Hot off the Press

In the Hot off the Press section of Molecular BioSystems members of the Editorial Board and their research groups highlight recent literature for the benefit of the community. This month the highlighted topics include high-throughput screening of mutant glycosyltransferases, the structure of a Mg⁺² channel/transporter and the use of t-RNA as a template for quantum dot synthesis and some items published recently in the RSC’s journals.

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Directed evolution of glycosyltransferases: a fluorescence-based high-throughput screening (HTS) methodology

Despite rapid developments in the combinatorial synthesis of peptides and oligonucleotides, the chemical synthesis of another important biopolymer—carbohydrates—remains challenging. Glycosyltransferases (GTs), enzymes that transfer an activated sugar group to a specific donor, are involved in a wide range of glycosylation reactions in the cell. Glycosyltransferases with improved stability, accelerated activity, or altered substrate specificity could be useful tools for the synthesis of carbohydrates, to study their biological roles. However, the directed evolution of glycosyltransferases has been hampered by the lack of efficient screens. The Withers lab has developed a high throughput screening methodology to facilitate the screening of large mutant libraries of glycosyltransferases and is particularly valuable for enriching and isolating rare mutants with beneficial activity.

By using a carefully designed fluorescently-labeled acceptor sugar and selectively trapping the fluorescent product in the cell, the formation of transfer product is directly correlated to the fluorescence of the intact Esherichia coli cell. The resulting cell population is then analyzed and sorted using a fluorescence-activated cell sorter (FACS). Using this new methodology, a new CstII variant was identified. CstII transfers sialic acid (Neu5Ac) from CMP-Neu5Ac to the acceptor saccharide (bodipy-lactose); the evolved variant accomplishes the reaction with 400 fold higher catalytic efficiency.

The crystal structure of the mutant F91Y in complex with CMP-3F-NeuAc, together with computational analysis of a model of bodipy-lactose bound to CstII, suggests that the improved activity of the CstII F91Y mutant is due mostly to the improved binding of the poor substrate bodipy-lactose. This fluorescence-based HTS method should allow for the evolution of GTs with new catalytic activities, providing new synthetic routes for complex carbohydrates and other glycoconjugates.

 

A. Aharoni, K. Thieme, C. P. Chiu, S. Buchini, L. L. Lairson, H. Chen, N. C. Strynadka, W. W. Wakarchuk, S. G. Withers, Nat. Methods, 2006, 3(8), 609–14.

Reviewed by: Yanqiu Yuan, Department of Chemistry and Chemical Biology, Harvard University

Bringing Mg²⁺ into cells

Three groups have determined the structure of CorA (Fig. 1), a protein involved in Mg²⁺ transport into the cytoplasm of bacteria, with homologs in mitochondria. Mg²⁺ is the last of the common biological ions for which the structure of a transporter or channel was lacking. The three structures are almost identical. CorA is a homopentamer. The C-terminal transmembrane domain consists of ten helices, two from each subunit. Five inner helices line the passage through the lipid bilayer. Constrictions in the passage suggest that the structures represent a closed form of CorA. The protein extends only small way into the periplasm, where a divalent metal-ion binding site with a conserved sequence motif is found, which is the site of Mg²⁺ entry and most likely responsible for the ion selectivity of CorA. By contrast, on the intracellular side, the major mass of the protein extends into the cytoplasm as a funnel of ~65Å, ending with a wide entrance 20Å in diameter. A remarkable α-helix, 100Å in length, begins as the inner transmembrane helix and spans the entire cytoplasmic domain. An αβα domain of unknown function, suggesting an unidentified regulatory activity, resides on the external surface of the cytoplasmic domain outside the wide entrance. Two Mg²⁺-binding sites are found between each subunit interface in the cytoplasm and may serve as sensors linked to the closure of CorA when the cell contains sufficient Mg²⁺. The distribution of charged side chains in the cytoplasmic domain is highly unusual and may be involved in transport and gating. The striking structure of CorA raises several questions. CorA looks like a channel and yet it has been implicated in the transport of Mg²⁺ into the cell against a concentration gradient.

The evidence supporting this idea must now be checked. If CorA is indeed a transporter, its energy source must be identified and the in vitro transport activity of the pure protein verified.

Fig. 1 Overall representation of the CorA. The position of the transmembrane region is indicated. From Said Eshaghi, Damian Niegowski, Andreas Kohl, Daniel Martinez Molina, Scott A. Lesley, and Pär Nordlund, Science, 2006, 313, 354–357. Reprinted with permission from AAAS.

 

Vladimir V. Lunin, Elena Dobrovetsky, Galina Khutoreskaya, Rongguang Zhang, Andrzej Joachimiak, Declan A. Doyle, Alexey Bochkarev, Michael E. Maguire, Aled M. Edwards, Christopher M. Koth, Nature, 2006, 440, 833–837.

