Final Report Summary - EAST-NMR (Enhancing Access and Services To East European users towards an efficient and coordinated panEuropean pool of NMR capacities to enable global collaborative research & boost technological advancements)
Executive Summary:
Nuclear magnetic resonance (NMR) spectroscopy is a key technology for research in the modern Life Sciences, with an increasing impact on human health. This technology is unique in new areas of molecular systems biology providing detailed insight into protein-protein and protein-ligand interactions.
In the face of increasing international competition, the composition of the consortium aimed on full exploitation of scientific and technological capabilities throughout the EU. This requires the full mobilization of all European countries, including Eastern Europe, where NMR applications are sparse and tackling challenging scientific projects is less common. Thus, Eastern and Western European Research Infrastructures teamed to address emerging technological and research frontiers.
The EAST-NMR project integrated pan-European potential for state-of-the-art NMR research by:
(i) Provision of transnational access to NMR instrumentation based in Eastern facilities. Beneficiaies in Ljubljana (SI), Brno (CZ), Warsaw (PL) and Debrecen gave 784 days of access to European guest scientists. Western beneficiaries offered 396 days which includes 328 days of access to solid-state NMR, an emerging technology at the international level. Altogether, 1180 days of access (out of stipulated 1060 days) were given to 150 European guest scientists.
(ii) The consortium educated and trained researchers in NMR’s potential and use, with special care for making aware for the scientific potential of Eastern Europeans. The project reached out to the Life Sciences community to attract new users in the general field of Structural biology at a total number of 105 events. Five major conferences with internationally reknown keynote speakers attracted more than 420 participants. 17 national/regional conferences raised local awareness for the benefits that bio-NMR can generate in their research. Overlapping with the Transnatioal Access endeavour, local operator or staff meetings, respectively, focused on skill enhancement as well as standard operation procedures to record the best possible NMR spectra at the available equipment.
(iii) Efforts of industrial and academic beneficiaries were joined and standard procedures for the production of proteins for solid-state NMR spectroscopy, including membrane and membrane associated proteins were optimized.
Project Context and Objectives:
Nuclear magnetic resonance (NMR) spectroscopy is a key technology for research in the modern Life Sciences, having proven to be invaluable for the investigation not only of the structure and dynamics of biomolecules, but also for their functional characterization. As a result, NMR is increasingly applicable to fields such as structural proteomics, molecular systems biology and metabonomics, and provides a basis for drug discovery. The maturation of NMR technology impacts high-performance biomedical applications from the advancement of research frontiers.
NMR Research Infrastructures (RIs) are of utmost importance in this process, as they offer the necessary facilities, resources and related services to conduct cutting-edge research, and to disseminate, exchange and preserve the generated knowledge to a broad group of European users. Substantial investments in new high-performance NMR facilities have taken place throughout the United States, Canada, and Asia. In response to such imminent competition, the scientific and technological capabilities of the EU Member States and Associated Countries must be mobilized in order to exploit their tremendous potential for the generation of knowledge and innovation. The potential for a corresponding impact on competitive sectors such as pharmaceuticals and biotechnology must not be ignored.
At present, pan-continental support for major European NMR investments is clearly imbalanced; the Eastern European countries have a significantly less-developed infrastructure than what is enjoyed by Western Europe. As a result, half of the human resources in European NMR are being dramatically underused. This is evidenced by a net knowledge flux by Eastern European scientists outside of Europe, and especially, at present, to the US and Canada. The massive investment levels in Asia threaten that a net flux in this direction may be coming as well, even when cultural and language barriers are taken into account. Without strengthening the role of the East-European scientific communities, Europe may in total loose its pace and impact in decades to come. Development of NMR methodology has been a stronghold of European, with three recent Nobel prizes (R. Ernst, P. Mansfield and K. Wüthrich), and we will only stay in this position, if the entire European human potential can be integrated and maintained.
The Eastern European countries have the potential to play a key role in raising the competitiveness of the European research area, especially in terms of human capital and openness to innovations. However, little Eastern European demand currently exists for accessing not only RIs in NMR spectroscopy, but RIs in general. This is a disturbing trend when the myriad benefits that a strong RI system can provide are considered. There are many possible reasons for this low demand – including an insufficient knowledge about the full potential of NMR spectroscopy for tackling problems in Molecular Biology, a perceived inability or lack of understanding about the potential for accessing RIs, or something as fundamental as difficulties in preparing NMR-ready samples. Some progress has been observed in the Eastern countries in the last ten years, and local investments in infrastructures have been made. However, few funds are available for research groups for national or transnational access.
