Chemical biology

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Chemical biology Paper alert A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in chemical biology. Current Opinion in Chemical Biology 2000, 4:475–484 Contents (chosen by) 475 Interaction, assembly and processing (Kappock) 476 Biocatalysis and biotransformation (Pohl) 476 Bio-inorganic chemistry (Cammack) 477 Combinatorial chemistry (Conn and Hall) 479 Next generation therapeutics (Projan) 480 Analytical techniques (Cass) 481 Mechanisms (Stewart) 482 Model systems (Cousins and Sanders) 482 Biopolymers (Flitsch, Lowden and Newman) • ••

of special interest of outstanding interest

Interaction, assembly and processing Selected by Joe Kappock Washington University, St Louis, Missouri USA

Structure of the cytoplasmic β subunit-T1 assembly of voltage-dependent K+ channels. Gulbis JM, Zhou M, Mann S, MacKinnon R: Science 2000, 289:123-127. •• Significance: Potassium channel α and β subunits, located respectively in the membrane and on the cytoplasmic face of the cell membrane, are tetramers with pores at their four-fold rotational symmetry axes. A flexible, positively charged inactivation peptide (a ball-and-chain like structure which may be fused to the amino terminus of either the α or β subunit) rapidly inactivates open channels by occluding the open ion channel. The cytoplasmic T1 domain of the α subunit contains a central pore too narrow and electropositive to admit cations (let alone the inactivation peptide or classic organic blocking agents), raising the question of how ions could traverse this middle segment of a channel shaped roughly like an inverted mushroom. Structure-based mutagenesis experiments presented here indicate that ions do not move up the center of T1 or the β subunit, and also locate a portion of the inactivation peptide binding site on the T1 domain. Findings: Mutations near the interface of the T1 domain and the β subunit do not affect inactivation except to interfere with the subunit association; rather, T1 contact loop residues appear to encode β subunit specificity. In contrast, inactivation is affected by mutations that remove negatively-charged residues at the opposite end of T1 (near the linker to the transmembrane region of the α subunit), or mutations that remove positively-charged residues in the inactivation peptide. Double-mutant and single-mutant analyses confirm that mutants of each type have coupled effects. Thus inactivation peptide and K+ ions are each proposed to approach the membrane pore through lateral openings between the T1 domain and the integral membrane part of the α subunit.

IscU as a scaffold for iron-sulfur biosynthesis. Sequential assembly of [2Fe-2S] and [4Fe-4S] clusters in IscU. Agar JN, Krebs C, Frazzon J, Huynh BH, Dean DR, Johnson MK: Biochemistry 2000, 39:7856-7862. AND

Transfer of iron-sulfur cluster from NifU to apoferredoxin. Nishio K, Nakai M: J Biol Chem 2000, 275:22615-22618. • Significance: Iron−sulfur cluster assembly and insertion into apoproteins (precursors of the mature holoproteins) is an essential step in diverse redox and metabolic pathways. NifS homologues, including the ubiquitous IscS proteins, mobilize sulfur from L-cysteine for use in cluster assembly by NifU homologues, which have ill-defined roles in target apoprotein maturation. These papers provide the first mechanism for cluster assembly within IscU (homologous to the amino-terminal domain of NifU) and evidence that NifU alone can reconstitute a target apoprotein. Findings: Agar et al. isolated and characterized three distinct iron−sulfur−IscU complexes formed in the presence of a catalytic amount of IscS. In each IscU homodimer, a single [2Fe−2S]2+ cluster forms first, then two [2Fe−2S]2+ clusters, which in turn are reductively converted into a single [4Fe−4S]2+ cluster. The lack of other forms of IscU and the observation that apo-IscU is not required for [4Fe−4S] cluster assembly suggest a concerted mechanism for cluster biosynthesis, rather than more complex alternatives. Using purified Synechocystis proteins, Nishio and Nakai demonstrate transfer of [2Fe−2S] from holo-NifU to apoferredoxin, resulting in partial functional reconstitution of the latter, in the absence of other proteins or small molecules. Synechocystis contains only one NifU protein, which apparently contains all of the machinery necessary for iron−sulfur cluster assembly in that organism. ATP for the DNA ligation step in base excision repair is generated from poly(ADP-ribose). Oei SL, Ziegler M: J Biol Chem 2000, 275:23234-23239. • Significance: Many cellular insults result in DNA nicking, which is repaired in mammalian cells by the base excision repair (BER) system. The final step in BER, an ATP-dependent DNA ligation reaction, is stimulated by poly(ADP-ribose) (PAR) or PAR polymerase (PARP-1), which converts NAD + into PAR. This paper provides an explanation for the previously obscure function of PAR/PARP-1 in DNA repair and evidence for functional association among the BER components: DNA polymerase β (pol β), DNA ligase III (lig III), X-ray repair cross-complementary protein-1 (XRCC1), and PARP-1. Findings: In the presence of nicked DNA, HeLa nuclear extract, and at least three dNTPs, α-32P labelled PAR is converted into ATP, which appears to be channelled to lig III. DNA ligases generally transfer the adenylyl moiety of ATP to the 5′-phosphoryl end of nicked DNA via an adenylated enzyme intermediate. Label from [32P]NAD+ was trapped as DNA-[32P]AMP and lig III-[32P]AMP adducts, indicating that PAR can function as an adenylyl donor (the chemical mechanism is not known). In the absence of added ATP, nicked circle DNA was converted to closed circle DNA by nuclear extracts supplemented with dNTPs and either NAD+ or PAR (but not ADP-ribose). PARP-1

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and pol β activities are transiently stimulated by induction of BER in an XRCC1 dependent process; thus PAR formation, DNA synthesis, and DNA ligation function in a concerted, NAD-dependent BER process when insufficient ATP is available.

Biocatalysis and biotransformation Selected by Nicola Pohl Iowa State University, Ames, Iowa, USA

A tyrosine-to-threonine mutation converts cycloartenol synthase to an oxidosqualene cyclase that forms lanosterol as its major product. Herrera JB, Wilson WK, Matsuda SPT: J Am Chem Soc 2000, 122:6765-6766. • Significance: The modification of a single amino acid in a terpene cyclase is enough to completely alter product distributions, thereby highlighting the potential power of protein engineering of this class of enzymes to form new terpene-like structures. Findings: A conserved tyrosine in the enzyme that converts oxidosqualene into the triterpene cycloartenol was mutated to a threonine conserved in a related enzyme that converts oxidosqualene to lanosterol. Instead of cycloartenol, the mutant produced lanosterol and a novel triterpene, which was identified as 9β-7-lanosterol. Enhanced metabolism of halogenated hydrocarbons in transgenic plants containing mammalian cytochrome P450 2E1. Doty SL, Shang TQ, Wilson AM, Tangen J, Westergreen AD, Newman LA, Strand SE, Gordon MP: Proc Natl Acad Sci USA 2000, 97:6287-6291. • Significance: Transgenic plants could be planted in chlorocarbon-contaminated areas for successful phytoremidation. Findings: Tobacco plants made to express mammalian cytochrome P450 2E1 metabolized trichloroethylene 640 times better than the control plants, in addition to debrominating ethylene dibromide. The native tobacco oxidoreductase successfully interacted with the mammalian P450. Enzymatic production of biohydrogen. Woodward J, Orr M, Cordray K, Greenbaum E: Nature 2000, 405:1014-1015. •• Significance: A high-yielding biotransformation process for generating hydrogen gas from glucose could lead to the efficient production of combustible fuel from renewable biomass. Findings: Inclusion of other enzymes from the oxidative branch of the pentose phosphate cycle with dehydrogenases from the cycle increased hydrogen production from glucose-6-phosphate in a coupled reaction with a bacterial hydrogenase and NADP+ nearly sixfold to 97% of the maximal limit. The duration of hydrogen production was also increased fourfold.

