Computing distance distributions from dipolar evolution data with overtones: RIDME spectroscopy with Gd(iii)-based spin labels

Abstract

Extraction of distance distributions between high-spin paramagnetic centers from relaxation induced dipolar modulation enhancement (RIDME) data is affected by the presence of overtones of dipolar frequencies. As previously proposed, we account for these overtones by using a modified kernel function in Tikhonov regularization analysis. This paper analyzes the performance of such an approach on a series of model compounds with the Gd(iii)-PyMTA complex serving as paramagnetic high-spin label. We describe the calibration of the overtone coefficients for the RIDME kernel, demonstrate the accuracy of distance distributions obtained with this approach, and show that for our series of Gd-rulers RIDME technique provides more accurate distance distributions than Gd(iii)-Gd(iii) double electron-electron resonance (DEER). The analysis of RIDME data including harmonic overtones can be performed using the MATLAB-based program OvertoneAnalysis, which is available as open-source software from the web page of ETH Zurich. This approach opens a perspective for the routine use of the RIDME technique with high-spin labels in structural biology and structural studies of other soft matter.

Pulsed EPR Dipolar Spectroscopy under the Breakdown of the High‐Field Approximation: The High‐Spin Iron(III) Case

“…For such spin pairs, the RIDME time trace is acquired on the slow relaxing spin label, whereas the fast relaxing metal centeri sf lippedb ys pontaneous relaxation. [25,53] The aim of the work presented here is to establish the PDSbased approach for distance measurements between ah ighspin Fe 3 + ion and an itroxide. Recent RIDME studies on metal ion/spin label andm etal ion/metal ion pairs, such as low-spinF e 3 + /nitroxide, [6,30] low-spinF e 3 + /trityl, [51] Cu 2 + /nitroxide, [20] Mn 2 + /nitroxide, [25] and Gd 3 + /Gd 3 + , [49,[52][53][54] confirmed these advantages and showedb etter signal-to-noise ratios and less orientation selectivity as compared to PELDOR.…”

Light‐Induced Pulsed EPR Dipolar Spectroscopy on a Paradigmatic Hemeprotein

“…This allows more than two well-resolved periods of the dipolar modulation to be observed, as seen in Figure 3 violet trace. [46] Distance analysis, together with the validation procedure, was performed for all data sets recorded on the ZnG19 protein using DeerAnalysis [45] or OvertoneAnalysis [46] in the case of the LiRIDME datasets, and the same most-probable distance (2.4 nm) and similar distance distributions were obtained in all cases. Each of the two methods has its own specific factors influencing the value of the modulation depth as previously discussed: it depends on the excitation efficiency of the pump pulse for LiDEER and on the laser excitation and quantum yield for (Re)LaserIMD.…”

Pulsed Dipolar EPR Spectroscopy and Metal Ions: Methodology and Biological Applications

“…Since the theoretical prediction of overtone coefficients appears to be difficult, Keller et al . determined them experimentally on a series of Gd 3+ ‐rulers with known Gd 3+ ‐Gd 3+ distance distributions . This calibration revealed that the overtone coefficients are independent from the mixing time and only weakly dependent on temperature and the spin‐spin distance within the range 3–6 nm.…”

“…Thus, as compared to Gd 3+ /Gd 3+ PELDOR, Gd 3+ /Gd 3+ RIDME might be less reliable for the measurement of long distances. The most significant challenge of Gd 3+ /Gd 3+ RIDME are the higher harmonics of the dipolar coupling frequency, namely 2 ν dd and 3 ν dd (Figure ) . These harmonics appear due to multiple quantum relaxation pathways and, together with the usual dipolar coupling frequency ν dd , modulate the RIDME time traces.…”

“…Such data analysis was developed by Keller et al . and released as the program OvertoneAnalysis . OvertoneAnalysis is based on the same data processing algorithm as DeerAnalysis, but uses a modified kernel function that consists of a linear combination of three terms corresponding to the first, second, and third harmonics of ν dd .…”