Ultrafast Bioorthogonal Spin-Labeling and Distance Measurements in Mammalian Cells Using Small, Genetically Encoded Tetrazine Amino Acids

Abstract

Site-directed spin-labeling (SDSL)in combination with double electron–electron resonance (DEER) spectroscopyhas emerged as a powerful technique for determining both the structural states and the conformational equilibria of biomacromolecules. DEER combined with in situ SDSL in live cells is challenging since current bioorthogonal labeling approaches are too slow to allow for complete labeling with low concentrations of spin label prior to loss of signal from cellular reduction. Here, we overcome this limitation by genetically encoding a novel family of small, tetrazine-bearing noncanonical amino acids (Tet-v4.0) at multiple sites in proteins expressed in Escherichia coli and in human HEK293T cells. We achieved specific and quantitative spin-labeling of Tet-v4.0-containing proteins by developing a series of strained trans-cyclooctene (sTCO)-functionalized nitroxidesincluding a gem-diethyl-substituted nitroxide with enhanced stability in cellswith rate constants that can exceed 106 M–1 s–1. The remarkable speed of the Tet-v4.0/sTCO reaction allowed efficient spin-labeling of proteins in live cells within minutes, requiring only sub-micromolar concentrations of sTCO-nitroxide. DEER recorded from intact cells revealed distance distributions in good agreement with those measured from proteins purified and labeled in vitro. Furthermore, DEER was able to resolve the maltose-dependent conformational change of Tet-v4.0-incorporated and spin-labeled MBP in vitro and support assignment of the conformational state of an MBP mutant within HEK293T cells. We anticipate the exceptional reaction rates of this system, combined with the relatively short and rigid side chains of the resulting spin labels, will enable structure/function studies of proteins directly in cells, without any requirements for protein purification.

Measuring conformational equilibria in allosteric proteins with time-resolved tmFRET

“…For tmFRET experiments, the expression and purification of MBP was done as described previously (11). For RIDME experiments, dual cysteine constructs of MBP-295C-211C and MBP-322C-278C with N-terminal 6×His tags were expressed from a pETM11 vector in E. coli C43(DE3) and subsequently purified by Co 2+ affinity chromatography as previously described (24). The 6xHis tag was removed by incubation with a 1:50 (TEV:MBP) weight ratio of TEV protease (4 h at room temperature, then 12 h at 4 °C) in K + -Tris buffer (130 mM KCl, 30 mM Tris, pH 7.4) containing 0.5 mM EDTA and 1 mM TCEP.…”

“…For these experiments, we introduced cysteine substitutions at both donor and acceptor sites in our previous MBP constructs, generating MBP-295C-211C and MBP-322C-278C. Nitroxide spin labels introduced at these site pairs have previously been employed to detect maltose-dependent conformational changes in MBP using DEER (24). To examine distance distributions obtained using the phenM label, MBP-295C-211C and MBP-322C-278C were labelled with phenM and subsequently labelled with Cu 2+ (Figure 6A).…”

From Stable Radicals to Thermally Robust High-Spin Diradicals and Triradicals

“…Aggregation (and/or association) leads to the very low electron spin T 1 (and T m ) observed for 15 mM gem - DiCarboxy in DNP juice, which is more than 2 orders of magnitude lower compared to Ox-063 in DNP juice or to 0.1 mM gem - DiCarboxy in trehalose/sucrose at a similar temperature. , Because of their electron withdrawing groups, gem - DiCarboxy and a related derivative are readily reduced, including rapid reduction by ascorbate; this is in contrast to tetraethyl pyrroline ( TEP ) nitroxides, which are among the most difficult to reduce nitroxide radicals. , Consequently, a derivative of TEP , such as sTEO-TEP , provided rapid and specific spin-labeling of proteins containing tetrazine amino acid both in vitro and in live cells. Resistance of TEP to reduction enabled EPR distance measurements on native (submicromolar) concentrations of labeled proteins in live eukaryotic cells …”

Cucurbit[7]uril Enhances Distance Measurements of Spin-Labeled Proteins

“…Tremendous efforts have been made to probe macromolecules in natural biological systems that can provide insight into their structures and functions. Site-directed spin labeling (SDSL) pulsed electron paramagnetic resonance (EPR) dipolar spectroscopy (PDS), which includes double electron–electron resonance (DEER) and double quantum coherence (DQC), is among the best techniques for accurate measurement of the conformations of flexible regions of biomolecules. − The advantages include high sensitivity, wide dynamic range, absolute distance distributions, and minimal structural perturbation from relatively small-size nitroxide spin labels. , The most widely used spin label for SDSL is methane thiosulfonate pyrroline nitroxide (MTSL), which selectively binds to cysteine, that can be introduced at selected locations by mutagenesis to provide doubly labeled proteins. , Using nitroxides, these measurements require cryogenic temperatures because of the dynamic averaging effects associated with methyl group rotation, which shorten the electron spin coherence time ( T m ) between about 80 and 300 K. − …”