A concurrent development in the state of NMR research is a significantly increased demand for solid-state NMR in the Life Sciences, especially for the study of membrane proteins. G-protein coupled receptors are the paradigm here – the first receptor-agonist structures were recently solved by solid-state NMR spectroscopy, a finding of tremendous importance for European pharmaceutical industries.
The new Integrated Activity EAST-NMR responds to the above-described situation by:
• Increasing awareness among researchers of the possibilities that NMR RIs offer for the advancement of their research and its applications and performing adequate training,
• Providing an integration of Eastern and Western European RIs, providing NMR measurement time and scientific expertise to researchers, who currently cannot access NMR RIs at a national or regional level,
• Providing NMR measurement time for solid-state investigations, including of membrane proteins and their complexes with agonists and antagonists, a new frontier at the international level,
• Performing joint research for the optimization of membrane protein samples for liquids and solids and appropriate dissemination of the results.
EAST-NMR will combine the following activities in a co-ordinated manner, based upon the Integrated Activity model: Networking, Trans-national access and Joint research activities, in order to achieve the following objectives:
• To promote the excellence of research in the Life Sciences by increasing the pool of users accessing NMR RIs,
• To strengthen the emerging field of solid-state NMR spectroscopy,
• To achieve advancements in special sample preparations of membrane proteins, necessary to fully exploit emerging opportunities in NMR.
Project Results:
Improving screening technologies for NMR Studies:
Extensive automation implemented by the combination of database tools, mechanization of key process steps and the use of micro-cryoprobes that give excellent data while requiring little optimization and manual setup. For screening NMR samples as part of a sample optimization process, the optimal construct design and solution conditions were assessed as well as for determining protein rotational correlation times in order to have an estimation about the protein oligomerization states. The use of databases allows for flexible implementation of new screening protocols and harvesting of the resulting output. The NMR screening pipeline also uses now detergent screening for membrane proteins.
Selective labelling techniques reduce the severe resonance overlap observed for many MPs and simplify spectra. The continuous-exchange cell-free expression (CECF) system offers full labelling flexibility with limited scrambling (some degree of scrambling has been observed in cases of aspartate, asparagine, glutamate, and glutamine). Apart from labelling single amino acids, sophisticated selective and combinatorial labelling schemes were developed. More elaborate descriptions are given below.
High-Throughput Screening Assays for Cys-mutated and metal-chelat tagged Proteins:
Protocols for testing the stability of Cys-mutated and metal chelated-tagged proteins compatible with a high-throughput screening assay were developed. The approach consists of several steps. In both cases the first step involves the selection of cysteine mutation sites, and the introduction of mutations using molecular biology techniques. The next step is the stability screening and sample condition optimization of proteins and protein domains, with temperature, denaturant and time as parameters. This step uses a microscale high-throughput UV absorbance and fluorescence assay based on the fluorescent dye SYPRO Orange or on direct tryptophan fluorescence. The setup uses 96-well plates, and can be scaled up for high-throughput analysis. The third step involves a scale up of soluble and stable candidates for screening of foldedness by NMR and activity assays.
Large Scale Screening of conditions for obtaining Microcrystalline Samples:
An integrated approach with the Grenoble High-Throughput Crystallization facility was developed for the setup of reliable protocols towards microcrystalline powders for NMR analysis. Strategies used in HTPX (High Throughput Protein Crystallography) were adapted to the case of Large Scale Screening of conditions for obtaining microcrystalline samples. This has resulted in a series of crystallisation plates now available to the NMR community for the automatic screening of NMR-compatible crystallogenic conditions (buffer, pH, precipitants). An automated protocol allows then monitoring of crystal growth, screening of scale-up conditions specific to NMR, and subsequent handling of microcrystalline powders with great efficiency.
