Wolfgang E. Trommer

Chemically and biologically synthesized CPP-modified gelonin for enhanced anti-tumor activity.

The ineffectiveness of small molecule drugs against cancer has generated significant interest in more potent macromolecular agents. Gelonin, a plant-derived toxin that inhibits protein translation, has attracted much attention in this regard. Due to its inability to internalize into cells, however, gelonin exerts only limited tumoricidal effect. To overcome this cell membrane barrier, we modified gelonin, via both chemical conjugation and genetic recombination methods, with low molecular weight protamine (LMWP), a cell-penetrating peptide (CPP) which was shown to efficiently ferry various cargoes into cells. Results confirmed that gelonin-LMWP chemical conjugate (cG-L) and recombinant gelonin-LMWP chimera (rG-L) possessed N-glycosidase activity equivalent to that of unmodified recombinant gelonin (rGel); however, unlike rGel, both gelonin-LMWPs were able to internalize into cells. Cytotoxicity studies further demonstrated that cG-L and rG-L exhibited significantly improved tumoricidal effects, with IC50 values being 120-fold lower than that of rGel. Moreover, when tested against a CT26 s.c. xenograft tumor mouse model, significant inhibition of tumor growth was observed with rG-L doses as low as 2 μg/tumor, while no detectable therapeutic effects were seen with rGel at 10-fold higher doses. Overall, this study demonstrated the potential of utilizing CPP-modified gelonin as a highly potent anticancer drug to overcome limitations of current chemotherapeutic agents.

Soluble Expression and Affinity Purification of Functional Domain of Human Acetylcholine Receptor α-subunit by the Modulation of Maltose Binding Protein

An open reading frame of the α-subunit 1-205 residues (α205) of human acetylcholine receptor (AchR) was amplified by PCR with pUC-AChRα205 as the template and inserted into vector pMAL-c2X. The constructed pMARα205 was transferred into E. coli BL21 which were then grown in LB medium. The amount of soluble MBP-AChRα205 protein reached about 25% of total soluble proteins from the cell lysate. Using amylose-affinity chromatography, about 35xa0mg MBP-AChRα205 could be obtained from 1 l culture. Western blot analysis and ELISA showed that immunoreactivities of both MBP-AChRα205 and AChRα205 were similar to that of AChR α-subunit from Torpedo.

Conformational changes of the chaperone SecB upon binding to a model substrate - bovine pancreatic trypsin inhibitor (BPTI)

Abstract SecB is a homotetrameric cytosolic chaperone that forms part of the protein translocation machinery in E. coli. Due to SecB, nascent polypeptides are maintained in an unfolded translocation-competent state devoid of tertiary structure and thus are guided to the translocon. In vitro SecB rapidly binds to a variety of ligands in a non-native state. We have previously investigated the bound state conformation of the model substrate bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin labeling and pyrene fluorescence methods. It was shown that SecB undergoes a conformational change during the process of substrate binding. Here, we generated SecB mutants containing but a single cysteine per subunit or an exposed highly reactive new cysteine after removal of the nearby intrinsic cysteines. Quantitative spin labeling was achieved with the methanethiosulfonate spin label (MTS) at positions C97 or E90C, respectively. Highfield (W-band) electron paramagnetic resonance (EPR) measurements revealed that with BPTI present the spin labels are exposed to a more polar/hydrophilic environment. Nanoscale distance measurements with double electron-electron resonance (DEER) were in excellent agreement with distances obtained by molecular modeling. Binding of BPTI also led to a slight change in distances between labels at C97 but not at E90C. While the shorter distance in the tetramer increased, the larger diagonal distance decreased. These findings can be explained by a widening of the tetrameric structure upon substrate binding much like the opening of two pairs of scissors.

Role of antioxidants in the O-hydroxyethyl-D-(Ser)8-cyclosporine A (SDZ IMM125)-induced apoptosis in rat hepatocytes.