Bio-inorganic chemistry Selected by Richard Cammack King’s College London, London, UK

Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 angstrom resolution. Toyoshima C, Nakasako M, Nomura H, Ogawa H: Nature 2000, 405:647-655. •• Significance: The ATP-dependent calcium pump of the sarcoplasmic reticulum membrane network is an essential component of muscle cells. After nerve-stimulated release of calcium induces muscle contraction, the calcium pump removes the calcium again, causing muscle relaxation. This is the first determination of the structure of such a P-type ATPase. Findings: The structure of the protein, embedded in a lipid bilayer, and in the presence of calcium, was determined by X-ray crystallography. It comprises a three-lobed structure on the cytoplasmic

side of the membrane, and a transmembrane helical structure. The phosphorylation and nucleotide-binding sites, identified by unreactive analogues, were present on different lobes of the cytoplasmic headpiece, separated by 2.5 nm. The structure binds two calcium ions at distinct sites. The hydrolysis of ATP and movement of calcium through the membrane-embedded section are proposed to involve large domain movements. Structure of a thioredoxin-like [2Fe-2S] ferredoxin from Aquifex aeolicus. Yeh AP, Chatelet C, Soltis M, Kuhn P, Meyer J, Rees DC: J Mol Biol 2000, 300:587-595. • Significance: Only two classes of [2Fe−2S] proteins with allcysteine coordination are known. Although much is known about the structures of the first class, such as plant-type ferredoxins, this is the first protein of the second class for which a structure has been determined. It is similar to ferredoxins associated with nitrogen fixation, and is the prototype of [2Fe−2S]-containing domains in complex iron−sulfur proteins such as hydrogenase and mitochondrial complex I. Findings: Apart from the [2Fe−2S] cluster, the structure shows no resemblance to the plant-type ferredoxins. The protein fold resembles the αβ architecture of thioredoxins and peroxiredoxins, which have active-site cysteines in homologous positions to those that bind the [2Fe−2S] cluster in this ferredoxin. The Crd1 gene encodes a putative di-iron enzyme required for photosystem I accumulation in copper deficiency and hypoxia in Chlamydomonas reinhardtii. Moseley J, Quinn J, Eriksson M, Merchant S: EMBO J 2000, 19:2139-2151. • Significance: This is an interesting example of the way in which genetic studies can predict the existence and function of new metalloproteins. Under conditions of copper deficiency, some algae, including Chlamydomonas reinhardtii, suppress the biosynthesis of the copper-containing electron-transport protein plastocyanin, and synthesize cytochrome c6 instead. Oxygen deficiency provokes a similar response, possibly caused by depletion of available copper under anaerobic conditions. Mutational analysis of this system revealed a novel gene that was predicted to encode a protein containing a dinuclear iron centre. Findings: Mutants of the green alga were screened for a copper-conditional phenotype. Mutations in the gene CRD1 were found to produce cells that had little of the photosynthetic apparatus, until traces of copper were added. The gene, which is activated under copper-defficient or oxygen-deficient conditions, is homologous to those containing carboxylatebridged di-iron centres. A possible electron-transfer function was suggested for the product of this gene. Magnetite defines a vertebrate magnetoreceptor. Diebel CE, Proksch R, Green CR, Neilson P, Walker MM: Nature 2000, 406:299-302. •• Significance: Candidate receptor cells for magnetic navigation have been observed in a layer of nasal cells in the fish Oncorhynchus mykiss (rainbow trout). Magnetite crystals, each representing an individual magnetic domain, were laid down in chains of about 1 µm length, which would be large enough to align with an external magnetic field. Findings: The cells were examined in fixed specimens, by a combination of magnetic force microscopy and confocal laser scanning microscopy. A linear arrangement of magnetic crystals was observed in specific, multilobed cells, and they were identified as magnetite by their response to an applied magnetic field.

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Bacterial metabolism — phosphite oxidation by sulphate reduction. Schink B, Friedrich M: Nature 2000, 406:37. •• Significance: Although traces of phosphines have long been known to be produced in marshes under anaerobic conditions, the biochemistry of reduced phosphorus compounds is almost completely unknown. Here, it is shown that a bacterium can use phosphite as an electron donor, in a chemolithotrophic mode of growth. Findings: The bacterial strain FiPS-3 was isolated from marine sediments near Venice, by selection for growth on phosphite and sulfate, which it converted to phosphate and sulfide. CO2 was used as the carbon source. This new process implies the existence of pathways of new phosphorus redox chemistry.

Combinatorial chemistry Selected by M Morgan Conn Amherst College, Amherst, Massachusetts, USA

Enantioselective magnetochiral photochemistry. Rikken GLJA, Raupach E: Nature 2000, 405:932-935. • Significance: To date, only extraterrestrial photochemistry with chiral polarized light and the chiral electroweak interaction have shown any capability to generate enantiomerically enriched molecules. Given the total domination of enantiopure chiral molecules in biology, the pre-biotic synthesis of enantiomeric molecules presents an ongoing puzzle. Findings: The authors report small enantiomeric excesses (~1 × 10−4) in the photochemical racemisation of Cr(III)trisoxalato complexes when performed with unpolarized light in a magnetic field parallel to the direction of irradiation. This magnetochiral effect, although quite small, shows in principle the potential for enantioselective chemistry. A DNA enzyme with N-glycosylase activity. Sheppard TL, Ordoukhanian P, Joyce GF: Proc Natl Acad Sci USA 2000, 97:7802-7807. •• Significance: The repertoire of chemical mechanisms that nucleic-acid-based enzymes can perform continues to grow. Complementing a recent report on the ability of the hammerhead ribozyme to catalyse a deglycosylation reaction (Hamm ML, Schwans JP, Piccirilli JA: J Am Chem Soc 2000, 122:4223-4224.), the authors report the ability of a DNA enzyme to perform backbone-modifying chemistry on nucleic acids. Findings: The structurally complex 93-nucleotide DNA enzyme accelerates 106-fold the deglycosylation of a specific guanosine residue with kobs = 0.2 min−1. This DNA enzyme was isolated from an in vitro selection protocol designed to elicit O-glycosidase activity by cleavage of lactose inserted into a DNA sequence. The emergence of this alternative activity provides a reminder, once again, how easily selection systems can find alternative solutions to the designed selection scheme. Molecular recognition of cAMP by an RNA aptamer. Loizumi M, Breaker RR: Biochemistry 2000, 39:8983-8992. • Significance: The ability of RNA to recognise cellular messengers such as cAMP could lead to ribozymes sensitive to changes in cellular signalling pathways. Findings: Using in vitro selection, the authors report the isolation of an RNA aptamer that specifically recognises 3′,5′-cAMP, a cellular signalling messenger molecule, with a dissociation constant of 10 µM. In particular, this aptamer is distinguished from previous adenosine-binding aptamers by its ability to discriminate between cAMP and other 5′-functionalized adenosine derivatives such as ATP, 5′-AMP, and 3′-AMP. The key to this

