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

Peter, Martin F.; Gebhardt, Christian; Maechtel, Rebecca; Munoz, Gabriel G. Moya; Glänzer, Janin; Narducci, Alessandra; Thomas, Gavin H.; Cordes, Thorben und Hagelüken, Gregor (2022): Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET. In: Nature Communications, Bd. 13, Nr. 1, 4396

Abstract

Pulsed electron-electron double resonance spectroscopy (PELDOR/DEER) and single-molecule Forster resonance energy transfer spectroscopy (smFRET) are frequently used to determine conformational changes, structural heterogeneity, and inter probe distances in biological macromolecules. They provide qualitative information that facilitates mechanistic understanding of biochemical processes and quantitative data for structural modelling. To provide a comprehensive comparison of the accuracy of PELDOR/DEER and smFRET, we use a library of double cysteine variants of four proteins that undergo large-scale conformational changes upon ligand binding. With either method, we use established standard experimental protocols and data analysis routines to determine inter-probe distances in the presence and absence of ligands. The results are compared to distance predictions from structural models. Despite an overall satisfying and similar distance accuracy, some inconsistencies are identified, which we attribute to the use of cryoprotectants for PELDOR/DEER and label-protein interactions for smFRET. This large-scale cross-validation of PELDOR/DEER and smFRET highlights the strengths, weaknesses, and synergies of these two important and complementary tools in integrative structural biology. Pulsed electron-electron double resonance spectroscopy (PELDOR/DEER) and single-molecule Forster resonance energy transfer spectroscopy (smFRET) are used to determine conformational changes and probe distances in biological macromolecules. Here the authors compare the methods on a large set of samples.

• Dokumententyp: Zeitschriftenartikel
• Fakultät: Biologie
• Themengebiete: 500 Naturwissenschaften und Mathematik > 570 Biowissenschaften; Biologie
• Sprache: Englisch
• DFG: Gefördert durch die Deutsche Forschungsgemeinschaft (DFG) - 111166240
• DFG: Gefördert durch die Deutsche Forschungsgemeinschaft (DFG) - 252961272