Hot off the press

Hot off the Press highlights recently published work for the benefit of our readers. Our contributors this month has focused on in situ membrane protein sythesis and protein labelling in living systems. New contributors are always welcome. If you are interested please contact molbiosyst@rsc.org for more information, we’d like to hear from you.

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Incorporation of in vitro synthesized GPCR into a tethered artificial lipid membrane system

Reviewed by: James R. Thompson, Physical and Theoretical Chemistry Laboratory, University of Oxford, UK.

 

G-protein coupled receptors (GPCRs) serve as signal transducers in membranes sensing the arrival of a ligand molecule and triggering a subsequent signal cascade within the cell. They are integral to many signal transduction processes and are ubiquitous in eukaryotes. GPCRs and processes in which they are involved are also implicated in many human diseases and as such are subjects of much research in academic and pharmaceutical laboratories. Unfortunately GPCRs are notoriously difficult to refold into artificial lipid bilayers for study. Researchers at the Max Planck Institute for Polymer Research in Mainz and their colleagues now describe a technique for the vectorial insertion of a recombinant GPCR into a tethered artificial lipid bilayer using an in vitro transcription and translation (IVTT) system. Using a rabbit reticulocyte extract expression system the researchers show data suggesting that the odorant receptor OR5 becomes inserted in a single orientation when expressed in the presence of a tethered lipid bilayer. The researchers may therefore have facilitated one of the most difficult tasks in studying membrane proteins in vitro. By examining fluorescent antibody binding using surface plasmon enhanced fluorescence spectroscopy they show that an N-terminal VSV-tag on the OR5 protein construct is present above the bilayer immediately after the IVTT reaction. A C-terminal VSV-tagged construct does not show antibody binding, suggesting that the C-terminus of OR5 is threaded across the supported lipid bilayer. The researchers then probe the conformation of the receptor by incubating the membrane with a ligand, Lilial. Through SEIRAS (surface enhanced infra-red absorption spectroscopy), a difference spectroscopy method using evanescent infra-red waves, they show an increased absorption of the amide I band of the protein, presumably due to a conformational change in the protein upon binding, suggesting that indeed the ligand may bind to the protein. This result could indicate that the GPCR has the correct conformation in the bilayer, although further analysis of the activity of the protein must be made to verify this result. This technique of in situ protein expression and incorporation into artificial lipid bilayers would represent a major breakthrough if it could be generalised for use with other challenging proteins. Firstly however, the results the authors present here would be bolstered by further studies on this system, for example with single-molecule fluorescence imaging of ligand binding, hopefully better exploring the nature of the interaction between the expressed protein and its ligand.

Incorporation of In Vitro Synthesized GPCR into a Tethered Artificial Lipid Membrane System, Rudolf Robelek, Eva S. Lemker, Birgit Wiltschi, Vinzenz Kirste, Renate Naumann, Dieter Oesterhelt and Eva-Kathrin Sinner, Angew. Chem., Int. Ed., 2007, 46(4), 605–608

A new addition to the repertoire of techniques for protein labeling in living systems

Reviewed by: Bo Liu, Division of Translational Research, University of Texas Southwestern Medical Center, USA.

 

Green fluorescence protein (GFP) is widely used to study protein functions in living systems when fused to target proteins genetically. However, chemical methods for site-specific protein labeling in situations where the size of GFP (∼27 kDa) causes structural perturbation is still in demand. Finding short peptides possessing orthogonal reactivity with chemical probes remains a formidable challenge. One outstanding example is that a short tetra-cysteine motif (CCXXCC) binds selectively to biarsenical fluorescein derivatives. Recently a new method of protein labeling has been explored by using enzymatic reactions to conjugate chemical-probe-tethered substrates to short target peptides. For example, Sfp phosphopantetheinyl transferase conjugates probe-tethered CoA to an 11-mer peptide and biotin ligase BirA conjugates biotin and its derivatives to a 15-mer peptide. Now Ploegh and co-workers add another weapon to the arsenal by using bacterial sortase A to conjugate various chemical probes to a 5-mer peptide.

