Contents: Metalloproteins, Peptide-membrane interactions
Metalloproteins
The special properties of transition metals, allowing them to assume different oxidation states and to form a variety of coordination compounds, turn them into central components in many enzymes involved in essential functions in biological systems are such electron transfer, oxygen transport and activation, redox catalysis and structure stabilization. Thus, there is a vital interest to unravel the fundamental chemical mechanisms by which metalloenzymes selectively and cleanly convert educts to products under the mildest possible chemical conditions of ambient temperature, pressure and neutral pH. Understanding these catalytic functions on a molecular level is a key to drugs design and to development of low-molecular weight catalysts.
The geometric and electronic structure of the metal site, determined by the metal ion properties and its unique interaction with the protein matrix, are usually closely related to its specific activity. The details of this relation are, however, not fully comprehended. Therefore, a complete characterization of such sites is essential. If the metal ion is paramagnetic, which is frequently met in the field of metalloprotein biochemistry, EPR is often the method of choice.
The various EPR experiments provide information on the close environment of the paramagnetic metal center via a variety of magnetic interactions of the unpaired electron(s) with its environment. Relating these to detailed spatial and electronic structure is not trivial, but recent development Density Functional Theory (DFT) has provided the missing link and we are using it through collaboration with Prof. Frank Neese, University of Bonn.
The systems that are studied in the lab are:
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cd1NiR catalyses the one-electron reduction of the nitrite anion to nitric oxide, NO-2 + e + 2H+NO + H2O, as part of the bacteria’s nitrogen-based energy conversion. The enzymes isolated from the different bacteria are all homodimers, each monomer containing a covalently linked c-type heme and one noncovalently bound d1-type heme. Both have low spin Fe(III) in the oxidized state (native). The c-heme acts as the electron-accepting site while the d1-heme provides the nitrite binding and reduction site. The reaction is intriguing because it involves a transfer of a reducing equivalent into an electron-rich species, e.g. the nitrite anion, possibly followed by protonation and dehydration. Moreover, a considerable complication is the possibility that product inhibition may occur because NO forms a highly stable complex with ferrous d1-heme and, at acidic pH, also with the c-heme. Although a number of suggestions on how this fine control takes place have been put forward, the experimental evidences from enzymes from different sources still do not provide one consistent picture. We are investigating spectroscopic properties of the two heme centers in the wild type and several mutants in the resting state and trying to trap intermediates and to obtain new insight into the mechanism of action |
Structures of the Fe(III) heme centers in cd1 nitrite reductase from Pseudomonas aeruginosa (PDB code 1NIR Nurizzo et al Structure, 1997, 5, 1157) |
(Collaboration with Francesca Cutrozulla and Murizio Brunori, U. of Rome, Israel Pecht, Weizmann Institute of Science, Ole Farver, University of Copenhagen, Peter M. H. Kroneck, University of Konstanz.)
DEAD box protein A (DbpA) is one of five Escherichia coli DEAD-box helicases. Helicases bind and remodel nucleic acids or nucleic acid – protein complexes in an ATP-dependent manner. They are encoded by virtually all organisms from viruses and bacteria to humans and constitute one of the largest classes of proteins. DEAD-box helicases are part of superfamily 2 (SF2) The name of this family is derived from the conserved DEAD motif (Asp Glu Ala Asp. Members of the DEAD-box RNA helicase family were found to participate in a variety of biological processes such as translation initiation, ribosome biogenesis, RNA splicing, microRNA function, RNA transport, viral RNA replication, and many other process that were recently reviewed. DbpA was proposed to take part in ribosome biogenesis, but to date its biological function is unclear.
DbpA from Escherichia coli and its Bacillus subtilis homolog, YxiN, are unique among the DEAD proteins because their ATPase activity is strongly enhanced by the presence of an RNA molecule that contains the hairpin 92 of 23S ribosomal RNA (HP92) with either 3' or 5' extensions. DbpA was shown to unwind short <9bp (base pairs) RNA duplexes positioned 3' or 5' to the HP9213 as well as long duplex RNA stretches in an ATP-dependent manner. Despite some recent progress in the field9, including detailed elucidation of the DbpA kinetic pathway leading to ATP hydrolysis and RNA unwinding, the fine molecular and chemical mechanisms associated with the different states DbpA adopts during the catalytic cycle are not yet fully understood. Although the protein itself does not carry any paramagnetic center, paramagnetic reporters can be introduced externally. For studies of the active site we substitute Mg2+ by Mn2+ to focus on the role of the metal ion in the catalysis. Such substitution preserves the catalytic activity. For studies of long-range interaction we introduce the spin labeleds into the protein and RNA and measure the distance between them. This work is done in collaboration with Prof. Irit Sagi.
Peptide-membrane interactions
Protein-membrane interactions are of fundamental importance to a wide range of cellular processes. However, the detailed molecular basis for the organization of the protein(s) in the membrane not fully understood. Improving our understanding requires the application of better physical methods that can provide structural details on the system. One of the techniques used to study membrane-protein assemblies is electron paramagnetic resonance (EPR). We focus on the development of an integrated EPR approach which combines classical continuous wave EPR spectroscopy with high resolution pulse EPR techniques and cryogentic-transmission microscopy (cryo-TEM). To establish this approach we first focus on simpler related systems; the interaction of host defense (anti-microbial) spin labeled peptides, specifically melittin with phospholipid model membranes in the form of vesicles.
DEER (double electron-electron resonance) is a method that can provides distances between paramagnetic centers in the range of 1.5-8 nm and it is used to obtained the peptide conformation. This is complimented by the application of ESEEM (electron spin echo envelope modulation) and ENDOR (electron-nuclear double resonance) techniques, which provide electron-nuclear distances and thereby can probe specific interactions between the spin labeled peptide and the membrane. Here we use isotopically labeled membranes.
Study of Fusion Process fo HIV-1
HIV infection requires fusion of the viral membrane to the target cell membrane and the understanding of the infection process on the molecular level is essential for the design of inhibitors (drugs). The fusion process is catalyzed by the HIV envelope glycoprotein gp160. This protein is composed of two noncovalently associated subunits - gp120 and gp41. Following the interaction of gp120 with the T-cell receptor (TCR) co-receptors on the target cell, gp41 undergoes conformational changes that mediate the fusion between the viral and the cellular membranes or between infected and healthy cells. The N-terminus of gp41 contains a hydrophobic, glycine-rich fusion peptide (FP) that is essential for membrane fusion.
We investigate the conformation and location of the gp41 FP once incorporated into the membrane and look for possible interactions with the e trans membrane domain of the TCR. Here understanding of the molecular interactions mediating the immunosuppressive activity of FP: (i) Facilitates the evaluation of its contribution to HIV pathology and its exploitation as an immunotherapeutic tool, ii) Sheds light on the nature of interaction inside the TCR complex itself. iii) Provides a clue about the T cells' regulatory mechanism, thus revealing some critical requirements of protein-protein interaction.
(Collaboration with Yechiel Shai, Biological Chemistry, Weizmann Institute of Science, Herbert Zimmerman, MPI for Medical Research, Heidelberg).