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Dr. Lars Kuhn, Technical University of Denmark

Hyperpolarization-enhanced 2D NMR observation of protein folding in real-time
Wann 10.05.2019
von 14:15 bis 15:15
Wo Hörsaal Physikalische Chemie
Termin übernehmen vCal

Although mainly applied to the study of protein structure at equilibrium, nuclear magnetic resonance (NMR) spectroscopy affords the opportunity to directly follow changes in the local environment of individual atoms ‘in situ’ during protein folding, i.e. while the native backbone fold is formed and the compact hydrophobic core is established. When applied in a one-dimensional fashion, however, these real-time NMR methods suffer from low spectral resolution and the acquisition of kinetic spectra of higher dimensionality is often limited by relatively long acquisition times and/or lower sensitivity.

In this presentation, a side-chain-selective and highly sensitive way of conducting heteronuclear 2D NMR experiments repetitively to monitor protein folding processes in real-time is presented. This is achieved by combining three key methodologies: i) rapid mixing of sample solutions to trigger protein refolding ‘in situ’; ii) photo-Chemically Induced Dynamic Nuclear Polarization (photo-CIDNP), a nuclear spin hyperpolarization phenomenon highlighting solvent-exposed tyrosine, tryptophan, and histidine side chain nuclei by means of a laser light-induced reaction of spin-correlated radical pairs; to this, we add here (iii) acquisition of heteronuclear 1H-15N HMQC photo-CIDNP data in real-time featuring a specifically tailored two-dimensional NMR pulse scheme based on the SOFAST method. Employing the apo-form of the protein bovine alpha-lactalbumin as a model system, it will be demonstrated how this approach can be used to productively combine the advantages associated with the photo-CIDNP technique as applied to amino acids and proteins with the benefits of 2D NMR spectroscopy.

It is believed that these kinetic experiments can be employed in the future as a complementary tool to existing one- and two-dimensional NMR analogues providing a side-chain selective, atomic-level insight into the structural transformations occurring during (multi-step) protein folding processes. Owing to the chemical signal amplification associated with the technique, these methods might prove particularly suitable for the identification and structural examination of lowly concentrated intermediate structures – occurring, for example, in a transient fashion as folding progresses – or ill-defined protein oligomers often populated to a relatively limited extent and thus difficult to trace using standard approaches.

Physikalisch-Chemisches Kolloquium
Institut für Physikalische Chemie