Stephan Klauser

The Drosophila melanogaster sex-peptide: A molecular analysis of structure-function relationships

Abstract In Drosophila melanogaster a 36 amino acid sex-peptide elicits the characteristic postmating behavior of females: rejection of males and induction of ovulation/oviposition. Here we report additional evidence by chemical peptide synthesis. To correlate peptide structure and function, a full length peptide and fragments thereof were synthesized, and chemically and enzymatically modified. A Staphylococcus aureus protein A-sex-peptide fusion protein was synthesized in E. coli . Based on our results obtained from bioassays we conclude: (1) The N-terminal 7 amino acids are not needed for sex-peptide function, whereas the disulfide bridge appears essential. (2) The C-terminal region (encoded by the second exon) is not sufficient. (3) No amino acid modifications are needed for sex-peptide action. (4) The potential trypsin cleavage site is not essential. (5) Synthetic fragments elicit either both or neither of the reactions, i.e. the two functions are not separable. (6) The same critical concentration of 0.6 pmol sex-peptide/female is needed to elicit rejection and ovulation. (7) All results are consistent with the assumption of only one target molecule for both reactions which is accessible via hemolymph.

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.

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.

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.

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.

9 – Design of Polypeptides

Polypeptide design has become significant in dealing with new epidemics, increasing concentrations of anthropogenic toxins, and other health and environmental problems, considering the immediate requirement for virus repressors, enzyme inhibitors, and pesticide-degrading polypeptides. A number of researchers are attempting to design novel functional polypeptides despite the fact that natural proteins are plentiful. At present, there is a significant gap between rationale and experimental results of design. There are three approaches to design artificial polypeptides: modification of natural proteins or peptides by replacement, deletion, and insertion of amino acid residues and shuffling of structural modules; rational or de novo design that yields sequences that inherently show little, if any, similarity with those of natural products; and construction of hybrid polypeptides from designed and authentic structural elements. Investigators have used these approaches to construct stable scaffolds for subsequent functionalization, to mimic or modify the structure and function of natural proteins, and to design novel ion channels, enzymes, and repressors that possess unique mechanisms and specificities. This chapter discusses many trends in polypeptide design based on a survey of the literature.

Reverse Phase High-Performance Liquid Chromatography for Protein Purification: Insulin-Like Growth Factors, Ca2+- Binding Proteins and Metallothioneins

Utilization of high-performance liquid chromatography (HPLC) in peptide/protein chemistry has become widespread. The technique is being used for amino acid analysis (1,2,3), for peptide isolation from natural sources (4,5) or from enzymatic/chemical cleavages (6,7), for the identification of products arising from Edman degradations (8,9) and for determining the specificity of chemical cleavage methods (10). Its application in the chromatography of larger peptide fragments or proteins has been limited, in part, by the lack of suitable column packings. Until only recently most commercially available reverse phase packings exhibited nonspecific adsorption and/or reduced resolution. However, some proteins have been chromatographed under a variety of conditions on quite different column supports.

Artificial 64-Residue HIV-1 Enhancer-Binding Peptide Is a Potent Inhibitor of Viral Replication in HIV-1-Infected Cells

An artificial HIV-1 enhancer-binding peptide was extended by nine consecutive arginine residues at the C-terminus and by the nuclear localization signal of SV40 large T antigen at the N-terminus. The resulting synthetic 64-residue peptide was found to bind to the two enhancers of the HIV-1 long terminal repeat, cross the plasma membrane and the nuclear envelope of human cells, and suppress the HIV-1 enhancer-controlled expression of a green fluorescent protein reporter gene. Moreover, HIV-1 replication is inhibited by this peptide in HIV-1-infected CEM-GFP cells as revealed by HIV-1 p24 ELISA and real-time RT-PCR of HIV-1 RNA. Rapid uptake of this intracellular stable and inhibitory peptide into the cells implies that this peptide may have the potential to attenuate HIV-1 replication in vivo.