Orthogonal Tyrosine and Cysteine Site-Directed Spin Labeling for Dipolar Pulse EPR Spectroscopy on Proteins

Abstract

Site-directed spin labeling of native tyrosine residues in isolated domains of the protein PTBP1, using a Mannich-type reaction, was combined with conventional spin labeling of cysteine residues. Double electron-electron resonance (DEER) EPR measurements were performed for both the nitroxide-nitroxide and Gd(III)-nitroxide label combinations within the same protein molecule. For the prediction of distance distributions from a structure model, rotamer libraries were generated for the two linker forms of the tyrosine-reactive isoindoline-based nitroxide radical Nox. Only moderate differences exist between the spatial spin distributions for the two linker forms of Nox. This strongly simplifies DEER data analysis, in particular, if only mean distances need to be predicted.

A Gadolinium Spin Label with Both a Narrow Central Transition and Short Tether for Use in Double Electron Electron Resonance Distance Measurements

“…[2][3][4][5] For example: greater stability within biological cells, enabling the use of EPR for incell structural biology; [6][7][8][9] improvements in the sensitivity of measurements due to their high spin properties without adding the complication of orientation selection; 2,10 and providing a paramagnetic center with complementary magnetic properties to nitroxides, so that distance measurements may be made between selected spin labels in a multi-labeled sample, in a strategy termed "spectroscopically orthogonal" labeling. 3,[11][12][13][14][15][16][17] In this work we show the rational design and subsequent synthesis of a nine-coordinate Gd(III) ligand for utilization as a spin label in EPR for distance measurements. We focus on keeping the lineshape of the Gd(III) EPR spectrum narrow, which should afford better concentration sensitivity and signal-to-noise, and minimising the linker length between the Gd(III) and the biomolecule.…”

Improving the accuracy of Cu(ii)–nitroxide RIDME in the presence of orientation correlation in water-soluble Cu(ii)–nitroxide rulers

“…Their strength, however, lies in the possibility of spectroscopic selection in systems of several interacting molecules, and they thus may well become a method of choice in the studies of intermolecular biomolecule interactions. [40][41][42][43][44][45][46][47][48][49] Both mentioned fields of application require an appropriate choice of spectroscopic method, which depends on the strength of the spin-spin interaction of interest. Interactions that are typically relevant in EPR spectroscopy can differ by orders of magnitude, depending on the coupled spins.…”

Two-Dimensional Distance Correlation Maps from Pulsed Triple Electron Resonance (TRIER) on Proteins with Three Paramagnetic Centers

“…Two soluble proteins that are well-characterized by X-ray structure analysis [56,57], NMR [58] and EPR spectroscopy [59][60][61] served as application model systems for TRIER: the heat-stable complex of subunits Rpo4 and Rpo7 (also known as F and E, respectively) of the archeal RNA polymerase of M. jannaschii [57,62] and the isolated RNA recognition motif 1 (RRM1) of the alternative splicing regulator polypyrimidine-tract binding protein 1 (PTBP1) [59,60]. The labeling sites for the Rpo4/7 complex (Rpo4: C36, G63C; Rpo7: V49C) were selected from a larger set of sites reported in [61], where all pairwise distances from DEER experiments have been measured.…”

“…RRM1, encoding an N-terminal chitin binding domain as affinity tag, was overexpressed in E. coli and afterwards purified by affinity and size-exclusion chromatography following previously published protocols [59,60].…”