Automated Cell-free Scanning Methodologies:
For NMR spectroscopy, cell-free expression has the additional benefit that it allows us to label proteins with isotope-labelled amino acids in almost any combination due to the almost complete absence of metabolic scrambling. These labelling schemes include combinatorial labelling that enables the amino acid specific assignment of resonances and the Transmembrane Segment- (or short: TMS)-labelling scheme that makes use of the fact that transmembrane helices consist to a large extent of only six different amino acid types. It was found at several test protein constructs that using the 6 different amino acids G, A, V, L, I and F turned out to be the most useful one. Obtaining the backbone assignment of the protein turned out to be sufficient to calculate structures based on the measurement of distance restraints via paramagnetic relaxation enhancement (PRE) measurements, proving that the concept of backbone assignment in combination with PREs can provide structural information.
The use of cell-free methodologies to express metalloproteins or proteins involved in metal ions trafficking avoiding heterologous expession in bacteria were developed. Several protein targets differing by molecular weight, origin (human or bacterial), toxicity, solubility in water envinroment, presence of disulfide bonds were expressed using the cell-free strategy.
Support for the automated cell-free protein preparation by the software tool “UPLABEL”:
The choice for the most-efficient labelling scheme is supported by a software tool “UPLABEL” developed in a collaboration. UPLABEL is an algorithm for finding optimal labelling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional methods. Following an approach published recently in the literature, types of amino acids are labelled with 13C or/and 15N such that cross peaks result only for pairs of sequentially adjacent amino acids of which the first is labelled with 13C and the second with 15N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labelled protein samples that have to be prepared while obtaining an optimal extent of labelled unique amino acid pairs. The UPLABEL algorithm for optimal unique pair labelling uses combinatorial optimization to find for a given amino acid sequence labelling patterns that maximize the number of unique pair assignments with a minimal number of differently labelled protein samples. Various auxiliary conditions, including labelled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labelling patterns.
Development of a Software “Protein Dynamic Center”:
The software contributes to the automation process in order to evaluate the quality of the protein sampes made. The software exploits hetero nuclear relaxation (NOE, T1, T2, T1rho, Rex) recorded from the test samples. The obtained parameters are sensitive indicators for protein dynamics on different time scales. Global isotropic or anisotropic motion and local motions need to be included in the formalism to calculate the individual relaxation parameters. By fitting calculated to experimental data, relevant quantities like correlation times and order parameters can be determined. The aggregation status can be monitored by determination of these quantities at different sample concentrations. The first part of the software development focuses on the basic analysis of the corresponding experiments and covers all steps from sample setup, data selection, analysis, viewing, report and export. These steps are offered in a method oriented Protein Dynamic Center. The user gets graphical access to all data and numeric quantities in textual or Excel format.
Development of a Sample Changer for Solid-State NMR
The developed sample changer for Solid-State NMR applications with biological tissues, the SamplePro HR-MAS hallmarks a great improvement at the intended automation process. This changer is a combination of a robot and a transfer system. The robot is a third party liquid handler equipped with a sucking sprout that positions the sprout on top of a MAS rotor located in a cooling rack and sucks it to the transfer system called “FEU - Fast Exchange Unit”. From there the rotor is transferred to the probe inside of the NMR magnet for measurement. The system is equipped with two barcode readers, one for the rack barcode and one for the rotor itself in the Fast Exchange Unit.
Pilot study for a commercial service of protein production:
The purpose of study was to develop an on-demand protein production service and standardise the methods necessary for protein expression, labelling, and purification to prepare samples for NMR spectroscopy analysis, and to provide a plan for price estimates for protein production under standardised conditions. The study allowed the classication into two groups:
• Group A: proteins with a high variation in expression yield at the different isotopic labelling schemes, but can be purified by the same standard scheme. Here, the price is influenced by the specific label and the yield during the product price formation. For the fixed production cost model, it is assumed in this study that each protein production requires at least sixteen hours of preparation in the early stages and eight hours in the final stage. This twenty-four hour minimum spent on any of the targets used in this study depends neither on the amount of product prepared nor on the complexity of the manipulations and is accepted as being equal for all proteins. For simplification purposes, it is assumed that on average, the preparation and use/depreciation of equipment and materials total €40 per hour. In total, 24 hours of preparation result in €960 of fixed costs involved in the production and sample preparation process. At the variable production cost model, the expenses of the production process depend on the choice of the specific label used for protein expression.
