Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET

Abstract

Pulsed electron-electron double resonance spectroscopy (PELDOR or DEER) and single molecule Förster resonance energy transfer spectroscopy (smFRET) are recent additions to the toolbox of integrative structural biology. Both methods are frequently used to visualize conformational changes and to determine nanometer-scale distances in biomacromolecules including proteins and nucleic acids. A prerequisite for the application of PELDOR/DEER and smFRET is the presence of suitable spin centers or fluorophores in the target molecule, which are usually introduced via chemical biology methods. The application portfolio of the two methods is overlapping: each allows determination of distances, to monitor distance changes and to visualize conformational heterogeneity and -dynamics. Both methods can provide qualitative information that facilitates mechanistic understanding, for instance on conformational changes, as well as quantitative data for structural modelling. Despite their broad application, a comprehensive comparison of the accuracy of PELDOR/DEER and smFRET is still missing and we set out here to fill this gap. For this purpose, we prepared a library of double cysteine mutants of three well-studied substrate binding proteins that undergo large-scale conformational changes upon ligand binding. The distances between the introduced spin- or fluorescence labels were determined via PELDOR/DEER and smFRET, using established standard experimental protocols and data analysis routines. The experiments were conducted in the presence and absence of the natural ligands to investigate how well the ligand-induced conformational changes could be detected by the two methods. Overall, we found good agreement for the determined distances, yet some surprising inconsistencies occurred. In our set of experiments, we identified the source of discrepancies as the use of cryoprotectants for PELDOR/DEER and label-protein interactions for smFRET. Our study highlights strength and weaknesses of both methods and paves the way for a higher confidence in quantitative comparison of PELDOR/DEER and smFRET results in the future.

Quantitative Description of Intrinsically Disordered Proteins Using Single-Molecule FRET, NMR, and SAXS

“…Attachment points were read from the respective PDB files of the conformational ensemble in an automated way using in-house software and were then used for the calculation of AVs and distance histograms. Parametrization for Alexa647 was adapted from Peter et al PDB files containing full side chains were used for AV calculation. Conformer-wise FRET files were then generated as an input for ASTEROIDS , selection, containing the different FRET distances used in the selection.…”

N-Methyl deuterated rhodamines for protein labelling in sensitive fluorescence microscopy

“…smFRET: labelling of SBD2 and analysis by msALEX spectroscopy We followed our published protocols for labelling and imaging of SBD2. 31,32,51 In brief, His-tagged SBD2 was incubated in buffer containing 1 mM DTT to keep all cysteine residues in a reduced state. Subsequently, SBD2 was immobilized on a Ni Sepharose 6 Fast Flow resin (GE Healthcare) and then incubated overnight at 4 C with 25 nmol of each uorophore dissolved in labelling buffer (50 mM Tris-HCl pH 7.6, 150 mM NaCl).…”

Multi-parameter photon-by-photon hidden Markov modeling

“…The protein variant YopO L113C/L497C is stochastically-labelled with fluorophorelinked maleimide derivatives, as described previously [82]. Briefly, 200 µg of protein is incubated with 5 mM DTT at 4°C for 30 minutes, to prevent oxidation of the cysteine thiol groups.…”

“…Peter et al and deBoer et al[17,82]. The His 6 -MalE double variant (200 µg) is incubated with 1 mM DTT and loaded immediately after on 200 µL (wet volume) Ni-Sepharose 6 Fast Flow resin, pre-equilibrated with labelling buffer 1 (50 mM Tris-HCl pH 7.4, 50 mM KCl).…”