CEA Direction des sciences du vivant : New isotopic labelling strategy for studying biomolecular assemblies

Thursday February 18 2010

New isotopic labelling strategy for studying biomolecular assemblies

Stereospecific Isotopic Labeling of Methyl Groups for NMR Studies of High Molecular Weight Proteins. Pierre Gans, Olivier Hamelin, Remi Sounier, M. Asunción Durá, Marjolaine Noirclerc-Savoye, Carlos D. Amero, Bruno Franzetti, Michael J.Plevin& Jérôme Boisbouvier.

CEA

Chemists from the Life Sciences Division (iBiTec-S) working alongside physicists from the Physical Sciences Division (Iramis) specialized in nuclear magnetic resonance (NMR) have developed a new solid-state NMR technique for measuring large distances between atoms. This technique, built around the use of an ultra-sensitive isotope, will make it possible to determine the conformation of small molecules bound to their biological receptors, which is essential information for understanding the modes of action of these molecules as a platform for designing new simpler-structured medical drugs that are easier to synthesize.

Functional and structural study of biological macromolecules is made difficult by the dimensional scale and flexibility of the target objects. NMR spectroscopy is the first-choice method for studying the structure and dynamics of biological macromolecules in solution, down to atomic scale. NMR spectrometers operating at intensely-high magnetic fields have brought major improvements in terms of analytical resolution. However, to extend the boundaries of this method to encompass the analysis of biomolecular assemblies up to a megaDalton, the protocol needs to employ a technique enabling isotopic labelling of proteins. This technique begins by replacing 100% of the natural hydrogen atoms contained in the proteins with deuterium. Next, a handful of deuterium atoms at specific strategic positions in the protein are then re-replaced by hydrogen atoms. This technique thus simplifies the structural analysis by focusing exclusively of the hydrogen atoms of interest.

Scientists from the IBS teamed up with the iRSTV to develop a protocol making it possible to stereoselectively incorporate 13CH3 methyl groups directly into the amino acids leucine and valine during protein synthesis. This labelling protocol, which allies organic synthesis with enzyme biotransformation required organic synthesis modes to be engineered and optimized to produce different precursors specifically enriched in stable deuterium and carbon-13 isotopes. This labelling scheme significantly enhances the quality of the NMR output data, and provides a simple yet efficient platform for applying NMR techniques to complex supramolecular frameworks.

Stereoselective protonation: The leucine and valine molecules inside the protein possess two end-chain methyl groups. The protonation step becomes stereoselective if it can specifically introduce protons into just one of the two methyl groups.

Nuclear Magnetic Resonance: phenomenon where a nucleus of the target atom absorbs electromagnetic radiation at a specific frequency when exposed to a strong magnetic field. The applications for NMR span physics, chemistry, medical imaging and structural biology. The IBS platform is officially accredited as national and European hub for very-high-field NMR.

MegaDalton: The Dalton is a fairly accurate descriptor of the atomic mass unit of a hydrogen atom. A protein amino acid represents about 110 Da, and a one-megaDalton macromolecule assembly contains around 9000 amino acids

Front cover of the academic journal Angewandte Chemie International Edition: Isotopic labelling strategy to improve the NMR study of molecular complexes

Research teams – References:
- Nuclear magnetic resonance laboratory (‘LRMN’), Jean-Pierre Ebel Institute of Structural biology (IBS, Joint-run CEA-CNRS- Joseph Fourier University institute), Grenoble.
- Molecular biophysics laboratory (‘LBM’), Jean-Pierre Ebel Institute of Structural biology (IBS, Joint-run CEA-CNRS- Joseph Fourier University institute), Grenoble.
- Macromolecular engineering laboratory (‘LIM’), Jean-Pierre Ebel Institute of Structural biology (IBS, Joint-run CEA-CNRS- Joseph Fourier University institute), Grenoble.
- Laboratory of Metals in Biological and Chemical Systems (‘LCBM’) at the Institute of Life Sciences Research and Technologies (iRTSV, joint-run CEA-CNRS-Joseph Fourier University institute), Grenoble.