Tandem mass spectrometry of intact GroEL-substrate complexes reveals substrate-specific conformational changes in the trans ring

It has been suggested that the bacterial GroEL chaperonin accommodates only one substrate at any given time, due to conformational changes to both the cis and trans ring that are induced upon substrate binding. Using electrospray ionization mass spectrometry, we show that indeed GroEL binds only one molecule of the model substrate Rubisco. In contrast, the capsid protein of bacteriophage T4, a natural GroEL substrate, can occupy both rings simultaneously. As these substrates are of similar size, the data indicate that each substrate induces distinct conformational changes in the GroEL chaperonin. The distinctive binding behavior of Rubisco and the capsid protein was further investigated using tandem mass spectrometry on the intact 800-914 kDa GroEL-substrate complexes. Our data suggest that even in the gas phase the substrates remain bound inside the GroEL cavity. The analysis revealed further that binding of Rubisco to the GroEL oligomer stabilizes the chaperonin complex significantly, whereas binding of one capsid protein did not have the same effect. However, addition of a second capsid protein molecule to GroEL resulted in a similar stabilizing effect to that obtained after the binding of a single Rubisco. On the basis of the stoichiometry of the GroEL chaperonin-substrate complex and the dissociation behavior of the two different substrates, we hypothesize that the binding of a single capsid polypeptide does not induce significant conformational changes in the GroEL trans ring, and hence the unoccupied GroEL ring remains accessible for a second capsid molecule.     

Towards future reference systems for GM analysis

Despite the fact that the measurement unit for the quantification of GMOs in food and feed products has not yet been unambiguously agreed upon in Europe, international trade requires reliable GMO analysis measuring comparably the GMO content of products. The two reference systems, based either on mass fractions or on copy number ratios, and their metrological traceability chains are presented and discussed. It is concluded that, properly established and expressed, measurement results in copy number ratios can provide a metrologically sound reference system. In this case, certified reference materials used for calibration and quality control can be independent of each other and the uncertainty derived from calibration can correctly be included in the overall uncertainty of the GMO measurement. However, further efforts are required to establish this metrological system.     

Training Course “Selection and Use of Reference Materials”, IRMM, Geel, 3–4 October 2006

The training course “Selection and use of reference materials” held at IRMM is described. This course dealt with practical and theoretical issues in the use of reference materials.     

Improving small laser light sheets by means of a diffractive optical element

Conventional optical elements like lenses and stops for the generation of light sheets in experimental fluid mechanics are challenged by modern diffractive optical elements (DOE) made to the specifications of the user. We report on a first application in connection with droplet investigations suggesting an enormous potential of the new technique.     

Intercomparison on measurements of PCBs in pork fat during the Belgian PCB-crisis

During the Belgian PCB crisis (1999/2000) the quality of the data from the PCB monitoring were studied with a proficiency testing experiment. Pork fat that was spiked at the Institute for Reference Materials and Measurements (EC-JRC-IRMM) was sent out as unknown to all laboratories that participated in the monitoring. In parallel, the material was certified on the basis of the results of several leading PCB laboratories throughout Europe that did not participate in the monitoring. During the first round 15 to 25% deviation was experienced, while in the end of this multistep intercalibration procedure the average deviation for the respective PCBs was 10.9% (PCB 28), 13.1% (PCB 52), 10.1% (PCB 101), 10.7% (PCB 118), 10.7% (PCB 138), 9.1% (PCB 153), 8.1% (PCB 180) and 8.2% for the sum of the 7 PCBs. The concentrations measured for the higher volatile PCB 28 had a tendency to show lower levels, while for PCB 180 this was less pronounced. On the other hand, PCB 153 showed results rather to the higher side. During this exercise the material was also tested for stability by one of the certifying laboratories.     

Production of certified reference materials for mycotoxins: IRMM’s view on the assessment of uncertainties

Analytical difficulty and the economic importance of controlling mycotoxin levels in food and feed led the Community Bureau of Reference (BCR) to prepare a series of certified reference materials (CRM) for various mycotoxins. Because of the wide acceptance of these CRM and the need to ensure the comparability and traceability of measurements in the future it is necessary to prepare and certify new batches of mycotoxin reference materials (RM). In the following text two different approaches for evaluation of the characterisation uncertainty of CRM will be compared using the certification of aflatoxin M₁ (AfM₁) in milk powder as an example. The conventional approach is based on evaluation of characterisation exercise data; the alternative approach is based on measurement uncertainties of the employed analytical methods. Because laboratories are using totally different approaches to estimate the measurement uncertainties, combination of the uncertainties obtained from the participating laboratories was not recommended. Therefore, a new integrated approach for assessment of the measurement uncertainties of the analytical methods on the basis of additional data collected during the characterisation exercise will be described. The conventional approach was found to be the most appropriate and economical approach to evaluate the characterisation uncertainty as a characterisation exercise must be performed anyway to establish the property values of candidate (C)RM, irrespective of whether or not reliable measurement uncertainties can be provided by the laboratories. An integrated approach for assessment of measurement uncertainties based on additional characterisation data as applied here to enable use of an uncertainty-based approach provides more information but is too time-consuming and cost-intensive to become common practice.     

Block and graft copolymers based on cationic ring-opening polymerizations

The highly living character of a number of cationic polymerizations has been used to synthesize new, well defined, segmented copolymers. This has been achieved by sequential monomer additions, by grafting reactions, by macromonomer copolymerizations as well as by transfer to polymer. In the present report, a number of examples of such polymers will be presented. The new materials are based on tetrahydrofurane (THF), alkyloxazolines (ROx), cyclic amines, cyclic acetals and cyclic sulfides.     

New materials from telechelic polyacetals

Telechelic polyacetals, obtained by cationic ring-opening polymerization of 1,3-dioxolane (DXL) and 1,3-dioxepane (DXP), have been used as building blocks for polymer materials. In the first part of this paper, the synthesis and the properties of networks based on polyDXL α,ω-bis(methacrylates) are discussed. The second part deals with the synthesis and the properties of polyacetal polyurethanes. A thermoplastic polyurethane was prepared with poly(DXL-co-DXP) α,ω-diol as soft segment and the combination butane-1,4-diol and hexamethylene diisocyanate as hard segment. Polyurethane networks were obtained from polyacetal polyols and diphenylmethane diisocyanate (MDI). Some physical properties of these new materials are reported.     

Stability of thermocapillary-driven flow in rectangular cavities

The flow in an infinitely extended cavity with rectangular cross section is considered. The upper boundary is a free surface where thermocapillary forces are driving a circulation within the cavity (Marangoni effect). If the driving force (temperature difference) exceeds a critical threshold, the two dimensional flow becomes unstable towards an infinitesimal three-dimensional perturbation. The critical mode depends on the aspect ratio and may be steady or oscillatory. Results are presented for vanishing Prandtl number and varying aspect ratios of the cavity. These are compared to the classical lid-driven cavity problem and the mechanism leading to instability is discussed. (© 2005 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim)