Forschungs- & Entwicklungsinformationsdienst der Gemeinschaft - CORDIS

FP6

LOW CCR NMR Berichtzusammenfassung

Project ID: 515039
Gefördert unter: FP6-MOBILITY
Land: Italy

Final Activity Report Summary - LOW CCR NMR (Cross Correlated Relaxation and Insensitive Nuclei Direct Detection for the Study of Biomolecular Assemblies)

During the last decade, Nuclear magnetic resonance (NMR) spectroscopy has become a leading technique to study biomolecules. Nowadays, its versatility allows the three-dimensional structure determination of biological macromolecules, the characterisation of protein dynamics, kinetic and thermodynamic information on protein folding, as well as on molecular interactions. The interface between methodological advancements in NMR spectroscopy, bio-computing and structural biology provides a comprehensive approach to address biological problems.

The major limitation of NMR arises from the increase of transverse relaxation rates which prevents the investigation of molecules with large molecular weights. In the last few years, cross correlation rates have been shown to be a way to investigate molecules with large molecular masses. Another potential breakthrough is the direct detection of 13C and / or 15N nuclei.

This project has developed new approaches based on the direct-detection of 13C to study fast relaxing nucleus systems by NMR. The new methodologies here tuned up has being applied both to paramagnetic metalloproteins, Calbindin D9K (publications 1, 2), and to medium size metalloproteins, CutA1 (publication 3), but it has being also proved to work on diamagnetic metalloproteins, c2 SOD (publication 4).

In this project, we have accomplished both methodological aims: pushing NMR frontiers in the detection of resonance belonging to fast relaxation nuclei (publications 1 and 3), developing new pulse sequences to obtain new structural constraints (publication 2); and also biological objectives (publications 3, 4 and 5).

Here, bellow it is included a brief summary of the articles published during the development of the project:
- Publication 1: Balayssac, S., Jiménez, B., Piccioli, M. 'Assignment Strategy for Fast Relaxing Signals: Complete Aminoacid Identification in Thulium Substituted Calbindin D9K' J. Biomol. NMR 2006, 34, 63-73.
We have used a set of NMR experiments optimised to capture data from these various concentric regions in assigning the signals from a paramagnetic Calbindin D9K derivative in which one of the two calcium ions has been replaced by thulium(III). Normal double- and triple-resonance experiments with 1H detection were used in collecting data from nuclei in the diamagnetic-like region; these approaches identified signals from fewer than 50 % of the amino acid residues. Using customised 1H detected experiments and specially designed heteronuclear (13C and 15N) detected ones, we were able to of at least one resonance per amino acid residue, including those in the thulium(III) coordination sphere. NMR signals were assigned on the basis of NOEs, through-bond correlations and, for signals closest to the metal, pseudocontact shifts.
- Publication 2: Balayssac, S., Jiménez, B., Piccioli, M. '13C Direct Detected COCO-TOCSY: a Tool for Sequence Specific Assignment and Structure Determination in Protonless NMR Experiments' J. Magn. Reson. 2006, 182, 325-329.
We have designed a novel experiment which provide inter-residue sequential correlations among carbonyl spins in 13C detected, protonless NMR experiments. The COCO-TOCSY experiment connects, in proteins, two carbonyls separated from each other by three, four or even five bonds. The quantitative analysis provides structural information on backbone dihedral angles as well as on the side chain dihedral angles of Asx and Glx residues. This has been the first dihedral angle constraint that could be obtained via a protonless approach. Additionally, long range homonuclear coupling constants, 4JCC and 5JCC, could be measured for some side chains. This constitutes an unprecedented case for proteins of medium and small size.
- Publication 3: Balayssac, S., Jiménez, B., Piccioli, M. 'Protonless 13C direct detection NMR: characterisation of the 37 kiloDalton trimeric protein CutA1' Proteins 2007, in press.
In this work, we have studied the functional properties of CutA1 protein from E. coli. CutA1 are a family of copper binding proteins, occurring in different organisms which are supposed to fulfil different functions in bacteria and in mammals.

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