Speaker
Description
The application of hydrostatic high-pressure (HP) is now a recognized tool to modify biomolecular conformational landscapes in a mild and largely reversible manner. HP combined with NMR spectroscopy (HP-NMR) has been extensively used over the years to characterize the folding properties and the dynamics of soluble proteins by stabilizing conformational states that are lowly populated at ambient pressure and characterized by lower partial volumes. On the other side, paramagnetic NMR is a well-established method to probe the molecular structure by grafting compounds, such as lanthanides, on proteins and is also central for the development of contrast agents used for MRI. As an example, the Pseudo-Contact Shifts (PCSs) generated by lanthanides such as Yb3+, Tb3+ or Tm3+ lead to spectral shifts allowing obtaining precise relative positions of the various atoms with respect to the lanthanide probe. Nevertheless, to date, how pressure affects the paramagnetic properties of lanthanides and whether PCS can be used to probe protein structure/dynamics at high pressure is unknown.
Here, we will describe recent progress to assess the pressure effects on PCS collected on ubiquitin protein and a contrast agent. We show that up to 2500 bars, the paramagnetic probe remains chemically intact and that the change in paramagnetic susceptibility tensor is significant but limited. We could detect at ultra-high precision the minute conformational deformation of the contrast agent by a fraction (~1%) of Å. Using Tb3+ and Tm3+ grafted at various positions on ubiquitin we could detect high quality PCSs for backbone 1H/15N atoms from 1 to 2500 bars. We detected a small displacement (~0.5 Å) of the probe from the protein at high pressure and demonstrated that PCSs can be used to probe the structure/dynamics of proteins under high-pressure conditions. Taken together, we established that paramagnetic NMR is a viable tool to characterize molecular structures under high pressure.