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selectivity was the use of C8-linked cAMP-agarose to bind to library members, followed by elution with cAMP. Biosynthetic phage display: a novel protein engineering tool combining chemical and genetic diversity. Dwyer MA, Lu W, Dwyer JJ, Kossiakoff AA: Chem Biol 2000, 7:263-274. •• Significance: The combinatorial possibilities of biological systems are limited to the use of naturally occurring monomers. Methods to incorporate a greater variety of building blocks into proteins and nucleic acids would allow exploration of structure and function in ways both subtler and more grand. Findings: This paper describes methodology whereby small peptides containing unnatural modifications can be ligated to peptides displayed on the surface of phage to allow simultaneous selection for natural and unnatural protein modifications. In the proof-of-concept example described here, the amino-terminus of the protease inhibitor eglin c was synthesised chemically with unnatural residues replacing a critical, internal Phe25. The carboxyl terminus of this protein was expressed on the surface of M13 phage as a library with randomised sidechains at positions that contact the former-phenylalanine (Val52, Val54). Selection by affinity capture yielded several proteins with mutations at positions 52 and 54 that compensated for the perturbation introduced at position 25. One sequence, two ribozymes: implications for the emergence of new ribozyme folds. Schultes EA, Bartel DP: Science 2000, 289:448-452. •• Significance: The paradigm that a given biopolymer primary sequence will lead only to a single stable three-dimensional structure takes another hit. Findings: By comparing the sequences and folds of two different ribozymes, the authors designed an RNA sequence that is capable of folding into both distinct structures with no common base pairs. Namely, a single ribozyme was synthesised that was capable of independently adopting the fold of the class III ligase, which joins an oligonucleotide substrate to it’s 5′ terminus, and the HDV ribozyme, which performs a site-specific selfcleavage. The ribozyme displayed both ligase and cleavage activity, although with reduced efficiency. Negative selectivity and the evolution of protein cascades: the specificity of plasmin for peptide and protein substrates. Hervio LS, Coombs GS, Bergstrom RC, Trivedi K, Dorey DR, Madison EL: J Am Chem Soc 1999, 121:5856-5859. • Significance: While proteolysis is an important method for the control and modification of protein activity, very precise control is necessary to avoid unintended damage. Findings: Substrate phage display was used to determine the optimal substrate for the protease plasmin by requiring cleavage of a randomised string of six amino acids connecting the phage coat protein and antibody epitopes. Only phage particles displaying plasmin substrates avoided antibody capture. Despite sharing the same residue at the cleavage site, the selection revealed that the optimised sequences for plasmin have very different sequences and are cleaved up to 710,000-fold more efficiently than the cleavage site in the plasmin precursor, plasminogen. This negative selectivity prevents plasmin from participating in autocatalytic activation by cleaving plasminogen. Selected by Dennis Hall University of Alberta, Edmonton, Alberta, Canada

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reactions, reflecting the solution-like behaviour of these types of supports as compared with standard cross-linked polystyrene resins. In addition, results from a study of solvent effects confirmed the importance of resin swelling on maintaining a solution-like chemical environment for the resin-bound substrate. Overall, this study lends support to the idea that the polymeric matrix plays an important role as ‘co-solvent’ in solid-phase chemical reactions.

Total synthesis of distamycin A and 2640 analogues: a solution-phase combinatorial approach to the discovery of new, bioactive DNA binding agents and development of a rapid, high-throughput screen for determining relative DNA binding affinity or DNA binding sequence selectivity. Boger DL, Fink BE, Hedrick MP: J Am Chem Soc 2000, 122:6382-6394. •• Significance: The development of small molecules capable of regulating gene expression is a promising therapeutic approach that is, however, thwarted by the difficulty of rationally designing tight-binding, sequence selective agents. This paper demonstrates that combinatorial high-throughput approaches can help accelerate the discovery of DNA-binding molecules. Findings: Two solution-phase libraries of 1320 compounds each were designed based on the DNA-binding antibiotic distamycin A as lead structure. To this end, the authors employed a linear strategy using the coupling of t-butyloxycarbonyl-protected amines to assemble trimeric compounds made of heterocyclic subunits A, B and C, randomized with 10, 11, and 12 components respectively. Compound isolation was performed using acid/base liquid−liquid extraction techniques. Library members were made in 264 mixtures of 10 compounds on reaction scales allowing multiple assays and were obtained with 95% purity. For screening, a rapid high-throughput assay for DNA-binding was developed based on the loss of fluorescence from a target oligonucleotide presaturated with ethidium bromide. Deconvolution of the most potent mixtures by resynthesis and evaluation of individual compounds ultimately revealed ligands 1000 times more cytotoxic than distamycin A. Selected library members were also found to bind poly[dA]–poly[dT] with comparable affinity, and one compound demonstrated high affinity (Ka = 4.5 micromolar) to the ARE-consensus sequence, a five base-pair AT-rich site relevant to the expression of a gene implicated in cases of prostate cancer that are irresponsive to hormone antagonists. The screening technique developed therein is also applicable to the evaluation of a single compound against libraries of hairpin oligonucleotides, hence providing qualitative and possibly quantitative determination of both binding affinity and sequence selectivity.

Spatially addressed synthesis of amino- and amino-oxysubstituted 1,3,5-triazine arrays on polymeric membranes. Scharn D, Wenschuh H, Reineke U, Schneider-Mergener J, Germeroth L: J Comb Chem 2000, 2:361-369. • Significance: There is significant interest in the development of new library formats that can combine the productivity of splitpool strategy with the advantages of spatially addressed parallel libraries to make non-peptidic compounds. This work on SPOT synthesis, a technique for the rapid generation of parallel libraries on two-dimensional surfaces, makes a significant step in this direction. Findings: Libraries of trisamino- and amino-oxy 1,3,5-triazines were elaborated by using amine and phenolate ions as building blocks, and by applying the SPOT synthesis technique on cellulose and polypropylene membranes. This required the development of an appropriate linker strategy, and the optimization of the chlorotriazine substitution chemistry by microwave activation. The resulting library members, identifiable by the position of their spot on the membrane, were cleaved with trifluoractic acid vapor, and remained adsorbed to the membrane for purpose of analysis and screening. Each spot affords enough material (50−250 nanomoles) for HPLC−MS analysis. A model library of 8000 1,3,5-triazines was synthesized and evaluated for binding to the anti-transforming growth factor-α-monoclonal antibody Tab2 as a means to identify epitope mimics. To this end, a secondary antibody assay was employed in conjunction with the detection of a chemoluminescent substrate. Five hits were found and the compounds were re-synthesized then tested in solution using a competition ELISA assay, revealing two library members displaying upper micromolar binding.

