Measurement of Protein Dynamics from Site Directed Cu(II) Labeling

Abstract

This review describes the use of Electron Paramagnetic Resonance (EPR) to measure residue specific dynamics in proteins with a specific focus on Cu(II)-based spin labels. First, we outline approaches used to measure protein motion by nitroxide-based spin labels. Here, we describe conceptual details and outline challenges that limit the use of nitroxide spin labels to solvent-exposed α-helical sites. The bulk of this review showcases the use of newly developed Cu(II)-based protein labels. In this approach, the strategic mutation of native residues on a protein to generate two neighboring Histidine residues (i.e., the dHis motif) is exploited to enable a rigid site-selective binding of a Cu(II) complex. The chelation of the Cu(II) complex to dHis directly anchors the Cu(II) spin label to the protein backbone. The improvement in rigidity expands both the spin-labeling toolkit as well as the resolution of many EPR measurements. We describe how EPR measurements of the Cu(II) label directly reflect backbone motion and fluctuations. The EPR are complemented by Molecular Dynamics simulations. Finally, the dHis motif provides access to the measurement of site-specific dynamics at both α-helices and β-sheets. The review outlines the limitations of the dHis method and provides an outlook for future developments.

Efficient sampling of molecular orientations for Cu(ii)-based DEER on protein labels

“…The ϕ angle of a spin dictates the effective g and A -values, as follows: 48 We used the g ‖ = 2.277, g ⊥ = 2.057, A ‖ = 162 G, and A ⊥ = 10 G, which are canonical for the dHis-Cu( ii ). 3,49…”

“…We used the g 8 = 2.277, g > = 2.057, A 8 = 162 G, and A > = 10 G, which are canonical for the dHis-Cu(II). 3,49 In addition to f, we tabulated the orientation of r with respect to the applied magnetic field, h. The angle y affects the magnetic dipolar interaction of each spin-pair, as follows:…”

“…Specifically, Continuous Wave (CW) EPR experiments on Cu(II) labeled biomolecules can measure site-specific dynamics when the Cu(II) label is attached to a selected site on a protein. 2,3 Additionally, when two or more sites are labeled, distance measurements can be obtained between these sites using pulsed dipolar spectroscopy. [4][5][6][7][8][9][10] While these experiments are routinely conducted using many other spin labels, [11][12][13] Cu(II) is unique due to its labeling strategy.…”

“…15 As a result, room temperature CW-EPR experiments on dHis-Cu(II) are incisive probes of backbone dynamics and are sensitive to time scales as small as 50 ps. 2,3 Furthermore, distance measurements between two dHis-Cu(II) sites are up to five times narrower than commonly used nitroxide labels. 14 Such narrow distributions enable the resolution of multiple conformations that otherwise would be indistinguishable using other labels.…”

Integrating Electron Paramagnetic Resonance Spectroscopy and Computational Modeling to Measure Protein Structure and Dynamics

“…When the dHis-Cu(II) labels experience faster motions, the CW-EPR spectra exhibit partially-to fully-averaged g and hyperfine splitting, with simulated spectra at the intermediate (purple spectrum) and isotropic regime (red spectrum) plotted in Figure 1B, respectively. [28][29][30][31] In addition to CW-EPR, 2D-pulsed EPR methods primarily using nitroxide probes can be used to access dynamics on the 100 s of nanoseconds to milliseconds timescales. [32][33][34][35][36][37] On the other hand, pulsed-dipolar spectroscopy (PDS) [38][39][40][41][42][43][44] can measure sparse distance constraints in the range of 2 to 8 nm, [42,44,45] and even up to 16 nm [46] for fully deuterated systems.…”