Isotopic labelling

Presentation

In order to increase the size limitation of molecules that one can feasibly study by NMR, the laboratory has invested significant effort in establishing protocols for isotopic labelling (15N, 13C and 2H) of proteins. The preparation of a number of perdeuterated samples, for which the effects of transverse relaxation improve spectral quality, has allowed us to study biologically interesting systems that are greater than 20kD.

Production of Isotopically labeled biomolecules for NMR

Production of Isotopically labeled biomolecules for NMR

Contact: Isabel Ayala, Lionel Imbert, J. Boisbouvier Collaboration : T. Vernet, B. Gallet, M. Noirclerc-Savoye, D. Blot, Laboratoire d’Ingénierie des Protéines, IBS, Grenoble

Automated protein production and solubility screening

We have interfaced the RoBioMol platform at the IBS (html link) with the NMR spectrometer to speed up the initial steps of an NMR study and the evaluation of the feasibility of a new structural biology project. RoBioMol is an automated platform for the production of recombinant proteins that carries out all tasks from oligo-design and PCR to expression and purification tests using robots and dedicated software. Adaptation of expression and purification conditions to NMR requirements leads to the automated production of initial 15N-labeled samples that are screened directly in the NMR spectrometer. The resulting 1H,15N-HSQC spectra provide a fingerprint of the protein that reports on the folding and the homogeneity of the protein sample. We also developed a protein solubility test using the IBS crystallization robot. Micro-drops are prepared in a fully automated manner in NMR-compatible buffers at varying pH, ionic strengths and with different additives. After the concentration of the protein by vapor diffusion, photos are taken and automatically analyzed to identify optimal solubility conditions for a given protein.

Development of isotopic-labeling techniques

Modern NMR techniques require isotopic labeling of biomolecules (15N, 13C, 2H). This is mainly achieved by growing bacteria (Escherichia coli) or unicellular eucaryotes (Pichia pastoris) in defined culture media containing isotopically labeled compounds as sole carbon and/or nitrogen sources. For large biomolecules, unfavorable relaxation properties can be overcome by the systematic replacement of 1H by 2H. However, the presence of individual protons at well-defined locations is indispensable for obtaining long-range distance and orientational information, especially in > 20 kDa molecules or molecular complexes.

In our wetlab located in the Carl-Ivar-Branden Building, we develop new techniques for the highly selective reintroduction of protons in otherwise 100% deuterium-labeled proteins and are now able to produce milligram quantities of 100% deuterated proteins selectively protonated on the methyl groups of either leucine/valine, isoleucine or alanine residues. This strategy allows us to expand the molecular-weight limit of structural NMR to several hundreds of kilodaltons. In addition, a part of our wetlab is dedicated to the large scale production and purification of isotopically labeled RNA, a prerequisite for the study of biologically important RNAs or protein-RNA complexes. Finally, in order to obtain structural and dynamic informations on heparane sulfate (HS)-protein interactions, we are currently setting up a protocol for the production of 13C-labeled HS. This will allow faster determination of more accurate structural constraints needed to determine 3D structures of HS-protein complexes.