Probing the reactivity of solid supports via Hammett relationships. Gerritz SW, Trump RP, Zuercher WJ: J Am Chem Soc 2000, 122:6357-6363. • Significance: Little is known on the role of the insoluble resin matrix in solid-supported chemical reactions. Precise knowledge on the relative reactivity of the different supports used in solid-phase synthesis, as well as their sensitivity to reaction conditions and substrate substituent effects, would help in optimizing the choice of resin and building blocks for the synthesis of combinatorial libraries. Findings: A comparison on the displacement of solid-supported pentaflurophenyl esters from a variety of popular supports was performed with five electronically different parasubstituted anilines using competition experiments. A high-throughput strategy was employed in which data was collected from single experiments carried out with seven different and visually recognizable supports simultaneously in each vessel. The generation of Hammett plots revealed the strong influence of the nature of the support and the solvent on the Hammett constant ρ. In particular, it was found that ρ values for poly(ethylene glycol)-grafted polystyrene supports are comparable to those obtained in solution–phase

A mass spectrometry screening method for antiaggregatory activity of proteins covalently modified by combinatorial library members: application to sickle hemoglobin. Park S, Wanna L, Johnson ME, Venton DL: J Comb Chem 2000, 2:314-317. • Significance: Covalent modification of target proteins and enzymes is a valuable approach in drug design. There is a need for efficient analytical methods to identify combinatorial library members that can selectively modify biological receptors. Mass spectrometric methods such as the one described in this article have potential to be generalized to several systems of clinical interest. Findings: The authors developed a homogeneous assay to identify compounds from a small model library of isothiocyanates that covalently modify hemoglobin and inhibit the aggregation phenomena characteristic of sickle hemoglobin. In this assay, electrospray mass spectrometry was used to measure the distribution of modified proteins in the supernatant as compared with the aggregate. Mass measurements and validation of this assay by the saturation concentration method confirmed the antisickling activity of specific isothiocyanates providing the highest supernatant/aggregate ratio. Although

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the current resolution of the instrumentation used requires library members to be differentiated by at least 16 mass units, more sensitive spectrometers coupled to automation should extend applications of this technique to other protein systems of pathological interest. Designing small-molecule switches for protein-protein interactions. Guo Z, Zhou D, Schultz PG: Science 2000, 288:2042-2045. • Significance: New approaches are required to selectively regulate protein−protein interactions with the help of small molecules. As shown in this work, the combination of mutagenesis techniques and the use of small-molecule libraries can be a powerful tool to tackle this problem. Findings: A two-step approach was employed toward a new strategy for modulating protein−protein interactions in a model system based on the human growth hormone (hGH) and its associated receptor (hGHbp). A cavity was first introduced at the protein−protein interface by appropriate mutations (Thr175 to Gly−hGH and Trp104 to Gly−hGHbp). Next, a screening assay was developed to find compounds from a 200-member library of indole derivatives that would complement the interface cavity and restore the high association of the protein−protein complex. To this end, phage-displayed mutant hGH was panned with hGHbp mutant immobilized onto magnetic beads in the presence of library members as mixtures of about 10 compounds. Phage recovery ratio following extensive washes revealed one very promising pool of compounds. Re-evaluation of individual compounds from this pool was carried out, followed by surface plasmon resonance measurement of the binding increase of mutant hGH and hGHbp provided by the most promising ligands. One library member was found to increase the association of mutants hGH and hGHbp by more than a 1000-fold. Euclidean shape-encoded combinatorial chemical libraries. Vaino AR, Janda KD: Proc Natl Acad Sci USA 2000, 97:7692-7696. • Significance: The powerful split-pool method for library synthesis has become a popular technique in combinatorial chemistry. This method, however, requires rapid and efficient ways of encoding library members, which would circumvent the disadvantages of currently available encoding strategies such as the need for costly analytical instrumentation. Findings: This paper describes an encoding method based on the use of small Euclidean shapes from insoluble gel-type polymeric support. The monoliths are fashioned from lightly cross-linked polystyrene that was cut into circles, triangles, squares, pentagons, and hexagons reminiscent of ‘Lucky Charms’ breakfast cereals. The shapes have an identical surface area to volume ratio, display good swelling and mechanical properties, and are large enough to provide milligram quantities of product. The potential utility of shape encoding was demonstrated by the synthesis of a model split-pool library of 25 ureas from five amines and five isocyanates reacted onto bromoacetylated, Wang linker-derived monoliths. The identity of the library members was made possible by shape discrimination of the monoliths, thereby indicating the nature of the amine component used in the first synthetic operation. Although few compounds can be encoded using the current approach, the addition of more shapes combined with the use of color dyes could allow the endoding of much larger libraries.

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Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Harris JL, Backes BJ, Leonetti F, Mahrus S, Ellman JA, Craik CS: Proc Natl Acad Sci USA 2000, 97:7754-7759. •• Significance: Genome sequencing efforts are revealing numerous genes that encode for new protease enzymes with the potential of becoming targets for drug development. This creates a need for general combinatorial methods enabling the rapid determination of substrate specificity for these numerous proteases, and consequently the design of selective substrates and inhibitors. Findings: The use of the bifunctional 7-amino-4-carbamoylmethylcoumarin (ACC) in place of the more commonly used 7-amino-4-methylcoumarin (AMC) allowed the preparation of an ACC-based resin from which peptide substrates can be elaborated via Fmoc-based solid-phase synthesis. This flexible strategy enables the incorporation of all 20 proteinogenic amino acids at every desired position including the P1 position. The fluorogenic substrates made this way are cleaved from the resin with standard acidolytic conditions. Interestingly, the fluorogenic ACC leaving group shows added advantages over AMC. Its quantum yield is three times greater, permitting the use of lower concentrations of enzyme and substrates. Positional scanning libraries of as many as 137,180 P4–P3–P2–P1 tetrapeptide protease substrates were prepared and screened in solution for profiling the specificity of several different serine proteases and two cysteine proteases. The use of this rapid and general method to obtain a ‘fingerprint’ of the specificity of a large number of enzymes provides a means to design potent inhibitors with high selectivity. Linkers and cleavage strategies in solid-phase organic synthesis and combinatorial chemistry. Guillier F, Orain D, Bradley M: Chem Rev 2000, 100:2091-2157. • Significance: The choice of linker is a crucial element of a successful strategy for the elaboration of small molecule libraries by solid-phase strategies. The availability of up-to-date and comprehensive documentation on this topic can be of great help. Findings: This comprehensive review article is divided mainly by type of conditions required for linker cleavage (electrophilic, photochemical, etc.). There are over 500 cited references and several useful tables are included.

