In-Cell Trityl–Trityl Distance Measurements on Proteins

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. T he cellular environment is complex and differs consid- erably in terms of viscosity, confinement, and composition from solution conditions under which protein structure and dynamics are commonly studied. The different conditions may affect conformational equilibria and the stability of proteins, and therefore, 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, also called PELDOR). 1-4 DEER measures the dipolar interaction between two paramagnetic centers, which is proportional to 1/r 3 , r being the distance between the two paramagnetic centers. 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. The insensitivity of DEER to the molecular size and the lack of an environmental background signal make it an attractive method for in-cell structural studies. The transition from solution to in-cell measurements is however challenging because it requires the conjugation between the label and the protein residue, as well as the spin label itself, to be stable in the reducing environment of the cell. Moreover, the measurement sensitivity should be high enough to allow access to protein concentrations close to the physiological range.

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