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.

Gd(III)–Gd(III) distance measurements with chirp pump pulses

“…Distance measurements involving Gd spin labels have been successfully demonstrated on a number of systems, including model compounds, peptides, nanoparticles, proteins and DNAs [7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. Moreover, a promising biochemical property of Gd spin labels is the stability under reducing conditions, which recently enabled Gd-Gd distance measurements of peptides and proteins embedded in cellular environments [22,23]. Because Gd(III) ions are in an S = 7/2 high spin state, high magnetic fields above 1 T are required to diminish unwanted contributions from the zero-field splitting (ZFS) to the obtained distance information [7].…”

Conformational dynamics of nucleic acid molecules studied by PELDOR spectroscopy with rigid spin labels

“…A rigid spin label will not only prolong the transversal relaxation time, but also allow collection of information on dynamics of biomolecules that occur on the nano-to microsecond timescale. Because the intrinsic high sensitivity of EPR, which allows experiments to be performed also in whole cells [91][92][93][94], investigations under physiological conditions (room temperature and in-cell) might be feasible in the future.…”

Combining NMR and EPR to Determine Structures of Large RNAs and Protein–RNA Complexes in Solution

“…If measurements need to be performed under reducing conditions, for instance in living bacterial cells, the redox lability of the nitroxide group also poses problems. In such cases, Gd(III) labels are advantageous (Martorana et al, 2014;Qi, Gross, Jeschke, Godt, & Drescher, 2014).…”