Final Report Summary - SBMPS (Structural Biology of Membrane Proteins)
Project context and objectives
This European network was devoted to developing new approaches and strategies for analysing the structure (dynamics) and functional relationships of membrane proteins (MPs).
MPs are of central importance in many fundamental cellular processes, such as signal transduction, ion exchange and the transport of metabolites. Despite their importance, detailed information about their function at a molecular level is scarce because they are difficult to study: there are substantial hurdles to extract them from their natural environment and to purify them in a native conformation. At the same time, it transpired from many studies, that even atomic details from high-resolution structures are not sufficient to understand the function of MPs in their cellular environment. More information is needed on the identification and characterisation of different conformations, on the dynamics related to conformational changes, on how MPs are inserted in their natural environment and on how they are organised within the membrane.
Work performed
Our consortium organised a joint training effort that involved the major biophysical methods which are used in the field of MPs structural biology: efficient in vivo and in vitro (cell-free) MP expression systems, X-ray crystallography, liquid and solid-state nuclear magnetic resonance (NMR), electron microscopy (EM), atomic force microscopy (AFM), single molecule force spectroscopies (SMFS), numerical simulations and advanced biochemical/biophysical and functional characterisation strategies. It was enriched by participation from several private companies, both from SMEs and from large industrial groups. Our network has trained 36 young academic researchers (PhD and young post-doctorate fellows) to the highest possible level on the theoretical and practical aspects of this wide range of approaches to structural biology. Through their research projects they could further acquire a broad scientific knowledge of membranes and membrane proteins, their functions and their applications in biotechnologies and pharmacology.
Firstly, a new generation of researchers (23 doctoral students and 13 young post-docs) was trained on the major biophysical methods used in the structural biology of membrane proteins. They learned a specific methodology from their hosting lab but were trained in other fields by their collaboration within and outside the network, including lab visits and exchanges. They had the opportunity to attend the 11 workshops organised by the network and to present their results at our four annual meetings. They also participated in external international conferences and external workshops to complete their training. In addition, two complementary skills workshops, three conferences and contacts with the private sector were also organised within the network for the members. All the workshops were open to external students, who also benefited from the presentations and discussions with the experts. The project fellows were also involved in organising the annual meetings; they participated in choosing the speakers to be invited and chaired scientific sessions. Within the network, the fellows had feedback on their research from the experienced scientists of the network (scientists-in-charge, associated partners and visiting scientists) through questions and discussions around their presentations at the annual meetings (oral and poster presentations) and through annual reports and a tutorship system.
Secondly, an active network of European structural biologists was set up; this collaborative approach is key for future breakthroughs in the membrane protein field. The 13 scientists in charge (including one from Zobio, an SME in drug development), the two associated partners (Sanofi and JPK), the 13 visiting scientists and all our external invited speakers contributed to the success of the network by developing scientific and face-to-face relationships though collaborations, visits, workshops and annual meetings.
The scientific results from the various research projects will lead to new knowledge and technical improvements in the field of membrane protein structure and study, now visible in terms of new publications, exploitable foreground, instrumentation and conceptual advances.
Main results
The research projects developed in the network covered various aspects of MPs studies, including the development of new methodologies, the biophysical characterisation of membrane proteins in their functional environment (structures, dynamics, oligomerisation, ligand and/or lipid interactions, etc.), determining their structures and exploring the role of the surrounding membrane. The projects were designed to involve multiple teams, so that students were trained in the respective fields. A wide range of membrane proteins was investigated that covered the major biological functions. So far, the project has published about 35 peer-reviewed publications, but many others should follow during the coming years.
The results obtained by the various partners (fellow-scientist in charge) contributed to different levels of the membrane protein characterisation:
- Expression-purification of membrane proteins in their active form (using both heterologous expression and cell-free systems) (Kai-Bernhard [1-5]), Proverbio-Bernhard [2, 5-7], Ghoshdastider-Bernhard-Filipek [5, 8, 9], Rath-Milon [10]);
- Reconstitution in a natural environment such as nanodiscs (Zocher-Müller [11]), lipidic cubic phases, amphipols, or directly with native lipids (Rath-Milon [10]) or in native membranes (Kawamura-Müller [12], Bosshart-Engel [13], Franks-Oschkinat [14, 15]) and the study of the folding/unfolding process necessary during the reconstitution of the membrane protein;
- Structures of new proteins; new features concerning an already known structure such as the oligomeric state, dynamics of the extracellular loops, mechanical properties [16], stabilisation or destabilisation of a membrane protein by specific mutations, characterisation of the minimal domain for interaction with a partner (Iordanov-Milon, Bosshart-Müller [17, 18], Kawamura-Müller [12, 19], Kai-Bernhard [4], Ghoshdastider-Filipek [20] Moiseeva-Pebay-Peyroula [21], Ghoshdastider-Filipek [22], Gradmann-Baldus [23]);
- Dynamics of a structural domain when interacting with its ligand (Flayhan-Breyton-Popot [24], Zocher-Müller [25], Kawamura-Müller [19], Ghoshdastider-Filipek [26]) or when being activated (Yuan/Ghoshdastider-Filipek [27]);
- New ligand screening and enzymology (Chen-Siegal [28, 29]) for a specific membrane protein (GPCR);
- Technical improvements such as the development of a zeptolitre volume meter (Sanden-Vogel [30]) for biochemical characterisations of unlabelled proteins, the development of a new platform for high-throughput drug screening (Kai/Proverbio-Bernhard [2, 5], Ghoshdastider-Filipek [5]), new strategies in solid-state NMR such as dynamic nuclear polarisation (Franks-Oschkinat [14, 31-36]), the study of complex large proteins by NMR (Renault-Baldus [37, 38], Chowdhury/Retel-Oschkinat [39]), automatic assignment strategies in ssNMR;
- Direct structural characterisation of a molecule in its natural environment by in-cell NMR (Renault-Baldus [40, 41]) or by single cell force microscopy.
