Two-Dimensional Distance Correlation Maps from Pulsed Triple Electron Resonance (TRIER) on Proteins with Three Paramagnetic Centers

Abstract

Recently, we introduced the pulsed triple electron resonance (TRIER) experiment, which correlates dipolar frequencies of molecules with three electron spins. These correlation patterns contain valuable information: in combination with double electron-electron resonance (DEER) they allow to interpret distance distributions of biological systems that exist in more than one conformation. Together with an improved sequence and new data processing, we were now for the first time able to obtain two-dimensional distance correlation maps of the previously investigated model compounds as well as of spin-labeled proteins. For this we applied twodimensional approximate Pake transformation to TRIER data. This enabled us to get distance correlation plots from two triple-labeled protein samples that were in good agreement with DEER data and simulations. With such information it should then be possible to assign peaks in DEER distance distributions for macromolecules that can exist in more than one conformation.

Dipolar pathways in multi-spin and multi-dimensional dipolar EPR spectroscopy

“…6). 15,52 The experiment was originally designed for the study of frequencydomain correlations between the different dipolar frequencies in a multi-spin system. Its complexity is reflected in the large number of multi-spin pathways that can contribute to its signal.…”

“…The TRIER experiment aims to record the contributions arising from the t 1 -modulated pathway refocusing at t 1 = τ 1 and the t 2 -modulated pathway refocusing at t 2 = τ 3 . The TRIER model proposed originally 15,52 contained both the two-spin contributions (modulated along each time dimension) as well the three-spin contributions (modulated along both dimensions) arising from pathways and . Under typical experimental conditions, both pathways will provide the largest contributions to the TRIER signal.…”

“…Next, we analyzed the TRIER data from the triply labeled Rpo47 protein complex previously published by Pribitzer et al 52 The dataset presents additional noise along the t 1 -dimension due to technical issues with the hardware of the home-built spectrometer at the time when this data was acquired. We employed the same TRIER model as with the oligoPPE radicals, including pathways to and a three-dimensional homogeneous background model.…”

“…While TRIER data have been analyzed in the frequency domain, 15 the multi-pathway model provides the first reliable approach for analyzing TRIER data directly in the distance domain. Our previous work 52 relied on an incomplete model of the dipolar signal as well as on a data analysis method, which we now consider to be inappropriate. This method was based on a simplistic background correction, which fails to account for important background features, 65 the application of a smoothing filter that can enhance correlations or possibly even introduce spurious ones, and the use of a two-dimensional approximate Pake transformation based on the two-spin dipolar kernel, which is inaccurate for the multi-spin case.…”

Pulsed EPR Methods to Study Biomolecular Interactions

“…correlation map using the Triple Electron Resonance (TRIER) experiment. [108,109] Our labelling strategy for this study is shown in Fig. 4(a).…”