In-Cell Trityl-Trityl Distance Measurements on Proteins.

Yin Yang,Feng Yang,Bin-Bin Pan,Xiaoli Tan,Yangping Liu,Xun-Cheng Su,Daniella Goldfarb

doi:10.1021/acs.jpclett.9b03208

Abstract

Double-electron electron resonance (DEER) can be used to track the structural dynamics of proteins in their native environment, the cell. This method provides the distance distribution between two spin labels attached at specific, well-defined positions in a protein. For the method to be viable under in-cell conditions, the spin label and its attachment to the protein should exhibit high chemical stability in the cell. Here we present low-temperature, trityl–trityl DEER distance measurements on two model proteins, PpiB (prolyl cis–trans isomerase from E. coli) and GB1 (immunoglobulin G-binding protein), doubly labeled with the trityl spin label, CT02MA. Both proteins gave in-cell distance distributions similar to those observed in vitro, with maxima at 4.5–5 nm, and the data were further compared with in-cell Gd(III)–Gd(III) DEER obtained for PpiB labeled with BrPSPy-DO3A-Gd(III) at the same positions. These results highlight the challenges of designing trityl tags suitable for in-cell distance determination at ambient temperatures on live cells.

Highlights

Double-electron electron resonance (DEER) can be used to track the structural dynamics of proteins in their native environment, the cell
The different conditions may affect conformational equilibria and the stability of proteins, and tracking proteins conformations in their native environment, the cell, though challenging, is of current high interest. This has been addressed by a number of biophysical methods, among them double-electron electron resonance (DEER, called PELDOR).[1−4] DEER measures the dipolar interaction between two paramagnetic centers, which is proportional to 1/r3, r being the distance between the two paramagnetic centers.[5]
Since most proteins are diamagnetic, DEER relies on introducing spin labels at well-defined positions in the protein and it provides the distance distribution between the two labels

Summary

Introduction

Double-electron electron resonance (DEER) can be used to track the structural dynamics of proteins in their native environment, the cell. DEER obtained for PpiB labeled with BrPSPy-DO3A-Gd(III) at the same positions. We report on W-band (94.9 GHz) in-cell trityl−trityl DEER measurements on two proteins, the K25C/ E153C mutant of prolyl cis−trans isomerase B from E. coli (PpiB) and the T11C/V21C mutant of immunoglobulin Gbinding protein (GB1) labeled with the CT02MA tag[30]

Results

Conclusion