Said Eshaghi, Damian Niegowski, Andreas Kohl, Daniel Martinez Molina, Scott A. Lesley, and Pär Nordlund, Science, 2006, 313, 354–357.

Payandeh and Pai, EMBO J., 2006, 25, 3762–3773.

Reviewed by: Ellina Mikhailova, University of Oxford, UK

Giant leaps for microtubules

Microtubules are highly dynamic cytoskeletal polymers that consist of tubulin-dimers. They are key players in cell functions such as trafficking and mitosis, and they are important for the shape and organization of cells. One interesting characteristic of microtubules is that they continuously alternate between periods of growth and shrinkage. The average growth and shrinkage rates have been quite well characterized in the absence and in the presence of associated proteins that regulate their dynamics, however only little is known about the growth and shrinkage mechanisms on a molecular scale.

Jacob Kerssemakers, Marileen Dogterom and colleagues now studied the dynamics of growing microtubules on the scale of individual dimers. They let single microtubules nucleate from axonemes, and polymerize against a microfabricated wall from purified tubulin. The growth steps were measured using a so-called "keyhole" optical trap, which also directed the microtubule against the wall (Fig. 2). Using an original step finding algorithm, they showed that growth occurs with steps of about 20 nm, which is larger than the maximum step-size of 8 nm expected for growth from individual dimers. Interestingly, when the same experiments were conducted in the presence of purified XMAP215, a protein that enhances the growth dynamics, the average step length became about 50 nm. The authors suggest two possible mechanisms to explain the increased step-size in the presence of XMAP215: XMAP215–tubulin complexes, preformed in solution, might attach at once at the growing end of the microtubule, or XMAP215 might first binds to the growing end of the microtubule and thereby enhance the rate of binding of tubulin dimers.

In vivo the growth dynamics of microtubules is determined by a variety of interacting microtubule-associated proteins. Understanding the molecular details of the interactions should be an important step forward for the understanding of the regulation of microtubule dynamics in cells.

Fig. 2 Schematic view of a growing microtubule, and schematic and DIC images of a ‘keyhole’ optical trap holding a microtubule in front of a microfabricated barrier. High-resolution details of growth and shrinkage events in the presence of XMAP215. Arrows indicate steps. The insets show schematic pictures of an XMAP215 molecule (left) and a microtubule (right) at the same vertical scale as the length data. Reprinted by permission from Macmillan Publishers Ltd: Nature (2006, 442, 709), copyright (2006)

 

Jacob W. J. Kerssemakers, E. Laura Munteanu, Liedewij Laan, Tim L. Noetzel, Marcel E. Janson and Marileen Dogterom, Nature, 2006, 442, 709.

Reviewed by: Benoit Sorre and Gerbrand Koster, Physicochimie Curie, Institut Curie, Paris

Antibody nanoarrays

In the past decade, properties of DNA were successfully used for the immobilization of biomolecules and for the design of a range of nanoarchitectures. For example, DNA arrays served as templates for nanoparticle and macromolecule organization enabling thrombin and streptavidin organization in 1D and 2D arrays. However, the questions remained if enzymes and antibody arrays, which would have a significant applications in immunodiagnostics, could also be produced using DNA templates.

Chendge Mao and his coworkers from Purdue University have now answered that question in a proof of concept experiment by which they designed 2D antibody arrays based on a fluorescein antigen. They assembled nine DNA strands in a 2D array with a repeating distance of 19 nm. Prior to assembly, fluorescein moieties were covalently coupled to the central DNA strand in such way that the final 2D array contains 2 fluorescein molecules at each cross motif. They can then bind to 2 sites of Y shaped anti-fluorescein monoclonal antibody (IgG). AFM measurements showed that fluorescein modified motifs retained their ability to form a 2D array in a same manner as non modified do and an IgG array was then obtained after self assembly of such motifs and incubation with IgG. Interestingly the number of fluorescein (antigen) moieties at a cross motif plays an important role in a formation of array. When only one molecule is present, there is no appreciable antigen–antibody binding, which leads to random distribution of antibodies relative to DNA array.

This proof of concept paper does not have yet any practical application, but it rather elegantly describes a use of DNA templating, covalent DNA modification and antibody–antigen interaction to build well defined 2D arrays. Authors believe that using this type of arraying single molecule studies of antigen–antibody interactions could be made feasible and the assembling procedure, which is done in the solution, might hold great promise fro future in vitro and in vivo studies.

 

Y.He, Y. Tian, A.E. Ribbe, C. Mao, J. Am. Chem. Soc., 2006, 128, 12664–12665.

Reviewed by: Ljiljana Fruk, Universität Dortmund, Germany

t-RNA aids the nanocrystal synthesis

Development of novel methods of nanomaterial design is one of the current hot topics in nanotechnology. Several successful methods are making use of high programmability and well described three dimensional properties of biomolecules (proteins, DNA) to design nanomaterials of defined size and structure.

