The structure of Escherichia coli succinate dehydrogenase (SQR), analogous to the mitochondrial respiratory complex II, has been determined, revealing the electron transport pathway from the electron donor, succinate, to the terminal electron acceptor, ubiquinone. It was found that the SQR redox centers are arranged in a manner that aids the prevention of reactive oxygen species (ROS) formation at the flavin adenine dinucleotide. This is likely to be the main reason SQR is expressed during aerobic respiration rather than the related enzyme fumarate reductase, which produces high levels of ROS. Furthermore, symptoms of genetic disorders associated with mitochondrial SQR mutations may be a result of ROS formation resulting from impaired electron transport in the enzyme.

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The structure of the membrane protein formate dehydrogenase-N (Fdn-N), a major component of Escherichia coli nitrate respiration, has been determined at 1.6 angstroms. The structure demonstrates 11 redox centers, including molybdopterin-guanine dinucleotides, five [4Fe-4S] clusters, two heme b groups, and a menaquinone analog. These redox centers are aligned in a single chain, which extends almost 90 angstroms through the enzyme. The menaquinone reduction site associated with a possible proton pathway was also characterized. This structure provides critical insights into the proton motive force generation by redox loop, a common mechanism among a wide range of respiratory enzymes.

A phage display system for the selection of peptides binding to heterologously expressed human melanocortin receptor 1 on the surface of insect cells has been established. It could be shown that phage particles displaying the natural ligand ␣-melanocyte-stimulating hormone bind selectively to cells expressing this receptor and that these phages exhibit biological activity on mouse B16F1 melanoma cells. Insect cells were superior to other cell lines tested and have been used to select binders from a small library, in which critical determinants (Phe 7 -Arg 8 -Trp 9 ) were kept, whereas the flanking regions where allowed to variate freely. One peptide displaying little similarity with native hormone was found that binds to the receptor also in its free form with an affinity of 7 nM. It showed a remarkable selectivity for this receptor, because it binds to the other melanocortin receptor subtypes with a maximum affinity of 21 M. This is the first time phage display has been used successfully with G-protein-coupled receptors lacking an extracellular binding domain.Phage display techniques, i.e. the display of libraries of peptides, enzymes, antibodies, and other proteins on the surface of bacteriophages and selection of functional sequences thereof, have undergone a rapid development (1-3). Originally used as selection systems to identify peptide epitopes (4, 5), phage display is today in use for almost any kind of problem that involves the interaction of peptides and proteins with other materials. Most of the published literature deals with the development of antibodies (6 -8). With the use of this technique one comes close to the power of the human immune system. Other milestones were the display of enzymes (9), enzyme inhibitors (10), hormones (11), and cloning of active protein domains (12), to list only a few applications. Among the numerous publications about the display of hormones there are only a few dealing with the bioactivity of the phage (for examples see Refs. 13 and 14), because in almost all cases purified domains of cell surface receptors are used for the selection of binders from libraries (for examples see Refs. 11 and 15). Only a very few successful experiments are published with receptors displayed on living cells and in all such cases the receptors bind the ligands with extracellular domains (16 -18).The selection of random libraries of small peptides is naturally rendered more difficult when working with impure target proteins. Libraries of larger protein ligands usually contain common structural features directing and restricting binding of phages to the intended target. Especially G-protein-coupled receptors for small ligands that bind the ligands within their transmembrane helices have been the exclusive target for chemical peptide and other compound libraries (19 -23). These can be analyzed and deconvoluted by other means than just the affinity of the compounds, as has been shown for example for the MC1 receptor (24), but they lack the complexity of bacteriophage displayed libraries. Becau...

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