Probing Protein Conformation in Cells by EPR Distance Measurements using Gd³⁺ Spin Labeling

Abstract

Protein structure investigations are usually carried out in vitro under conditions far from their native environment in the cell. Differences between in-cell and in vitro structures of proteins can be generated by crowding effects, local pH changes, specific and nonspecific protein and ligand binding events, and chemical modifications. Double electron-electron resonance (DEER), in conjunction with site-directed spin-labeling, has emerged in the past decade as a powerful technique for exploring protein conformations in frozen solutions. The major challenges facing the application of this methodology to in-cell measurements are the instabilities of the standard nitroxide spin labels in the cell environment and the limited sensitivity at conventional X-band frequencies. We present a new approach for in-cell DEER distance measurement in human cells, based on the use of: (i) reduction resistant Gd(3+) chelates as spin labels, (ii) high frequency (94.9 GHz) for sensitivity enhancement, and (iii) hypo-osmotic shock for efficient delivery of the labeled protein into the cell. The proof of concept is demonstrated on doubly labeled ubiquitin in HeLa cells.