Bernd Gutte

An artificial crystalline DDT-binding polypeptide

A hydrophobic 24‐residue polypeptide that could potentially form a four‐stranded antiparallel β‐pleated sheet and bind the insecticide DDT was designed and synthesized. The synthetic peptide aggregated in 1 M acetic acid but was monomeric in aqueous 50% ethanol. In the latter solvent the 24‐residue polypeptide and DDT formed a complex with an apparent dissociation constant of ≈2 × 10−5 M. The DDT binding of an analogue of this peptide possessing the same amino acid residues in a random sequence was more than 2 orders of magnitude and that of bovine serum albumin at least 3 orders of magnitude weaker. The designed polypeptide could be crystallized.

Preparation and characterization of polyclonal and monoclonal antibodies against the insecticide DDT

A synthetic DDT derivative in which the molecular structure of DDT was completely retained was coupled to bovine serum albumin. Animals were immunized with the DDT-bovine serum albumin conjugate and polyclonal and monoclonal antibodies against the insecticide were isolated. These antibodies seemed to be the first true anti-DDT antibodies and distinguished much better between DDT and DDT metabolites than previously prepared anti-DDT antisera. In competitive solid phase radioimmunoassays, DDT concentrations as low as 10 nM or 0.0035 mg/1 were detectable. The anti-DDT antibodies can be used for environmental analyses and lend themselves to the elucidation of the structure of the DDT binding site.

The leucine zipper domains of the transcription factors GCN4 and c-Jun have ribonuclease activity.

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.

Conformation of a synthetic 34-residue polypeptide that interacts with nucleic acids

In [l] the design, synthesis and properties of a 34-residue polypeptide with ribonuclease activity were studied. Based on secondary structure prediction rules [2] and model building, an attempt was made to build a threedimensional configuration capable to bind a trinucleotide. The amino acid sequence is given in table 1. In the sequence an intrachain disulfide bridge between halfcystines 10 and 33 is intended to stabilize the structure, and to bring potential active site residues (Asp’, Thr12, His32) into spatial proximity [ 11. In addition to the given 34-residue polypeptide, a 6%residue dimer and higher oligomers with interchain cystine bridges are obtained. The respective [Ala”, Ala33]analogue which cannot form disulfide bonds was prepared as a reference, in order to investigate the role of the covalent crosslink in stabilizing the peptide fold. As shown by difference spectra and enzymatic assays, the 3 peptides all interact with cytidine phosphate or single-stranded DNA, showing significant ribonuclease activity with preference for cleavage at the 3’end of cytidylate. The nuclease activity of all 3 compounds suggests that the disulfide bond is not essential for the structure and function; obviously the noncovalent interactions are sufficient to establish a structure capable to bind and cleave the substrate [ 11. The present measurements are intended to characterize the 3 peptides by CD spectroscopy in order to prove or disprove the structure predicted on the basis of statistical and stereochemical methods. As a result the far-ultraviolet spectra of the 3 compounds all exhibit characteristic minima at 225 and 198 nm reflecting a considerable contribution of p-structure but disproving the presence of significant amounts of helix. The spectra are also very similar in the range of aromatic transitions(260-290 nm),pointing to anomalous absorption of the Phe and Tyr residues. CD spectral titration of the 68-residue polypeptide with 2’CMP lends itself to verify the binding constant determined by chromatographic techniques. Kawasfoundtobe2.0X lO’M_‘.

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.

An artificial HIV enhancer-binding peptide is dimerized by the addition of a leucine zipper

Abstract A 42 residue artificial peptide that binds to the HIV-1 enhancers has been described previously. The specificity of interaction of the peptide with its target DNA sequence has been demonstrated by a variety of techniques. Naturally occurring regulatory proteins frequently bind to DNA as dimers, thereby increasing the strength and specificity of the interaction, the dimer interface often being provided by a leucine zipper type coiled coil. As a suitable binding site for this kind of system is located to the 5′ end of the HIV enhancer region, it was decided to design and synthesize a fusion peptide that not only contained the DNA binding sequence of the original 42 residue peptide but also incorporated a leucine zipper based on that of the GCN4 transcriptional activator, that should, therefore, be capable of dimerizing. The resultant peptide, LZ66, has now been shown to be fully active in band shift and in vitro transcription assays and to exhibit about double the inhibitory activity of the parent 42 residue peptide. Preliminary CD measurements revealed that the peptide has a high α-helical content and that it adopts a stable conformation down to the low micromolar peptide concentration range. Sedimentation equilibrium studies confirmed that the principles involved in the design of the peptide are valid and that the peptide is indeed dimeric in solution.

The DNA-binding properties of an artificial 42-residue polypeptide derived from a natural repressor.

Bacteriophage 434 repressor recognizes the operator sequences ACAAG and ACAAT. As the same or similar sequences occur in the enhancer region of HIV‐1, 434 repressor was a potential HIV enhancer‐binding protein. We found that the interaction of the DNA‐binding domain of 434 repressor with a 57‐bp HIV enhancer DNA was very weak whereas a 42‐residue construct, comprising the recognition helix and four copies of a positively charged segment of the repressor, bound strongly. The results of footprint and cell‐free in vitro transcription studies showed that the 42‐residue peptide bound preferably to the enhancer region of HIV‐1 and acted as an artificial repressor. Replacement of an essential glutamine of the recognition helix by glutamic acid resulted in a partial shift of the sequence specificity of the 42‐residue peptide.

Structure-function studies of designed DDT-binding polypeptides

An artificial 24-residue DDT-binding polypeptide (Moser, R., Thomas, R.M., and Gutte, B. (1983) FEBS Lett. 157, 247-251) and several analogues of this peptide were characterized by ligand binding, spectroscopic, and immunological studies. Comparison of dissociation constants showed that Phe14 and His16 were important for DDT binding and that the designed peptide had noticeable ligand specificity. Measurement of the circular dichroism of the artificial DDT-binding peptide revealed a high proportion of beta-structure which was abolished only partly by 8 M urea. When Tyr15, Tyr17, and Phe3 whose side chains were on the same side of the proposed beta-sheet were replaced by non-aromatic amino acids, the cross-reactivity with antibodies against the original DDT-binding peptide decreased stepwise. In summary, the results of this study support essential features of our structural model of the designed 24-residue DDT-binding peptide.

Sepharose-supported DNA as template for RNA synthesis

A 96-bp DNA fragment containing the bacteriophage SP6 promoter was covalently coupled to EAH-Sepharose 4B in an orientation-specific manner. Here we show that the immobilized DNA was able to serve as template in in vitro transcription of RNA. This solid-phase approach allowed easy separation of the RNA products by centrifugation and made digestion of the template DNA by DNase superfluous. The Sepharose-bound DNA template was active in at least four consecutive cycles of in vitro transcription whose results were highly reproducible. The system may form a useful basis for the development of other molecular biology reactions on solid supports.

From Binding to Catalysis - Investigations Using Synthetic Peptides

Receptors, repressors, protein inhibitors, and antibodies are proteins that bind a ligand but do not alter it chemically. Receptors act by forming membrane channels (acetylcholine receptor), by activating membrane-bound enzymes (adrenaline receptor), or by regulating gene expression (nuclear estrogen receptor). Repressors can prevent transcription of bacterial operons by binding very tightly to the Dperator region of the operon. Repressor binding is controlled allosterically by metabolites.