Nanoparticles measure up

Förster resonance energy transfer (FRET) is a measurement of energy transfer between a donor and an acceptor fluorophore. The distance dependence of the energy transfer has allowed this technique to be used for monitoring biochemical events such as conformational changes and protein-protein interactions. However, the use of FRET is limited to short time periods, because of blinking and photobleaching, and short distances, because of low signal intensity. Gold and silver nanoparticles have a plasmon resonance that results in a visible light scattering, with a wavelength that is sensitive to the presence of other nearby nanoparticles. Taking advantage of this distance-dependent nanoparticle plasmon coupling, Alivisatos and colleagues have developed a molecular ruler for measuring distances on the single-molecule level. To demonstrate potential applications, the authors labeled single-stranded DNA (ssDNA) with nanoparticles. Silver nanoparticles were blue, showing a wavelength shift to green upon dimer formation, and gold nanoparticles were green with a dimer-induced shift to orange. Addition of complementary DNA (cDNA) to ssDNA caused formation of the longer double-stranded DNA. For ssDNA labeled with nanoparticles at both ends, addition of cDNA caused the expected wavelength shift, confirming that these nanoparticles can be used as molecular rulers. Because of the relatively large size of the nanoparticles, they will be limited to in vitro applications. However, the strong, stable signals of nanoparticles will permit their use for continuous monitoring over long times and large distances, making these 'plasmon rulers' a powerful complement to FRET. (Nat. Biotechnol. 23, 741–745, 2005) JK

Inhibiting the anthrax lethal factor

Anthrax is caused by a Gram-positive bacterium, Bacillus anthracis. After infection of a host, bacterial spores germinate and multiply rapidly, causing a typically fatal toxemia. Because of its development into a bioweapon, the possibility of a large-scale anthrax outbreak is a threat. If administered early enough, antibiotics, such as ciprofloxacin, can cure anthrax infections. Although the bacterium itself is susceptible to antibiotics, it secretes a zinc-dependent metalloprotease, termed lethal factor (LF), that causes widespread damage to host cells. By optimizing a lead inhibitor from Merck's compound archive, Shoop et al. have developed a potent LF inhibitor (LFI), which was shown to act competitively. A crystal structure of inhibitor-bound LF shows the hydroxamate group of LFI chelated with a zinc atom in the enzyme's catalytic center. The authors demonstrated that the inhibitor blocks LF's cytotoxic effects in mouse macrophages and LF-mediated death in mice. LFI treatment alone was shown to increase survival rates after anthrax infection in both mice and rabbits. In rabbits treated several days after infection with anthrax (at a point when 50% of the subjects survived after ciprofloxacin administration), dosing with a combination of LFI and ciprofloxacin resulted in 100% subject survival. This work shows how an anthrax infection and its LF legacy can be effectively treated with antibiotics combined with LFI. (Proc. Natl. Acad. Sci. USA 102, 7958–7963, 2005) GW

Filtering kinase inhibitors

Protein phosphorylation is one of the most common post-translational modification reactions. Because all protein kinases contain conserved ATP binding sites, the selective inhibition of unique kinases in a background of hundreds of related proteins has proven challenging. However, recent biochemical and structural studies have identified 'gatekeeper' amino acids that control inhibitor access to the ATP binding site. A threonine gatekeeper, for example, allows binding of large inhibitors, whereas bulkier gatekeepers (such as leucine or methionine) restrict access to smaller analogs. In a recent study, Cohen et al. identified other 'selectivity filters' that allowed the design of covalent kinase inhibitors with enhanced selectivity. The authors identified three kinases (RSK1, RSK2 and RSK4) with threonine gateway residues that also possessed a cysteine residue in the C-terminal kinase domain (CTD), which might react with an electrophilic inhibitor. They designed a bulky adenine analog (fmk) to capture the active-site cysteine. Fmk inhibited wild-type RSK2 activity in the low nanomolar range, and formed a covalent cross-link with the protein. In contrast, fmk produced only weak inhibition of RSK proteins that had mutated gatekeeper or cysteine residues. Using their expanded selectivity filter, the authors were able to generate fmk-sensitive variants of other kinases by inserting the gateway threonine and/or CTD cysteine residues. The selectivity filter concept may be extendable to diverse kinase families, offering the possibility that the development of selective kinase inhibitors may be less challenging than was previously thought. (Science 308, 1318–1321, 2005) TLS

A taste for fresh garlic

Garlic (Allium sativum), used in cuisines throughout the world, adds distinctive flavors to foods by virtue of its organosulfur chemistry. When a garlic clove is crushed or minced, the enzyme alliinase transforms alliin, a cysteine allyl sulfoxide, to an unstable compound called allicin, which reacts to produce organosulfide compounds. Any garlic lover has noted that raw garlic produces a biting sensation on the tongue, whereas roasted garlic has a milder flavor. How is it that we respond differently to raw and cooked garlic? To address this question, Macpherson et al. showed that allicin in raw garlic activates transient receptor potential (TRP) proteins, a class of ion channel (see also the Review by Voets et al., p. 85 in this issue) involved in sensing compounds found in spicy foods, such as capsaicin. Chinese hamster ovary cells or Xenopus laevis oocytes, engineered to express specific TRP channels, were used as model systems and were assayed by calcium imaging or electrophysiology. Raw garlic extracts and purified allicin activated cells containing TRPA1 and TRPV1 but not TRPM8, whereas baked garlic extracts had no effect on these TRP channels. NMR measurements on garlic extracts showed that raw garlic contained ∼10 μM allicin, whereas the primary component of baked garlic was alliin. Although further studies may be required to understand the molecular details of this process, the authors suggest that allicin induces the burning sensation of raw garlic and that it mediates its effect through TRPA1 and TRPV1 receptor activation. (Curr. Biol. 15, 929–934, 2005) TLS

Research highlights written by Greg Watt, Joanne Kotz and Terry L. Sheppard