• Group B: comprises proteins which exhibit basal expression level. The optimisation involves recloning as well as a better purification procedure (e.g. the number of chromatographic rounds, material losses during the purification processes, refolding of insoluble protein etc). Here, the price is dominated by the influence of the to-be-established purification schemes and number of cloning attempts used. The purity of the final product must be as high as possible, and therefore the use of multiple purification procedures is crucial for the price calculation. Usually, a purity of 90-95% or higher of the target protein is desired and sufficient for all subsequent NMR experiments. The purification process, however, involves two important aspects. Firstly, the more of the product that needs to be purified, the more it costs, and secondly, each purification stage implies loss in the final amount.
Potential Impact:
The impact of EAST-NMR project on the European Research Area evolves from the synergy of our three endeavours:
(I)Transnational Access Activities: 150 European Guest scientists originating from 16 countries were able to record quality spectra at high-field instruments. Usually, dissemination of knowledge takes place when the users interact with the local operators during the set up of the experiments. Further assistance might be given by the infrastructure operators as latest state-of-the-art experiments often require special approaches during the data analysis. The tight cooperation with world-wide e-Infrastructure project WeNMR (Grant agreement no: 261572) further strengthens the direct benefits for the users as the software platform integrates and streamlines the computational approaches necessary for the NMR data analysis.
(II) Dissemination Activities: (A) Three Transnational Access User Conferences attracted in total 427 participants at which a number of key-note speakers in the field of Bio-NMR from both Europe and overseas were featured. (B) With focus on the Eastern beneficiaries, two Young Investigator Meetings were organised in Istanbul, TR, and Bratislava, SK. Altogether, 94 Scientists from 16 coutries participated at the meetings which were rated as success both considering the scientific coverage and the promotion of further communication and contact between young and senior researchers. (C) 17 national/regional scientific events were held. Though they are not forums for major scientific discoveries, national and regional meetings had an important impact on NMR communities in their respective countries and regions. Altogether 880 scientist participated in the meetings. Besides the scientific presentations, they allowed enough space for informal discussions and exchange of ideas and experience. They also served as forums for discussing the scientific policies of the respective countries, which still differ greatly among the individual countries and regions. (D) Within the consortium, twelve times a staff exchange as ‚twinning‘ took place. Each twinning exchange based on a specific research topic, which was discussed and worked hands on at the visit. (E) Also in reference to the Transnational Access Activities, the exchange of protocols, standard operating procedures and best-practices was organised among the local operators of the distributed NMR infrastructure. The quality control test experimental set-ups at all RI were made publically available at the East-NMR home page in the section Exchange of best practices. The necessity as well as the possibility of the standard control experiments were debated at the User group meetings during Annual User Meetings as the best performing equipment should be offered to the Transnational Access guest scientists. Thus, technical knowledge was disseminated not only between the members of the consortium but also for the user group as part of the Transnational Access Activities.
(III) Outreach Activities: The project-website adressed the general public and was highly visited during the whole project duration. A total of 25.900 visitors (15.427 unique visitors) were registered by the statistic tools. The basic principle of biological NMR as well as its scientific benefit was disseminated to the youngest public by uploading a project movie to Youtube. In order to inspire university students for biological NMR, external workshops were held by the Eastern beneficiaries. A total of 12 events have been organised by partners (Tel Aviv, IL, Warsaw, PL, Tallinn, EE, and Riga, LV). 316 pre-doctoral students were informed about the NMR technology. The events included introductions to NMR spectroscopy, practical sessions at NMR spectrometers combined with exercises and analyses. If possible, a visit to biochemical laboratory was organized and techniques of protein expression briefly presented. In our strive to strengthen the awareness for bio-NMR across Europe (as well as India, Canada, Japan, China and the United States of America), our Principal Investigator’s informed the BioMed- or non-NMR Community about the scope oft he project. Partners participated to a total number of 105 events. The total audience was more than 25.000 people as the average audience of the visited events was around 250. Partners disseminated EAST-NMR as part of a lecture and by poster presentations.
For the Beneficiaries #3 (Masaryk University Brno, CZ), #5 (University of Warsaw, PL) and #12 (University of Patras, GR), the commitment to the EAST-NMR project helped to secure national funding for their own capital investments. A 950 MHz, an 850 MHz, an 800 MHz and a 750 MHz NMR spectrometer for high-resolution spectroscopy were recently purchased. This together with the fact that the vast majority of Networking Activities took place at the Eastern beneficiaries allows the conclusion that the EAST-NMR project indeed had impacts on their “Science and Society” and related policies.