The mechanisms underlying the apoptotic activity of the immunosuppressive drug cyclosporine A and its O-hydroxyethyl-D-(Ser)(8)-derivative SDZ IMM125 in rat hepatocytes are not yet fully understood. It was the purpose of the present study to investigate the role of anti- and pro-oxidants and of caspase-3 and intracellular Ca(2+) in SDZ IMM125-induced apoptosis in rat hepatocytes. SDZ IMM125 induced an increase in chromatin condensation and fragmentation, and the activation of caspase-3. Supplementing the cell cultures with the antioxidants, D,L-alpha-tocopherol-polyethylene-glycol-1000-succinate, ascorbic acid, and the reducing agent, dithiothreitol, significantly inhibited the SDZ IMM125-mediated increase in chromatin condensation and fragmentation, and caspase-3 activity. D,L-alpha-tocopherol-polyethylene-glycol-1000-succinate and dithiothreitol caused significant inhibition on SDZ IMM125-mediated cellular Ca(2+) uptake. The glutathione synthetase inhibitor, buthionine sulfoximine, increased SDZ IMM125-mediated caspase-3 action in parallel to chromatin condensation and fragmentation as well as Ca(2+) influx. Supplementation the culture medium with the intracellular Ca(2+) chelator bis-(o-aminophenoxy)-ethane-N,N,N,N-tetraacetic acid as well as omission of calcium in the medium reduced SDZ IMM125-induced apoptosis whereas the calcium supplementation of the culture medium elevated SDZ IMM125-induced apoptosis. Calcium antagonists inhibited SDZ IMM125-induced caspase-3 activation. Our data indicate that SDZ IMM125-mediated apoptosis in rat hepatocytes can be inhibited by antioxidants, and that the intracellular redox-state can act as a modulator of cytotoxicity and apoptosis. Further, the results suggest that SDZ IMM125-induced uptake of extracellular calcium is also a redox-sensitive process and that the increased intracellular calcium might directly cause apoptosis by increasing the caspase-3 activity as a central event in the cyclosporine-induced apoptotic mechanism.

Photoaffinity Spin Labeling

Over the last few decades affinity- and especially, photoaffinity labeling became a major tool for investigating the structure and interactions of biochemical macromolecules [1–3].

Synthesis of a pH-sensitive spin-labeled cyclohexylcarbodiimide derivative for probing protonation reactions in proton-pumping enzymes

Abstract The synthesis of a pH-sensitive spin-labeled carbodiimide, N-cyclohexyl-N′-(1-oxyl-2, 2, 3, 5, 5-pentamethylimidazolidine-4-yl)-methylcarbodiimide (1) is described. The compound is an analog of dicyclohexylcarbodiimide and reacts specifically with the membrane sector of the proton-pumping F1F0-type ATP synthase. This reaction of1 with the ATP synthase (and perhaps other proton-pumping enzymes) should enable studies of protonation reactions in the enzyme using ESR spectroscopy. The preparation of another potentially useful pH-sensitive spin-label, 4-isothiocyanatomethyl-2, 2, 3, 5, 5-pentamethylimidazolidine-1-oxyl (8) is also described. The synthesis of N-cyclohexyl-N′-(1-oxyl-2, 2, 3, 5, 5-pentamethyl-imidazolidine-4-yl)-methylcarbodiimide1, a pH-sensitive affinity spin label, and the reaction of1 with the proton-pumping ATP synthase enzyme is described. Download : Download full-size image

Advantages of 15N and Deuterium Spin Probes for Biomedical Electron Paramagnetic Resonance Investigations