Next generation therapeutics Selected by Steven Projan Wyeth–Ayerst Research, Pearl River, New York, USA

Reverse transcriptase-polymerase chain reaction validation of 25 ‘orphan’ genes from Escherchia coli K-12MG1655. Alimi J-P, Poirot O, Lopez F, Claverie JM: Genome Res 2000, 10:959-966. • Significance: As multiple bacterial (and other) genomes are being completely sequenced, a common finding is that a significant percentage of the open reading frames (ORFs) identified ‘in silico’ represent putative genes that have no homologue based on amino acid similarity. For a given genome, these can represent up to half of the ORFs and the large majority of these have not been characterized in any manner. In this study, the authors have chosen 25 such ‘orphan’ genes in Escherichia coli and sought to determine whether these are expressed at the level of transcription. Indeed most of these orphan genes appear to be expressed. Findings: From the 1393 E. coli ORF sequences annotated by Blattner et al., 25 ORFs, all greater than 300 nucleotides in

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length, were selected for study. RNA was harvested from E. coli K-12 in either exponential or stationary phase of growth using Luria−Bertani broth (a nutrient medium) at 37°C. Oligonucleotide primer pairs were prepared based on the published sequences for the 25 ORFs and reverse transcriptase-polymerase chain reactions (RT-PCRs) were performed. RT-PCR is perhaps the most sensitive method for the detection of transcripts. Expression was not detected for six of the ORFs in either exponential or stationary phase, two of the ORFs were expressed in only one of the two phases and 17 were expressed in both phases. The authors conclude that such ‘hypothetical ORFs’ represent ‘bona fide genes’ in E. coli. Given similarities in genome sizes and structures this conclusion could well be extrapolated to other bacteria. An essential two-component signal transduction system in Mycobacterium tuberculosis. Zahrt TC, Deretic V: J Bacteriol 2000, 182:3832-3838. AND

Molecular characterization of two-component systems of Helicobacter pylori. Beier D, Frank R: J Bacteriol 2000, 182:2068-2076. • Significance: Signal transduction across the bacterial membrane is often mediated by what are now commonly referred to as two-component regulatory systems (TCRs) or two-component signal transduction systems. These consist of a pair of proteins one of which, the sensor/membrane protein, is a histidine kinase and the second, a cytoplasmic protein, is referred to as the response regulator. The histidine kinase auto-phosphorylates in response to the binding of a ligand and then relays the phosphate to the response regulator that then either activates or represses the transcription of target operons. (For a review see Hoch JA: Curr Opin Microbiol 2000, 3:165-170.) Two recent papers have described similar findings in two divergent bacterial pathogens (M. tuberculosis and H. pylori) (i.e. that genetic inactivation of specific response regulator genes was not possible). In both cases the authors conclude that these TCRs are ‘essential’ and may represent targets for novel chemotherapeutic agents. Findings: Zahrt and Deretic report that the mtrA response regulator gene could be disrupted on the M. tuberculosis chromosome only in the presence of a plasmid carrying an intact copy of mtrA. They also demonstrate, with mtrA-gfp fusions (gfp encoding for the expression of the reporter ‘green fluorescent protein’) that mtrA is differentially expressed during growth of M. tuberculosis in macrophage. Beier and Frank identified three response regulators in H. pylori based on sequence similarity (HP166, HP1043 and HP1021). They found that disruption of these putative genes was lethal despite the fact that two of these genes bore non-conserved substitutions in nominally well-conserved ‘receiver domains’. The finding that certain TCRs cannot be genetically disrupted is becoming a common one. Similar findings have been reported for Bacillus subtilis, Camphylobacter jejuni and Staphylococcus aureus, among others. In each case, it has been the response regulator gene that could not be disrupted. Given that many response regulators serve as both transcriptional activators and repressors (in both the phosphorylated and non-phosphorylated states) it is probably more likely that the authors are observing a form of toxic de-repression rather than the loss of an essential function. Otherwise, the histidine kinase gene should be equally as ‘essential’, with assertions of ‘cross-talk’ with other TCRs not withstanding. If this is indeed

the case then, contrary to popular belief, these are unlikely to make for good drug targets.

Analytical techniques Selected by Tony Cass Imperial College of Science, Technology and Medicine, London, UK

High-performance affinity beads for identifying drug receptors. Shimizu N, Sugimoto K, Tang J, Nishi T, Sato I, Hiramoto M, Aizawa S, Hatakeyama M, Ohba R, Hatori H et al.: Nat Biotechnol 2000, 18:877-881. • Significance: High-throughput analysis of receptor ligand binding interactions is an important aspect of the drug discovery process. Many known drugs have not yet had their molecular targets identified and this can limit their therapeutic efficacy. In principle, affinity capture of receptors by immobilised drugs provides a general approach to target identification; however, non-specific binding by other proteins in cell extracts can compromise this strategy. Findings: Latex beads 200 nm in diameter with a glycidylmethacrylate surface react with amine derivatives of drugs to provide a high surface area (20 m2/g), low non-specific binding matrix for affinity extraction of receptors from crude cell extracts. The performance of these materials is demonstrated by the specific extraction of the FK506-binding protein onto FK506 modified beads. In a further illustration of the power of this approach, the beads were modified with E3330 (an antiinflammatory compound) and used to specifically extract a protein that bound this compound. The protein could be specifically eluted and subjected to partial amino acid sequencing that allowed its idnetification. Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Harris JL, Backes BJ, Leonetti F, Mahrus S, Ellman JA, Craik CS: Proc Natl Acad Sci USA 2000, 97:7754-7759. •• Significance: Rapid profiling of enzyme activity in combinatorial compound libraries can be used to both elucidate the nature of an enzyme activie site and to rapidly identify novel substrates or inhibitors. Where the enzymes are proteases, the specificity is determined by the amino acids to the amino-terminal side of the cleavage site (the so-called P1, P2, P3… positions). The number of potential specificity determining sequences is rather large so a very sensitive assay method is required to screen mixtures of potential substrates. Findings: A synthetic route to 7-amino-4-carbamoylmethylcoumarin (ACC) P1-peptide derivatives is described that is compatible with standard solid-phase synthesis methods. The ACC leaving group shows a large increase in fluorescence when hydrolysed and this has been used to elucidate primary and extended substrate specificities of various proteases. Second-site screening with a spin-labeled first ligand. Jahnke W, Perez LB, Paris CG, Strauss A, Fendrich G, Nalin CM: J Am Chem Soc 2000, 122:7394-7395. •• Significance: The past few years have seen the use of NMR spectroscopy as a screening tool for identifying molecular fragments that can act as ligands for target proteins. This so-called ‘SAR by NMR’ is based upon identifying two ligands (first and second site ligands) that although individually possessing modest affinity when joined in a single compound can have nanomolar affinities. There are a number of technical problems associated with identifying the second site ligand when the protein has the first site saturated and this paper proposes a method to overcome these.