Conclusions
The training provided in this project formed new high-level academic researchers who will become major actors in the development of European biotechnology and personalised medicine. These new researchers developed close ties within an international network of structural biologists and will spread their knowledge and culture throughout the scientific community. Their research projects led to novel results, which have contributed towards expanded knowledge and promising applications in biotechnologies and pharmacology.
Socio-economic impact of the project
The network has recruited 36 young researchers from 18 different countries, 36 % of whom were women and 55 % were nationals from European countries. The main nationality was German (five fellows), followed by Chinese, Indian and Italian (four fellows each). The network also recruited 13 highly experienced researchers as visiting scientist, 8 % of whom was female and 23 % of whom came from European countries. The network benefited mostly from experts across the Atlantic (USA/Canada) as they represented 46 % of the visiting scientists; 23 % came from Russia. This action thus gave a wide audience to the European strength in this field of important research.
The two associated partners, Sanofi-Aventis and JPK Instruments, one of the partners (Gregg Siegal - ZoBio) and one visiting scientist (Martial Pioto, Bruker Biospin SA) were all from private industry. They contributed to opening the network to the non-academic world, and taught the fellows how fundamental knowledge is being applied and translated into new therapeutic agents. In exchange, they were in close contact with the top-level scientific community and experienced cutting-edge research. The European industry will benefit from this action, either directly from the results already obtained (which will be turned into new products, equipment and methodologies made commercially available) or indirectly from the knowledge and networking that was developed.
Project website: http://www.sbmp-itn.eu/sbmps/home/
This European network was devoted to developing new approaches and strategies for analysing the structure (dynamics) and functional relationships of membrane proteins (MPs).
MPs are of central importance in many fundamental cellular processes, such as signal transduction, ion exchange and the transport of metabolites. Despite their importance, detailed information about their function at a molecular level is scarce because they are difficult to study: there are substantial hurdles to extract them from their natural environment and to purify them in a native conformation. At the same time, it transpired from many studies, that even atomic details from high-resolution structures are not sufficient to understand the function of MPs in their cellular environment. More information is needed on the identification and characterisation of different conformations, on the dynamics related to conformational changes, on how MPs are inserted in their natural environment and on how they are organised within the membrane.
Work performed
Our consortium organised a joint training effort that involved the major biophysical methods which are used in the field of MPs structural biology: efficient in vivo and in vitro (cell-free) MP expression systems, X-ray crystallography, liquid and solid-state nuclear magnetic resonance (NMR), electron microscopy (EM), atomic force microscopy (AFM), single molecule force spectroscopies (SMFS), numerical simulations and advanced biochemical/biophysical and functional characterisation strategies. It was enriched by participation from several private companies, both from SMEs and from large industrial groups. Our network has trained 36 young academic researchers (PhD and young post-doctorate fellows) to the highest possible level on the theoretical and practical aspects of this wide range of approaches to structural biology. Through their research projects they could further acquire a broad scientific knowledge of membranes and membrane proteins, their functions and their applications in biotechnologies and pharmacology.
Firstly, a new generation of researchers (23 doctoral students and 13 young post-docs) was trained on the major biophysical methods used in the structural biology of membrane proteins. They learned a specific methodology from their hosting lab but were trained in other fields by their collaboration within and outside the network, including lab visits and exchanges. They had the opportunity to attend the 11 workshops organised by the network and to present their results at our four annual meetings. They also participated in external international conferences and external workshops to complete their training. In addition, two complementary skills workshops, three conferences and contacts with the private sector were also organised within the network for the members. All the workshops were open to external students, who also benefited from the presentations and discussions with the experts. The project fellows were also involved in organising the annual meetings; they participated in choosing the speakers to be invited and chaired scientific sessions. Within the network, the fellows had feedback on their research from the experienced scientists of the network (scientists-in-charge, associated partners and visiting scientists) through questions and discussions around their presentations at the annual meetings (oral and poster presentations) and through annual reports and a tutorship system.
