In-cell structural measurements

Being able to determine the structure of proteins, on the atomic level, in their native environment, the cell, and follow structural transformation they undergo in the cell upon interaction with other cellular components is of major importance for understanding their function. Accordingly, considerable efforts are directed towards achieving this goal. Currently there are two major methods that are used to achieve this goal, one is NMR that suffers from relatively low sensitivity and limitation to small proteins and the other is FRET that while having very high sensitivity cannot provide atomic level structural information. DEER (also named PELDOR) is an EPR (electron paramagnetic resonance) method based technique that has emerged in the last decade as highly effective for obtaining structural information on the atomic level and is particularly useful for exploring functional structural dynamics in proteins. It is based on selectively labeling the proteins with spin labels (usually nitroxides) and measuring the distance between them. We are developing methods for carrying such measurements in cells at in cell concentrations < 10 uM. To achieve this we used a new family of spin labels, based on Gd3+ that unlike nitroxides are stable in the cell, we carry out the measurements at high magnetic fields using a home built spectrometer, that provided the needed sensitivity, and we developed a method to introduce the label protein into human cells while keeping the viability of the cells. Having such a methodology at hand opens now many new opportunities to explore the effects of confinement on structure, the effect of interaction with other proteins, the effect of aggregation of proteins involved in Alzheimer and Parkinson deceases, polymerization of uniquitin, the fate of intrinsically disordered proteins in the cell and many more. We are very excited about this new development and believe that the sensitivity can be improved by a factor of at least two by some instrumental developments.

 

References

  • Martorana, A.; Bellapadrona, G.; Feintuch, A.; Di Gregorio, E.; Aime, S.; Goldfarb, D. Probing Protein Conformation in Cells by EPR Distance Measurements using Gd(3+) Spin Labeling. Journal of the American Chemical Society 2014, 136, 13458-13465.