Observing protein’s conformational change in the cell

In-cell structural stability and the conformation of biomolecules might differ from in vitro conditions, owing to the complex, strong, or weak interactions they experience with other cell components as well as cellular crowding and post-translation modifications. Therefore, one has to ‘look’ at protein structure and dynamics inside the cell. Because most proteins in the cell are diamagnetic, DEER primarily detects the introduced spin labels grafted on the protein of interest, and therefore, it is a highly attractive method to probe biomolecules’ structural features in the cell with minimal background signals. Applying this technique in the cell requires challenging adaptations; the spin label and its conjugation bond should be resistant to the reducing cellular environment, the delivery methods for introducing the labeled protein into the cells should be efficient while maintaining the cell viability, and the measurement sensitivity should be high to allow close to physiological concentrations of the labeled biomolecules. We are using Gd(III) spin labels that so far have been shown to be highly effective for in-cell measurements at W-band frequencies because of their stability and high sensitivity. We employ them for Gd(III)-Gd(III) DEER distance measurements to access distances between 2-6 nm. To access shorter distances, we employ electron-nuclear double resonance (ENDOR) to measure Gd(III)-19F dipolar interactions, where the 19F is introduced at a specific amino acid in the sequence. Currently, we focus on the in-cell behavior of Hsp90 and development of in-cell Gd(III)-19F ENDOR.

                      

 

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