Secondly, an active network of European structural biologists was set up; this collaborative approach is key for future breakthroughs in the membrane protein field. The 13 scientists in charge (including one from Zobio, an SME in drug development), the two associated partners (Sanofi and JPK), the 13 visiting scientists and all our external invited speakers contributed to the success of the network by developing scientific and face-to-face relationships though collaborations, visits, workshops and annual meetings.
The scientific results from the various research projects will lead to new knowledge and technical improvements in the field of membrane protein structure and study, now visible in terms of new publications, exploitable foreground, instrumentation and conceptual advances.
Main results
The research projects developed in the network covered various aspects of MPs studies, including the development of new methodologies, the biophysical characterisation of membrane proteins in their functional environment (structures, dynamics, oligomerisation, ligand and/or lipid interactions, etc.), determining their structures and exploring the role of the surrounding membrane. The projects were designed to involve multiple teams, so that students were trained in the respective fields. A wide range of membrane proteins was investigated that covered the major biological functions. So far, the project has published about 35 peer-reviewed publications, but many others should follow during the coming years.
The results obtained by the various partners (fellow-scientist in charge) contributed to different levels of the membrane protein characterisation:
- Expression-purification of membrane proteins in their active form (using both heterologous expression and cell-free systems) (Kai-Bernhard [1-5]), Proverbio-Bernhard [2, 5-7], Ghoshdastider-Bernhard-Filipek [5, 8, 9], Rath-Milon [10]);
- Reconstitution in a natural environment such as nanodiscs (Zocher-Müller [11]), lipidic cubic phases, amphipols, or directly with native lipids (Rath-Milon [10]) or in native membranes (Kawamura-Müller [12], Bosshart-Engel [13], Franks-Oschkinat [14, 15]) and the study of the folding/unfolding process necessary during the reconstitution of the membrane protein;
- Structures of new proteins; new features concerning an already known structure such as the oligomeric state, dynamics of the extracellular loops, mechanical properties [16], stabilisation or destabilisation of a membrane protein by specific mutations, characterisation of the minimal domain for interaction with a partner (Iordanov-Milon, Bosshart-Müller [17, 18], Kawamura-Müller [12, 19], Kai-Bernhard [4], Ghoshdastider-Filipek [20] Moiseeva-Pebay-Peyroula [21], Ghoshdastider-Filipek [22], Gradmann-Baldus [23]);
- Dynamics of a structural domain when interacting with its ligand (Flayhan-Breyton-Popot [24], Zocher-Müller [25], Kawamura-Müller [19], Ghoshdastider-Filipek [26]) or when being activated (Yuan/Ghoshdastider-Filipek [27]);
- New ligand screening and enzymology (Chen-Siegal [28, 29]) for a specific membrane protein (GPCR);
- Technical improvements such as the development of a zeptolitre volume meter (Sanden-Vogel [30]) for biochemical characterisations of unlabelled proteins, the development of a new platform for high-throughput drug screening (Kai/Proverbio-Bernhard [2, 5], Ghoshdastider-Filipek [5]), new strategies in solid-state NMR such as dynamic nuclear polarisation (Franks-Oschkinat [14, 31-36]), the study of complex large proteins by NMR (Renault-Baldus [37, 38], Chowdhury/Retel-Oschkinat [39]), automatic assignment strategies in ssNMR;
- Direct structural characterisation of a molecule in its natural environment by in-cell NMR (Renault-Baldus [40, 41]) or by single cell force microscopy.
Conclusions
The training provided in this project formed new high-level academic researchers who will become major actors in the development of European biotechnology and personalised medicine. These new researchers developed close ties within an international network of structural biologists and will spread their knowledge and culture throughout the scientific community. Their research projects led to novel results, which have contributed towards expanded knowledge and promising applications in biotechnologies and pharmacology.
Socio-economic impact of the project
The network has recruited 36 young researchers from 18 different countries, 36 % of whom were women and 55 % were nationals from European countries. The main nationality was German (five fellows), followed by Chinese, Indian and Italian (four fellows each). The network also recruited 13 highly experienced researchers as visiting scientist, 8 % of whom was female and 23 % of whom came from European countries. The network benefited mostly from experts across the Atlantic (USA/Canada) as they represented 46 % of the visiting scientists; 23 % came from Russia. This action thus gave a wide audience to the European strength in this field of important research.
The two associated partners, Sanofi-Aventis and JPK Instruments, one of the partners (Gregg Siegal - ZoBio) and one visiting scientist (Martial Pioto, Bruker Biospin SA) were all from private industry. They contributed to opening the network to the non-academic world, and taught the fellows how fundamental knowledge is being applied and translated into new therapeutic agents. In exchange, they were in close contact with the top-level scientific community and experienced cutting-edge research. The European industry will benefit from this action, either directly from the results already obtained (which will be turned into new products, equipment and methodologies made commercially available) or indirectly from the knowledge and networking that was developed.
Project website: http://www.sbmp-itn.eu/sbmps/home/