Site Selective and Efficient Spin Labeling of Proteins with a Maleimide-Functionalized Trityl Radical for Pulsed Dipolar EPR Spectroscopy

Abstract

Pulsed dipolar electron paramagnetic resonance spectroscopy (PDS) in combination with site-directed spin labeling (SDSL) of proteins and oligonucleotides is a powerful tool in structural biology. Instead of using the commonly employed gem-dimethyl-nitroxide labels, triarylmethyl (trityl) spin labels enable such studies at room temperature, within the cells and with single-frequency electron paramagnetic resonance (EPR) experiments. However, it has been repeatedly reported that labeling of proteins with trityl radicals led to low labeling efficiencies, unspecific labeling and label aggregation. Therefore, this work introduces the synthesis and characterization of a maleimide-functionalized trityl spin label and its corresponding labeling protocol for cysteine residues in proteins. The label is highly cysteine-selective, provides high labeling efficiencies and outperforms the previously employed methanethiosulfonate-functionalized trityl label. Finally, the new label is successfully tested in PDS measurements on a set of doubly labeled Yersinia outer protein O (YopO) mutants.

Methanethiosulfonate Derivative of OX063 Trityl: A Promising and Efficient Reagent for Side‐Directed Spin Labeling of Proteins

“…One proposed way to overcome the problem of FTAM aggregation is to use a low concentration of both protein and spin label . This strategy resulted in narrow EPR spectra for HSA‐FTAM‐10 and HSA‐FTAM‐17 labeled by using 3 μ m of HSA and 4.5 labels per free ‐SH.…”

“…One proposed way to overcome the problem of FTAM aggregation is to use al ow concentration of both protein and spin label. [15] This strategy resulted in narrow EPR spectra for HSA-FTAM-10 and HSA-FTAM-17 labeled by using 3 mm of HSA and 4.5 labels per free -SH. However,t he labeling efficien-cy with HSA was as low as 64 %w ith more than 1/3 of free -SH groups unlabeled under these conditions (Tables 1a nd S1) rather than the 90 %a chieved [15] with other proteins.…”

“…[15] This strategy resulted in narrow EPR spectra for HSA-FTAM-10 and HSA-FTAM-17 labeled by using 3 mm of HSA and 4.5 labels per free -SH. However,t he labeling efficien-cy with HSA was as low as 64 %w ith more than 1/3 of free -SH groups unlabeled under these conditions (Tables 1a nd S1) rather than the 90 %a chieved [15] with other proteins. This approach to labeling also required meticulous preparation of solutions and laboriousp urification and concentration of the dilute labeled solution.…”

“…Reagent [a] Label [%] [b] Aggregate [%] [c] Cleaved [d] [a] Amount of spin label reagent: ++ shows large excess of reagent of 17.9 per free -SH group; + av alue of about4 .5; + *a dapted protocol of Jassoy et al [15] [b] Labeling efficiency:s pins per starting free -SH group.…”

“…Jassoy et al [15] virtually eliminated noncovalent attachment by carefullyo ptimized, low concentrationso fl abel for their proteins while achieving about 90 %l abeling efficiency.M oreover,t he optimized labeling protocol neededa dditional processing and purification.T hey concluded that "new labels should displayi ncreased water solubility,e .g.,b y functionalizingt he trityl OX063 instead of the Finland trityl." [15] We test here that strategy:r eplacing the FTAM core with the inherentlyh ydrophilic OX063, 2.I ts twelve hydroxyl groups and three carboxyl groups are expected to produce labels with enhanced hydrophilicity,g reatly reduced toxicity, and am uch lower tendency to aggregate. [16] We test the potential of OX063 for the next generation of spin labels using the popular methanethiosulfonate bioconjugate chemistry and we compare these prototype spin labels with similarf irst-generation FTAM labels on ar ather problematic protein:n atural human serum albumin (HSA).…”