV enhancer-controlled transcription (7). Because modeling studies suggested that the retro-sequence of the GCN4 leucine zipper also seemed to form a suitable dimerization module, a 35-residue retro-GCN4 was synthesized and characterized. Oxidized retro-leucine zipper extended with Cys-Gly-Gly, previously termed (r-LZ38) 2 (r-GCN4-p1Ј; Fig. 1A), crystallized and is now shown by x-ray structure analysis to fold into a parallel tetrameric coiled coil. Materials and MethodsUltracentrifugation. Sedimentation velocity experiments were performed with a Beckman-Spinco XL-A analytical ultracentrifuge. The peptide was dissolved in 20 mM Tris⅐HCl͞80 mM NaCl adjusted to pH 5.0, and measurements were made over a 10-to 100-M peptide concentration range. The centrifuge was operated at a speed of 50,000 rpm at 20°C. A partial specific volume, v 20 ϭ 0.74 cm 3 ⅐g Ϫ1, was calculated from the amino acid composition as was an estimate of the degree of hydration, d 1 ϭ 0.47 g water͞g protein. Sedimentation velocity traces were analyzed according to the method described in ref. 8, and a value of the sedimentation coefficient, s w,20 ϭ 1.77 Ϯ 0.5 Svedberg, was obtained by extrapolation to infinite dilution. The axial ratio was calculated as described in ref. 9.Crysta...

Basic-region leucine zipper (bZIP) proteins are one of the largest transcription factor families that regulate a wide range of cellular functions. Owing to the stability of their coiled coil structure leucine zipper (LZ) domains of bZIP factors are widely employed as dimerization motifs in protein engineering studies. In the course of one such study, the X-ray structure of the retro-version of the LZ moiety of yeast transcriptional activator GCN4 suggested that this retro-LZ may have ribonuclease activity. Here we show that not only the retro-LZ but also the authentic LZ of GCN4 has weak but distinct ribonuclease activity. The observed cleavage of RNA is unspecific, it is not suppressed by the ribonuclease A inhibitor RNasin and involves the breakage of 3′,5′-phosphodiester bonds with formation of 2′,3′-cyclic phosphates as the final products as demonstrated by HPLC/electrospray ionization mass spectrometry. Several mutants of the GCN4 leucine zipper are catalytically inactive, providing important negative controls and unequivocally associating the enzymatic activity with the peptide under study. The leucine zipper moiety of the human factor c-Jun as well as the entire c-Jun protein are also shown to catalyze degradation of RNA. The presented data, which was obtained in the test-tube experiments, adds GCN4 and c-Jun to the pool of proteins with multiple functions (also known as moonlighting proteins). If expressed in vivo, the endoribonuclease activity of these bZIP-containing factors may represent a direct coupling between transcription activation and controlled RNA turnover. As an additional result of this work, the retro-leucine zipper of GCN4 can be added to the list of functional retro-peptides.

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

Vibrational Raman optical activity (ROA) spectra have been measured for aqueous solutions of 4(R)-hydroxyproline at three different pH values and are compared with theoretical results calculated for several conformations of anionic, cationic and zwitterionic 4(R)-hydroxyproline using density functional theory (DFT) and the polarizable continuum model (PCM). The experimental ROA bands have been ascribed to the normal modes by comparison of the experimental and calculated vibrational frequencies and ROA intensities. Overall, using PCM for geometry optimization and force field calculations gives simulated Raman and ROA spectra that agree with the main features of the experimental spectra, whereas using PCM also in the calculations of optical tensors seems more problematic.

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.