Accessing protein conformational distributions in mammalian cells with fast, bio-orthogonal spin-labeling and DEER spectroscopy
Biophysical Journal(2022)
摘要
The ability to measure protein structure and dynamics directly within the cellular environment is fundamental to understanding the molecular mechanisms of protein function and dysfunction. Site-directed spin-labeling, in combination with double electron-electron resonance (DEER) spectroscopy, is a powerful method for determining both the structural states and the conformational equilibria of biomacromolecules. The application of DEER to expressed proteins within cells, however, has been hindered by issues of sensitivity, toxicity, specificity and extent of labeling, and spin-label stability. Here we combine genetic code expansion, bio-orthogonal “click” spin-labeling, and DEER spectroscopy to measure distance distributions of transiently-expressed proteins in intact mammalian cells. Using amber codon suppression, we site-specifically encoded novel, tetrazine-bearing non-canonical amino acids into green fluorescent protein (GFP) and maltose-binding protein (MBP) expressed both in E. coli and in cultured mammalian cells. Reaction with strained trans-cyclooctene (sTCO) nitroxides resulted in specific and quantitative spin-labeling, with reaction rates > 105 M−1 s−1. The remarkable speed and specificity of the sTCO/tetrazine reaction, combined with the permeability of the sTCO-nitroxides, enabled quantitative labeling of proteins in mammalian cells within minutes, requiring only nanomolar concentrations of spin-label added to the culture medium. Subsequent DEER measurements on intact cells revealed distance distributions in general agreement with those measured from samples purified and labeled in vitro. Our results demonstrate that fast, quantitative, and site-specific spin labeling of proteins can be achieved in living cells under conditions where both protein and spin-label concentration are low, and that DEER is capable of resolving conformational distributions of these proteins in the cellular context. We anticipate that this approach will facilitate structure/function studies of proteins under near-native conditions without need for purification.
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关键词
conformational distributions,mammalian cells,protein,bio-orthogonal,spin-labeling
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