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Findings: The method is based upon using a spin-labelled derivative of the first-site ligand, which results in an enhanced relaxation rate of nuclei in the second-site ligand when both are bound to the protein. The enhanced relaxation rate (a consequence of the unpaired electron) reduces the amount of protein required in second-site screening by one to two orders of magnitude and the strong distance dependence of the relaxation rate allows the realtive orientation of the two ligands in the protein to be deduced. This in turn can guide the choice of appropriate linkers between the two. Targeting Alzheimer amyloid plaques in vivo. Wengenack TM, Curran GL, Podulso JF: Nat Biotechnol 2000, 18:868-872. • Significance: The only definitive method currently available for the diagnosis of Alzheimer’s disease (AD) is the post mortem identification of neuritic plaques in brain sections. A method that could be used prior to behavioural changes becoming apparent would allow early diagnosis and the potential for therapy. Findings: Amyloid-β peptide (Aβ) was previously shown to bind to neuritic plaques in vitro and offers a potential diagnostic tool if a similar process occurred in vivo. A mouse model of AD was used to demonstrate that Aβ radiolabelled with 125I and modified with the polyamine putrescine could cross the blood-brain barrier following intravenous injection. Subsequent autoradiography showed that the Aβ bound at sites in the brain that also stained with thioflavin S and hence were amyloid plaques. A quantitative, high-throughput screen for protein stability. Ghaemmaghami S, Fitzgerald MC, Oas TG: Proc Natl Acad Sci USA 2000, 97:8296-8301. • Significance: The measurement of protein stability in a high-throughput fashion would have applications in screening for altered stability either as a consequence of mutations, misfolding or ligand binding. Traditional methods for assessing protein stability are generally slow and require significant quantities of pure protein; they are thus not suited to a high-throughput approach. Findings: A method based upon hydrogen/deuterium (H/D) exchange coupled to matrix assisted laser desorption ionisation mass spectrometry (MALDI-MS) is described that can be used to assess protein stability in crude extracts in a quantitative fashion. The high sensitivity of MALDI-MS means that it is suitable for bacterial cultures grown in 96-well microtitre plates. The method is demonstrated with the maltose binding protein (in the presence and absence of maltose) and with eight mutants of the monomeric λ repressor.

Mechanisms Selected by Jon D Stewart University of Florida, Gainesville, Florida, USA

A single-molecule study of RNA catalysis and folding. Zhuang X, Bartley LE, Babcock HP, Russell R, Ha T, Herschlag D, Chu S: Science 2000, 288:2048-2051. • Significance: This is the first time that single-molecule observation techniques have been applied to understanding the conformational changes associated with RNA catalysis. Because most ribozymes undergo structural changes during catalytic turnover, the methods described here should be generally useful. Findings: A derivative of the Tetrahymena ribozyme, which catalyzes guanosine-dependent cleavage of an exogenous RNA substrate, was tethered to a streptavidin-coated solid support

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and its 3′ end was labeled with a fluorescent dye. Kinetic measurements demonstrated that immobilization did not affect catalysis. To probe structural changes that occur during the catalytic cycle, the 3′ and 5′ ends were labeled with different fluorescent dyes, allowing fluorescence resonance energy transfer (FRET) measurements. By calculating the time-dependent distance between the two dye molecules, three substrate-dependent folding pathways were discerned and the relative fluxes through each channel were determined. Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries. Harris JL, Backes BJ, Leonetti F, Mahrus S, Ellman JA, Craik CS: Proc Natl Acad Sci USA 2000, 97:7754-7759. • Significance: The methods described here should be applicable to determining the substrate specificity of proteases that do not require substrate contacts beyond the cleavage site (P1′, P2′, P3′ etc.). This task will become even more common as proteases identified by genome sequencing projects are characterized. Findings: Methods for standard solid-phase synthesis of peptides terminating with the leaving group 7-amino-4-carbamoylmethylcoumarin (ACC) were developed. The quantum yield for fluorescence of free ACC is approximately three times that for the traditional fluorogenic leaving group, 7-amino-4-methylcoumarin (AMC), and peptides containing this moiety are accepted by a variety of proteases. Several peptide libraries were prepared in which residues at specific positions were derived from mixtures of 19 amino acids (cysteine was omitted) and these were used to probe a variety of common proteases. Determining the rates of cleavage allowed the authors to deduce consensus peptide substrates. For proteases with known sequence preferences, these deduced substrates had the expected sequences. Mutation of Arg-166 of alkaline phosphatase alters the thio effect but not the transition state for phosphoryl transfer. Implications for the interpretation of thio effects in reactions of phosphatases. Holtz KM, Catrina IE, Hengge AC, Kantrowitz ER: Biochemistry 2000, 39:9451-9458. • Significance: Thio effects (comparisons between reaction rates of analogous phosphate and phosphorothioate esters) are widely used to probe transition states of phosphoryl transfer reactions. These results show that extra caution is warranted when interpreting the effects of sulfur substitution on enzymatic reactions. Findings: Steady-state kinetic parameters for cleavage of p-nitophenylphosphate and p-nitrophenyl phosphorothioate by wild-type and a mutant Escherichia coli alkaline phosphatase were determined. For the wild-type enzyme, sulfur substitution decreased the second-order rate constant (kcat/KM) by a factor of 70; however, a mutant lacking an arginine sidechain that forms hydrogen bonds with the phosphate (Arg166Ala) gave a thio effect of only 3. The possibility that this change in thio effects was caused by a greater degree of bond cleavage between the phosphorus and the leaving group in the transition state was ruled out by the observation that both the mutant and wild-type enzyme gave identical correlations between log(kcat/KM) and the pKa of the leaving group (βlg). This implied that the nature of the transition states for the phosphate and phosphorothioate were very similar. The authors suggest instead that the thio effects differ because sulfur substitution alters substrate binding, perhaps by interacting strongly with the active site Zn(II) ion.

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Model systems Selected by Graham RL Cousins and Jeremy KM Sanders University of Cambridge, Cambridge, UK

Direct comparison of electron transfer properties of two distinct semisynthetic triads with non-protein based triad: unambiguous experimental evidences on protein matrix effects. Hu Y-Z, Takashima H, Tsukiji S, Shinkai S, Nagamune T, Oishi S, Hamachi I: Chem Eur J 2000, 6:1907-1916. AND

