Spatiotemporal Resolution of Conformational Changes in Biomolecules by Combining Pulsed Electron–Electron Double Resonance Spectroscopy with Microsecond Freeze-Hyperquenching

Abstract

The function of proteins is linked to their conformations that can be resolved with several high-resolution methods. However, only a few methods can provide the temporal order of intermediates and conformational changes, with each having its limitations. Here, we combine pulsed electron−electron double resonance spectroscopy with a microsecond freeze-hyperquenching setup to achieve spatiotemporal resolution in the angstrom range and lower microsecond time scale. We show that the conformational change of the C α -helix in the cyclic nucleotide-binding domain of the Mesorhizobium loti potassium channel occurs within about 150 μs and can be resolved with angstrom precision. Thus, this approach holds great promise for obtaining 4D landscapes of conformational changes in biomolecules.

Time-resolved DEER EPR and solid-state NMR afford kinetic and structural elucidation of substrate binding to Ca 2+ -ligated calmodulin

“…The shortest reaction time that can be achieved with the current apparatus is 1.5 ms. Although a more rapid mixing and freeze-quenching apparatus has recently been reported and demonstrated for DEER measurements with a very impressive dead time of just over 80 μs ( 24 ), the sample volumes are too small for ssNMR applications, and the operational time range is less than that of our current setup.…”

“…To address this knowledge gap, we have performed time-resolved electron paramagnetic resonance (EPR)-based double electron-electron resonance (DEER) and solid-state (ss) NMR studies that jointly elucidate the process of CaM/4Ca 2+ –peptide complex formation in quantitative kinetic and structural terms. This work relies on three technological advances: 1) rapid mixing and freeze quenching to sequentially trap the state of the reaction mixture on the millisecond timescale ( 17 – 24 ), thereby permitting time-resolved DEER EPR and ssNMR measurements of protein–substrate interactions; 2) the application of phase-memory time ( T m ) filtering ( 25 ) to quantitatively extract species populations from DEER data ( 26 ) so that time-dependent probability distance distributions between pairs of spin labels can be monitored; and 3) quantitative analysis of ssNMR 13 C- 13 C correlation spectra to provide residue-specific information on the appearance of structural order and the development of intermolecular contacts between substrate and protein ( 21 , 23 ).…”

Unraveling a Ligand‐Induced Twist of a Homodimeric Enzyme by Pulsed Electron–Electron Double Resonance

“…Pulsed electron–electron double resonance (PELDOR or DEER)[ 12 , 13 ] in combination with site‐directed spin labeling (SDSL) [14] is a powerful method to study conformational changes in proteins. [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ] In SDSL, spin labels are site‐specifically attached to the protein. Most commonly, the methanethiosulfonate spin label (MTSL)[ 22 , 23 ] is used, which reacts selectively with the thiol group of cysteines forming the so‐called R1 side chain containing a disulfide linkage (Figure 1 c ).…”

“…Pulsed electron–electron double resonance (PELDOR or DEER) [12, 13] in combination with site‐directed spin labeling (SDSL) [14] is a powerful method to study conformational changes in proteins [15–21] . In SDSL, spin labels are site‐specifically attached to the protein.…”

Entschlüsselung der ligandeninduzierten Verdrehung eines homodimeren Enzyms mit Hilfe der gepulsten Elektron‐Elektron‐Doppelresonanz‐Spektroskopie

“…Die gepulste Elektron‐Elektron‐Doppelresonanz (PELDOR oder DEER) [12, 13] in Kombination mit der ortsspezifischen Spinmarkierung (engl. Site‐Directed Spin Labeling , SDSL) [14] ist eine leistungsfähige Methode zur Untersuchung von Konformationsänderungen in Proteinen [15–21] . Bei der SDSL werden die Spinlabel ortsspezifisch an das Protein angebracht.…”