15N and deuterium spin labels significantly advanced the application of electron paramagnetic resonance (EPR) in the fields of biomedical science and molecular biology. Problems involving the motional dynamics of enzyme catalysis, protein/ protein interactions, and membrane structure, which heretofore could not be addressed with EPR techniques, can now be investigated with highly quantitative approaches. The remarkable improvements afforded by isotopic substitutions of 15N and deuterium are due to three factors: (1) simplification of EPR spectral line shapes by reduction in the number of peaks; (2) increased sensitivity of the spin labels as evidenced by the elevated amplitude of spectral peak heights; and (3) enhanced resolution caused by narrowing of peak widths and elimination of overlaps of peaks. These factors facilitate the accurate interpretation of EPR spectral data, particularly for structure-function studies of large proteins and membranes. The purpose of this review is to examine the extraordinary utility of the isotopically substituted spin labels by evaluation of selected EPR investigations which may have wide applicability for biomedical experimentation. Therefore, we shall not present a detailed review of recent literature, but rather outline the uses of these spin labels for structure-function studies of enzymes and for the interactions of proteins and membranes in normal and pathological states.

Journal of Membrane Biology: Editorial.

The New Year brings significant changes to JMBi. First, I have taken the helm, succeeding Dr. Tom Woolf, to become the third Editor-in-Chief in the journal’s history. In keeping with tradition, Tom will now be listed as Editor Emeritus on the masthead, in recognition of his years of service to the journal. Second, the journal has been re-structured to better reflect the scope of the field and the broadened areas we want to represent in the papers we publish. At launch, we have four new sections: Cell Biology, Biophysics, Biotechnology & Engineering, and Membranes in Disease, each one is being shaped by a newly appointed Section Head whose ideas and vision will build each section— and the journal—into an important place to publish. Respectively, the section heads are Diane Lidke, University of New Mexico; Sandro Keller, TU Kaiserslautern; Stavroula Sofou, Rutgers University; and Amithaba Chattopadhyay, Center for Cellular and Molecular Biology, Hyderabad, India. The Section Heads own Editorials follow, wherein each explains his or her vision for each section. Part of this vision includes a re-configuring of the traditional Editorial Board structure. Each Section Head will be bringing new Associate Editors on board, to intensify the focus on each section’s topic and to help handle submissions in a timely way. More changes to come—click here to visit and bookmark our website to keep up with what’s new, to browse new issues, to subscribe to our Tables of

Open and Closed Form of Maltose Binding Protein in Its Native and Molten Globule State As Studied by Electron Paramagnetic Resonance Spectroscopy

An intensively investigated intermediate state of protein folding is the molten globule (MG) state, which contains secondary but hardly any tertiary structure. In previous work, we have determined the distances between interacting spins within maltose binding protein (MBP) in its native state using continuous wave and double electron-electron resonance (DEER) electron paramagnetic resonance (EPR) spectroscopy. Seven double mutants had been employed to investigate the structure within the two domains of MBP. DEER data nicely corroborated the previously available X-ray data. Even in its MG state, MBP is known to still bind its ligand maltose. We therefore hypothesized that there must be a defined structure around the binding pocket of MBP already in the absence of tertiary structure. Here we have investigated the functional and structural difference between native and MG state in the open and closed form with a new set of MBP mutants. In these, the spin-label positions were placed near the active site. Binding of its ligands leads to a conformational change from open to closed state, where the two domains are more closely together. The complete set of MBP mutants was analyzed at pH 3.2 (MG) and pH 7.4 (native state) using double-quantum coherence EPR. The values were compared with theoretical predictions of distances between the labels in biradicals constructed by molecular modeling from the crystal structures of MBP in open and closed form and were found to be in excellent agreement. Measurements show a defined structure around the binding pocket of MBP in MG, which explains maltose binding. A new and important finding is that in both states ligand-free MBP can be found in open and closed form, while ligand-bound MBP appears only in closed form because of maltose binding.

A Partially Structured Molten Globule Protein

Maltose binding protein (MBP) from E. coli was shown to bind maltose even in its molten globule state, although with substantially reduced affinity. The native protein of which the X-ray structure is known, is devoid of cysteines. Seven different mutants with two cysteines each were labeled with the MTS-SL. Distances from the active site as derived from the X-ray structure vary from 14 to 31 A. DEER measurements have so far shown very good agreement between the X-ray data and the native structure. Here we compare distances in the native protein with those in the molten globule state.