Cyclodextrin-appended myoglobin as a tool for construction of a donor–sensitizer–acceptor triad on a protein surface. Hamachi I, Takashima H, Hu Y-Z, Shinkai S, Oishi S: Chem Commun 2000:1127-1128. •• Significance: Photosynthetic and mitochondrial respiratory systems rely on multi-step electron transfer processes facilitated by protein matrices. Fixed donor–acceptor distances, protein structural features and dipole moments induced by protein hydrogen bonding networks mediate these complicated electron transfer processes. Model systems are being developed with a view to mimicking and understanding such processes. The authors report the synthesis and study of donor–acceptor diads and triads hybridised to protein matrices — myoglobin and cytochrome b562 — to compare electron transfer events in a protein matrix to those in homogeneous systems. These semisynthetic systems, assembled with protein structures, may provide valuable insights into the roles of protein matrices in electron transfer processes and allow for the advancement of artificial photoreaction systems. Findings: The first article describes a covalent donor–sensitizer–acceptor triad incorporated into myoglobin and cytochrome b562. The triad employs a heme-based donor, a ruthenium bipyridyl complex as the sensitizer and a catenated cyclic viologen for the acceptor. Photoexcitation generated a charge-separated state via electron transfer from an excited triplet state of the heme-donor. In the absence of protein matrix, the charge separated state is still observed, although arises from an excited singlet state and not an excited triplet state. The lifetimes for the charge-separated states were significantly different for experiments with and without protein matrix. The protein matrix thus influences the electron-transfer pathway and the lifetimes of the charge separated states. The second article reports a donor–sensitizer–acceptor triad that is incorporated into the protein matrix via self-assembly facilitated by cyclodextrin binding to adamantane. Charge-separated states are observed for this system, but are not the direct result of electron transfer from the heme to cyclic viologen. Instead, they result from the photoexcitation of the ruthenium bipyridyl complex. However, this is a report of a non-covalently linked donor–acceptor triad on a myoglobin surface, which offers a new approach to novel semisynthetic electron-transfer systems.

demonstrates control over the folding of minimal parallel β-sheet model systems by manipulation of the local conformational preference of the linker segment. Findings: A study was performed of four tetrapeptide analogues each containing a proline-based core and L-valine and L-leucine strand residues connected via their carboxyl termini, which could form minimal parallel sheets should a hydrogen bond occur between the valine and leucine residues. Comparison of the D-Pro and L-Pro diastereomers in the linkers allowed the authors to probe the effect of local twist on parallel hairpin formation. Extensive NMR studies of these compounds confirmed that the D-Pro linker promotes β-sheet interactions whereas the L-Pro linker does not. The D-Pro influence over parallel sheet formation is also observed in N-to-N-linked tetrapeptide analogues. These results highlight the potential of proline configuration as a control for β-sheet formation. Phosphodiester bond cleavage mediated by a cyclic β-sheet peptide-based dinuclear zinc(II) complex. Yamada K, Takahashi Y, Yamamura H, Araki S, Saito K, Kawai M: Chem Commun 2000:1315-1316. • Significance: Molecules designed to function as models for enzymes are expected to increase our understanding of enzyme catalysis, especially the design of novel synthetic catalysts. Peptide and peptidomimetic structures have served as scaffolds for the synthesis of such enzyme models and the authors report the synthesis of a β-sheet peptide functionalised with two zinc(II) centers as a model for metal-center-catalysed phosphodiester bond cleavage. Many enzymes have been shown to exhibit active sites containing two metal ions, which operate cooperatively to effect catalysis — features that are mirrored in this model system. Findings: Cyclic peptide gramicidin S (GS) — which possesses a stable antiparallel β-sheet conformation — was functionalised through the sidechain amine groups, with two bis(2-pyridylmethyl) amino derivatives. This formed the basis of a dinuclear Zn(II) complex that accelerated the cleavage of the RNA model substrate 2-hydroxypropyl p-nitrophenyl hydrogen phosphate by a factor of 6500. A cooperative mechanism between the two metal centers is supported by comparison of relative rates of reaction with a mononuclear complex and a dinuclear complex not appended to the cyclic peptide scaffold. In both cases, the relative rate of reaction was much less than for the GS-dinuclear complex implying that the relative arrangement of the two metal centers, established by the cyclic peptide backbone, is integral to the catalysis observed. A pH dependence study suggested the mechanism involves Zn2+–OH– as a nucleophile, the other Zn(II) ion coordinates to the phosphate oxygen and thereby assists nucleophilic attack.

Biopolymers Control of hairpin formation via proline configuration in parallel β-sheet model systems. Fisk JD, Powell DR, Gellman SH: J Am Chem Soc 2000, 122:5443-5447. • Significance: Understanding β-sheet folding preferences is essential for predicting, modifying and controlling protein conformations. Model systems for antiparallel β-sheet conformations are beginning to provide insights into the covalent forces governing antiparallel β-sheet stability. Many β-sheet models are generated by linking two peptide strands with turninducing units. The authors report a study of proline-containing linkers for both N-to-N and C-to-C connection of peptide strands that assist parallel β-sheet formation. The article

Selected by Sabine Flitsch Edinburgh University, Edinburgh, UK

Fringe is a glycosyltransferase that modifies Notch. Moloney DJ, Panin VM, Johnston SH, Chen J, Shao L, Wilson R, Wang Y, Stanley P, Irvine KD, Haltiwanger RS, Vogt TF: Nature 2000, 406:369-374. AND

Glycosyltransferase activity of Fringe modulates NotchDelta interactions. Brueckner K, Perez L, Clausen H, Cohen S: Nature 2000, 406:411-415. •• Significance: The Notch receptor controls tissue patterning events in Drosophila. They are broadly expressed in animal

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development but are tightly regulated to allow formation of tissue boundaries. Members of the fringe gene family appear to be responsible for regulation but their mechanism of action had been a matter of speculation. The two papers define the function of Fringe and importantly suggest that glycosylation might be a novel and important process in signal transduction events. Findings: The mechanism by which Fringe can activate Notch is proposed to be as follows: Fringe acts in the Golgi as a β1,3 N-acetylglucosaminyltransferase that uses O-linked fucose residues on the epidermal growth factor modules of Notch as acceptor substrates. Glycosylation, which has been shown to be essential for Fringe action in vivo, alters the ability of Notch to bind its ligand (Delta). Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius has a unique Asn-linked highly branched hexasaccharide chain containing 6-sulfoquinovose. Zaehringer U, Moll H, Hettmann T, Knirel YA, Schaefer G: Eur J Biochem 2000, 267:4144-4149. • Significance: Isolation and structure determination of a novel N-linked hexasaccharide from archaebacterial glycoprotein with a number of unusual structural features. Findings: Cytochrome b558/566 is a membrane bound hemoprotein that is heavily glycosylated by O-mannosylation and N-glycosylation containing at least seven hexasaccharide N-glycans. The structure of the N-glycans was determined by spectroscopic methods and has a number of unusual features: firstly, N-glycosylation appears to be homogeneous, only a single hexasaccharide structure was isolated. Secondly the N-glycan contains a trisubstituted GlcNAc core and a rare acidic sugar, 6-sulfoquinovose. It is speculated that the sulfonate group on the N-glycan is important because it would be particularly stable and would remain charged at the low bulk phase pH of 2–2.5 the protein is likely to be exposed to. Highly efficient synthesis of β(1-4)-oligo- and -polysaccharides using a mutant cellulase. Fort S, Boyer V, Greffe L, Davies GJ, Moroz O, Christiansen L, Schuelein M, Cottaz S, Driguez H: J Am Chem Soc 2000, 122:5429-5437. • Significance: Selective mutagenesis of an endoglucanase resulted in a new enzyme that can catalyse the formation of a number of beta(1-4) glycans efficiently with high selectivity. Findings: Glycosidases have great potential in catalysing the formation of glycosidic bonds, even more so with the recent discovery that the glycosylation efficiency of an endoglucanase can be changed by site-directed mutagenesis of the active site carboxylate nucleophile. The present study shows how this can be applied to a cellulase. The resulting mutant enzyme can catalyse the formation of a range of β(1-4) glycans with high stereo- and regioselectivity and in high yields. Application of NMR based binding assays to identify key hydroxy groups for intermolecular recognition. Vogtherr M, Peters T: J Am Chem Soc 2000, 122:6093-6099. • Significance: Key hydroxyl groups often determine the selectivity of protein–carbohydrate interactions. Identifying such key hydroxyl groups can be a laborious affair because it requires synthesis of specific analogues. This paper points to a rapid nmr screening method of random analogue mixtures that can correctly identify the library members that bind to the target receptor. Findings: Sambucus nigra agglutinin binds the trisaccharide sialyllactose with micromolar affinity but has also millimolar