Now a group of scientists from Boston University has successfully used RNA as a template for CdS quantum dot synthesis. They have chosen t-RNA as a template because of its size (5 nm) and compact structure. The simple synthetic protocol was developed which involved mixing quantum dot precursor CdCl₂ with t-RNA template followed by a 10 min incubation and addition of another precursor Na2S. Analysis of the products showed that different types of quantum dots are obtained in the presence and absence of t-RNA. RNA assisted synthesis yielded a highly soluble material with average particle size of 6 nm which can be easy analysed by gel electrophoresis while non assisted synthesis resulted in poor solubility and larger distribution of particle sizes. To assess the influence of RNA structure on the properties of CdS particles, mutant RNA was designed in such way to disrupt the compact conformation of wild type RNA. In the presence of such mutant larger and less defined nanostructures were produced. On the other hand, wild type RNA yielded a single product type with a compact structure which pointed out the importance of the use of structurally well defined template. Generation of well defined single product in the combination with the simplicity of protocol makes t-RNA a very promising template for nanomaterials engineering.

 

N. Ma, C.J. Dooley, S. O. Kelley, J. Am. Chem. Soc., 2006, 128, 12598–12599

Reviewed by: Ljiljana Fruk, Universität Dortmund, Germany

Hot off the RSC Press

Monitoring cell survival in chips

A way of monitoring cell respiration in microchips could lead to better devices for fertility research.

Microfluidic devices are used to manipulate biological cells, for example, in sorting human sperm, but information on how well the cells are surviving within such microchips can be limited. Now, Hitoshi Shiku at Tohoku University, Sendai, Japan, and colleagues have used a technique called scanning electrochemical microscopy (SECM) to monitor individual cells in a microfluidic device by measuring their oxygen uptake.

Shiku and colleagues developed their method using a channel filled with Escherichia coli cells as a model of a microfluidic device. One face of the channel was formed from a membrane through which oxygen could flow freely and the team used SECM to measure the oxygen concentration along this face. Shiku’s co-worker Takeshi Saito said that because the measurement takes place on the other side of an oxygen-permeable membrane, disturbance to the cells is avoided.

Shiku found that oxygen uptake levels for the cells in his model device differed from values reported for similar cells trapped in collagen gels. This means the dimensions and material of a chip affect oxygen uptake and therefore how well the cells survive, said Shiku. Being able to monitor cell respiration in this way should lead to improved designs for chips for biological research, he added.

Commenting on the work, Salvatore Daniele, an expert in electrochemistry from the University of Venice, Italy, said that the main advantage of the approach was its potential for continuous monitoring of cell activity in very small volume samples.

Future work will involve applying the method to other cell suspensions such as semen and Saito says that this technology could have a role in studies aimed at improving human fertility.

 

T Saito, C-C Wu, H Shiku, T Yasakawa, M Yokoo, T Ito-Sasaki, H Abe, H Hoshi and T Matsue, Analyst, 2006, 131, 1006–1011.

Reviewed by: Michael Smith, Royal Society of Chemistry, Cambridge, UK.

Resistance tracks cell mobility

A downscaled geophysical technique could be used to study biological processes such as wound healing, according to Swiss bioengineers.

The resistance of cells to an alternating current can yield information about cell and tissue structure and movement, said Pontus Linderholm. With colleagues from the Swiss Federal Institute of Technology in Lausanne, he created an electronic device that measures this resistance in a layer of cells grown on top of it (Fig. 3). The system images vertical cross-sections of the cell layer, and provides more structural information than current electrical techniques.

According to the team, their method can gain information ranging from the density of cells in a tissue to the type of cells present. They successfully used the technique to study the migration of cells across a ‘cut’ made in a cell layer.

Linderholm said that the devices may be used to gain a better understanding of cell migration in the body, for instance in wound healing and the development of embryos.

He also sees potential for drug-screening: ‘It is important to know what effect drugs have on cell mobility,’ he said. He added that being able to screen hundreds of compounds simultaneously using electronics would save a lot of time compared to studying the effect of each compound on cell movement through a microscope.

The group borrowed their technique from geophysicians, who use resistance measurements on a much larger scale to study things like glaciers and groundwater flow. Although the idea of using this technique to image cells is about twenty years old, Brian Brown of the University of Sheffield, UK—himself a pioneer in the field—believes that this is the ‘first significant development of a microsystem able to record cell migration’.

Fig. 3 Skin cells growing on electrodes. Reproduced from Lab Chip, 2006, 6, 1155 by permission of The Royal Society of Chemistry.

 

P Linderholm, T Braschler, J Vannod, Y Barrandon, M Brouard, P Renaud, Lab Chip, 2006, 6, 1155.

Reviewed by: Danièle Gibney, Royal Society of Chemistry, Cambridge, UK.

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