Bacterial sortases are enzymes that covalently attach proteins to the bacterial cell wall. The enzymes recognize an LPXTG motif on various proteins and attach the proteins to a pentaglycine cell wall precursor. Popp et al. converted sortase A to a protein-labeling machine by fusing the LPXTG motif to proteins of interest and tethering various chemical probes to a pentaglycine peptide. In many cases, sortase A tolerated the modified pentaglycine peptide and conjugated the chemical probes to the proteins through the reaction between LPXTG and pentaglycine. The authors demonstrated that the enzymatic labeling strategy worked successfully both in test tubes and on mammalian cell surfaces. However, the authors pointed out that a large excess of sortase A was needed to drive the reactions to completion; so cleaning up the enzyme after reactions remains a concern.

M. W. Popp, J. M. Antos, G. M. Grotenbreg, E. Spooner and H. L. Ploegh, Nat. Chem. Biol., 2007, 3,707–708.

Hot off the RSC Press

Glutamate brainwave

Reviewed by: Emma Shiells, Royal Society of Chemistry, Cambridge, UK.

Selective detection of glutamate in brain tissue using microelectrode arrays has been achieved by scientists in the US.

Glutamate is an amino acid neurotransmitter and plays an important role in the mammalian central nervous system. A number of neurological and psychiatric disorders are thought to be linked to abnormalities in the transmission of glutamate.

Nigel Maidment and his colleagues at the University of California, Los Angeles, therefore developed ceramic-based platinum microelectrode arrays (MEAs) coated with an electropolymerized, overoxidized polypyrrole (OPP) that selectively detects glutamate in brain tissue.

The challenge has been for scientists to develop selective glutamate sensors with sensitive and rapid responses to glutamate. They should also not experience interference from other neurotransmitters, such as dopamine (DA) and ascorbic acid (AA). Interference is usually caused by the direct oxidation of DA or AA at the platinum electrodes.

Current methods use electrodes coated with Nafion, a well-known co-polymer, to overcome this problem. Maidment and his team showed that using their OPP-coated MEAs produced similar glutamate sensitivity and response times to those previously found for Nafion-coated MEAs. Unlike the Nafion-coated MEAs, their system is not affected by DA (at a concentration of 5 μM), which results in a highly selective glutamate detection.

Maidment’s future plans ‘are to selectively load different enzymes onto individual sites on multi-electrode arrays, thereby permitting detection of several neurotransmitters simultaneously’. But the team’s primary challenges are ‘improving sensitivity, longevity and further miniaturization [for these systems]’, said Maidment.

Eric Walker, Jianjun Wang, Naser Hamdi, Harold G. Monbouquette and Nigel T. Maidment, Analyst, 2007, 132, 1107.

Complex DNA binding unravelled

Reviewed by: Russell Johnson, Royal Society of Chemistry, Cambridge, UK.

 

Understanding how an anticancer complex binds DNA has brought metal–metal based antitumour drugs one step closer. Dirk Deubel at the Swiss Federal Institute of Technology Zurich (ETH Zurich), Switzerland, and Helen Chifotides at Texas A&M University, in College Station, US, have calculated the binding mechanism of complex dirhodium tetracarboxylate to the DNA base guanine.

Increasing attention is being paid to metal–metal based anticancer complexes as potential inhibitors of DNA replication. ‘DNA–complex binding is believed to be the key reaction responsible for the anticancer activity of these compounds,’ said Judit Šponer, a specialist in computational analysis of metallopharmaceuticals at the Academy of Sciences of the Czech Republic in Brno. Whilst the reactants and products in this step can be identified through conventional experiments, the mechanism of the interaction is not always clear. Clarifying the reaction mechanism is useful for redesigning ligands to improve the effectiveness of potential drugs.

Using a combination of computational approaches, Deubel and Chifotides managed to identify the possible intermediates in the DNA–complex binding reaction and the transition states between them. The team then calculated the free energies of these states to discover the lowest energy pathway. Their results showed that the reaction proceeds through five intermediates and involves guanine migrating from an axial to an equatorial position in the complex. This is a ‘very detailed description of the full reaction pathway,’ said Šponer.

Looking to the future, Deubel said that the challenge was to ‘tune the ligands at the metal–metal core to optimise the interplay between lability [of the ligands] and robustness of the di-metal core. Potentially, computational approaches could be used to screen ligands by predicting the energy of intermediates and transition states in systematic studies.’

Dirk V. Deubel and Helen T. Chifotides, Chem. Commun., 2007, 3438

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