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affinity for galactose itself. From a library of randomly methylated galactose derivatives and their isomers those compounds that bound were identified by saturation transfer difference nmr in combination with 13C isotope labelling protocols. The nmr experiments agreed with previous biochemical data that the C3 and C4 hydroxyl groups in galactose were critical for binding. Selected by Philip AS Lowden University of Exeter, Exeter, UK

Tecto-RNA: one-dimensional self-assembly through tertiary interactions. Jaeger L, Leontis NB: Angew Chem Int Ed 2000, 39:2521-2524. • Significance: This is the first example in which RNA tertiary structure motifs are used to assemble artificial nucleic acid nanostructures. These results provide the basis for a wide range of self-assembling three-dimensional structures. Findings: RNA hairpins were designed carrying two GAAA tetraloops and/or GAAA tetraloop receptor motifs. They were shown to undergo Mg2+-dependent dimerisation mediated by tetraloop−tetraloop receptor interactions. RNA four-way junctions were then designed, based on fusions of these hairpins, and demonstrated to undergo one-dimensional self-assembly into oligomers of at least 15 molecules. A single-molecule study of RNA catalysis and folding. Zhuang X, Bartley LE, Babcock HP, Russell R, Ha T, Herschlag D, Chu S: Science 2000, 288:2048-2051. • Significance: This is the first report of single molecule studies of a ribozyme. The discovery of a previously unobserved folding intermediate and a new fast-folding pathway demonstrate the power of single molecule analysis for the study of ribozyme catalysis and folding. Findings: The Tetrahymena group 1 intron ribozyme was immobilised and labelled at two positions with different fluorescent dyes. Fluorescence resonance energy transfer between dye molecules on individual ribozymes was measured as a reporter for conformational changes during folding. A reversible local docking step was directly observed and kinetically characterised, and the overall folding was analysed. Three separate folding pathways could be discerned, including a new fast folding pathway, proceeding through a rarely populated state, unobservable by ensemble methods. One sequence, two ribozymes: implications for the emergence of new ribozyme folds. Schultes EA, Bartel DP: Science 2000, 289:448-452. •• Significance: This is the first example of an RNA sequence that can adopt two distinct tertiary structures with independent catalytic activities. It has strong implications for the evolution of diverse RNA functions in a hypothetical ‘RNA world’. New folds and activities could arise from existing ones, without proceeding through inactive sequences, and perhaps preceding gene duplication. Findings: The authors examined the sequences of two unrelated ribozymes – the hepatitis delta virus (HDV) self-cleaving ribozyme and an artificially selected self-ligating ribozyme – and derived a single sequence that could satisfy the base-pairing requirements for both starting ribozymes. This ‘intersection sequence’ displayed catalytic activity for both cleavage and ligation reactions, and structural probing demonstrated that it adopted the tertiary folds of both starting ribozymes, with no base pairs in common. Small changes in the sequence greatly accelerated the rate of either reaction,

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and these variants could be transformed into the prototype ribozymes through a series of neutral mutations. Direct haplotyping of kilobase-size DNA using carbon nanotube probes. Woolley AT, Guillemette C, Cheung CL, Housman DE, Lieber CM: Nat Biotechnol 2000, 18:760-763. •• Significance: This is a remarkably direct and potentially very powerful technique for the analysis of single nucleotide polymorphisms. It could lead to a great improvement in high-throughput genetic screening of, for example, markers for disease susceptibility. Findings: DNA templates were hybridised under stringent conditions with probe sequences labelled with streptavidin or the fluorophore IRD800. The hybridised DNA molecules were then imaged by atomic force microscopy using singlewalled carbon nanotube tips and the location of the probe determined at ~10 base resolution. Because of the different sizes of the labels, two different target sequences could be detected on the same DNA molecule. After validation on the M13mp18 plasmid, this technique was applied to the glucuronosyltransferase gene UGT1A7, whose enzyme product is involved in carcinogen inactivation. Haplotypes of individuals heterozygous at two sites crucial for cancer risk could be directly visualised. Selected by Richard Newman Imperial Cancer Research Fund, London, UK

Rab1 recruitment of p115 into a cis-SNARE complex: programming budding COPII vesicles for fusion. Allan BB, Moyer BD, Balch WE: Science 2000, 289:444-447. •• Significance: Intracellular membrane traffic involves the sequential budding of vesicles from a donor membrane followed

by vesicle fusion with a specific target membrane. The molecular mechanism involved in generating vesicles that know what their target membrane should be has been shown to be linked to a protein, designated p115, which is specifically recruited to vesicles budding from the endoplasmic reticulum. The presence of p115 then helps the vesicle to recognise when it has reached the correct target Golgi membrane and permits subsequent fusion. Thus there is a molecular program directing budded vesicles so that subsequent targeting and fusion with the Golgi apparatus can occur. Findings: The guanosine triphosphatase (GTP) Rab1 regulates the transport of newly synthesized proteins from the endoplasmic reticulum to the Golgi apparatus through unknown molecular interactions with effector molecules. The authors identified Rab1 effector molecules that specifically bound Rab1 in the guanosine 5′-O-3′-thiotriphosphate(GTP)bound form. They identified p115, a factor that tethers membranes together before SNARE-regulated fusion, as a prominent protein retained by GST-Rab1-GTP beads. They identified the specific step in vesicle transport requiring p115 they used cell-free assays that separately reconstituted COPII vesicle budding and COPII vesicle fusion with Golgi membranes. The interaction between Rab 1 and p115 only occurs when Rab1 is bound to GTP. This observation is important because those proteins that bind exclusively to the GTP-bound form of Rab are known from previous studies to be critical for membrane targeting. Also p115 is found in a complex with a number of targeting SNAREs (membrin, rbet1, and syntaxin5) on vesicles travelling from the ER to the Golgi. So during budding the Rab protein directs the assembly of a tethering protein p115 into a complex of SNAREs that will subsequently be involved in the targeted fusion of the vesicle.