List of Websites:
Project website address: http://www.east-nmr.eu
Name, title and organisation of the scientific representative of the project's coordinator:
Dr. Harald Schwalbe
Johann Wolfgang Goethe-Universität Frankfurt am Main, Center for Biomolecular Magnetic Resonance (BMRZ)
Max-von-Laue-Strasse 7
D-60438 Frankfurt am Main, DE
Tel:+49 69 7982 9130
E-mail:schwalbe@nmr.uni-frankfurt.de
Number and organisation of the scientific representative of the other Beneficiaries:
Benef. No. 2 - National Institute of Chemistry, Slovenian NMR Centre (NIC), SI
Janez Plavec, janez.plavec@ki.si
Benef. No. 3 - Masaryk University Brno, National Centre for Biomolecular Research (MU), CZ
Vladimír Sklenář, sklenar@chemi.muni.cz
Benef. No. 4 - University of Debrecen, Institut of Chemistry (UD), HU
Katalin E. Kövér, kover@tigris.unideb.hu
Benef. No. 5 - University of Warsaw (UNIWAR), PL
Wiktor Koźmiński, kozmin@chem.uw.edu.pl
Benef. No. 6 - Universiteit Utrecht (UNIUT), NL
Boelens, Rolf, r.boelens@chem.uu.nl
Benef. No. 7 - Consorzio Interuniversitario Risonanze Magnetiche di Metalloproteine Paramagnetiche, Magnetic Resonance Center (CIRMMP), IT
Claudio Luchinat, luchinat@cerm.unifi.it
Benef. No. 8 - The Chancellor, Masters and Scholars of the University of Oxford, Department of Biochemistry (UOXF. AL), UK
Christina Redfield, christina.redfield@bioch.ox.ac.uk
Benef. No. 9 - Centre National de la Recherche Scientifique, Centre Européen de RMN à Très Hauts Champs de Lyon (CNRS-CRMN), FR
Emsley, Lyndon, Lyndon.Emsley@ens-lyon.fr
Benef. No. 10 - Asla Biotech Ltd. (ASLA), LV
Andris Zeltinsh, anze@asla-biotech.com
Benef. No. 11 - Bruker BioSpin GmbH (BRUKER), DE
Frank Engelke, Frank.Engelke@bruker.de
Benef. No. 12 - University of Patras, Department of Pharmacy, Laboratory of Pharmacognosy and Chemistry of Natural Products (UPAT), GR
Georgios A. Spyroulias, G.A.Spyroulias@upatras.gr
Benef. No. 13 - Eötvös Loránd University, Laboratory of Structural Chemistry and Biology (ELTE), HU
András Perczel, perczel@chem.elte.hu
Benef. No. 14 - Institute of Biochemistry and Biophysics Polish Academy of Sciences (IBB PAN), PL
Andrzej Ejchart, aejchart@ibb.waw.pl
Benef. No. 15 - Weizmann Institute of Sciences Department of Structural Biology (WEIZMANN), IL
Jacob Anglister, Jacob.Anglister@weizmann.ac.il
Benef. No. 16 - Tallinn University of Technology (TUT), EE
Ago Samoson, ago.samoson@gmail.com
Benef. No. 17 - Latvian Institute of Organic Synthesis, Department of Physical Organic Chemistry (LIOS), LV
Edwards Liepinsh, edv@osi.lv
Benef. No. 18 - Institute of Chemistry, Slovak Academy of Sciences (ICSAS), SK
Miloš Hricovíni, Milos.Hricovini@savba.sk
Benef. No. 19 - Institute of Organic Chemistry with Centre of Phytochemistry - Bulgarian Academy of Sciences (IOCCP-BAS), BG
Svetlana Simova, sds@orgchm.bas.bg
Benef. No. 20 - TÜBİTAK Marmara Research Center Food Institute (TUBITAK MAM GE), TR
Somer Bekiroglu, Somer.Bekiroglu@mam.gov.tr
Benef. No. 21 - eurelations AG (EUREL), CH
Katrin Reschwamm, KReschwamm@eurelations.com