Structure and Dynamics of Metal Ion Transporters using Solid-State Nuclear Magnetic Resonance at High Field and Fast Magic Angle Spinning
Objective
Funding Scheme
MC-IIF - International Incoming Fellowships (IIF)
Coordinator
CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Address
Rue Michel Ange 3
75794 Paris
France
Activity type
Research Organisations
EU Contribution
€ 202 405,80
Administrative Contact
Pascaline Toutois (Dr.)
Project information
MEM-MAS
Grant agreement ID: 624918
Status
Closed project
-
Start date
1 March 2014
-
End date
29 February 2016
Funded under:
FP7-PEOPLE
-
Overall budget:
€ 202 405,80
-
EU contribution
€ 202 405,80
This project is featured in...
Novel methodology unveils membrane protein structure
FUNDAMENTAL RESEARCH
HEALTH
Proteins constitute the major constituent of all living cells and play a central role in many important biological processes. Proteins located in the cell membrane are vital for mediating entry and exit of molecules across membranes, for signalling, and cell adhesion. Despite their importance, there is no 3D structure information on all membrane proteins because of the difficulty in obtaining crystal structures. The EU-funded MEM-MAS (Structure and dynamics of metal ion transporters using solid-state nuclear magnetic resonance at high field and fast magic angle spinning) project set out to develop nuclear magnetic resonance (NMR) spectroscopy methodology to determine the structure of membrane proteins. The consortium managed to overcome existing bottlenecks in NMR-based structure determination and improve sensitivity and reliability while maintaining spectral resolution. The advanced method was applied on two membrane proteins, one of which yielded well-resolved spectra. The development of new pulse sequences accelerated the time-consuming step of resonance assignment, and alongside the application of new instrumentation, it allowed magic angle sample spinning. This proved particularly suitable for larger proteins with less structural homogeneity such as the viral nucleocapsid protein. Overall, the MEM-MAS project results are expected to revolutionise the field of structure determination by solid state NMR. A higher throughput elucidation of membrane protein structure will improve our knowledge on their function and aid in the development of new treatments for human diseases.
Keywords
Membrane protein, structure, NMR spectroscopy, pulse sequence, magic angle sample spinning
Project information
MEM-MAS
Grant agreement ID: 624918
Status
Closed project
-
Start date
1 March 2014
-
End date
29 February 2016
Funded under:
FP7-PEOPLE
-
Overall budget:
€ 202 405,80
-
EU contribution
€ 202 405,80
This project is featured in...
Discover other articles in the same domain of application
Final Report Summary - MEM-MAS (Structure and Dynamics of Metal Ion Transporters using Solid-State Nuclear Magnetic Resonance at High Field and Fast Magic Angle Spinning)
Determination of protein structures is the first step toward an understanding of protein function, and can aid in the rational development of small molecules that modulate function. Due to the paucity of membrane protein structures reported to date, the project set out to develop Nuclear Magnetic Resonance (NMR) spectroscopy methodology and apply that methodology to the determination of membrane protein structure.
We acquired spectra for two membrane proteins, one of which yielded well-resolved spectra amenable to structure determination, while the other membrane protein requires further sample optimization for high-resolution structural measurements. We have also developed new pulse sequences that accelerate the often time consuming step of resonance assignment. A method for robust side-chain resonance assignment was developed, which was used in a structure calculation of a protein dimer within a large viral capsid. We determined the structure of this viral nucleocapsid protein of previously unknown structure by application of new instrumentation that allows magic angle sample spinning (MAS) at 100 kHz. This represents a major improvement in the methodology, which should allow many more proteins to be efficiently investigated by NMR in the future. For us, the successful structure determination also served as proof that the method is successful for larger proteins with less structural homogeneity, and is currently being applied to a membrane protein of unknown structure in the lab in Lyon. Of particular interest to advancing the field, we demonstrated improvements to the sensitivity and reliability of the method by extending the measurements to 100 kHz MAS, which allows more nuclear spins to be observed while maintaining spectral resolution, and eliminating major bottlenecks for NMR based structure determination. These developments were critical to the de novo structure determination of the viral nucleocapsid protein, and are set to revolutionize the process of structure determination by solid state NMR.
Successful development of techniques that accelerate protein structure determination by MAS NMR is expected to allow a higher throughput, which will improve the understanding of membrane proteins, with extensive downstream societal benefits, such as the development of new treatments of human diseases.
Project information
MEM-MAS
Grant agreement ID: 624918
Status
Closed project
-
Start date
1 March 2014
-
End date
29 February 2016
Funded under:
FP7-PEOPLE
-
Overall budget:
€ 202 405,80
-
EU contribution
€ 202 405,80
This project is featured in...
Deliverables
Deliverables not available
Publications
Project information
MEM-MAS
Grant agreement ID: 624918
Status
Closed project
-
Start date
1 March 2014
-
End date
29 February 2016
Funded under:
FP7-PEOPLE
-
Overall budget:
€ 202 405,80
-
EU contribution
€ 202 405,80
This project is featured in...
Project information
MEM-MAS
Grant agreement ID: 624918
Status
Closed project
-
Start date
1 March 2014
-
End date
29 February 2016
Funded under:
FP7-PEOPLE
-
Overall budget:
€ 202 405,80
-
EU contribution
€ 202 405,80
