Abstract
Double electron-electron resonance(DEER) spectroscopy applied to orthogonally spin-labeled biomolecular complexes simplifies the assignment of intra- and intermolecular distances, thereby increasing the information content per sample. In fact, various spin labels can be addressed independently in DEER experiments due to spectroscopically nonoverlapping central transitions, distinct relaxation times, and/or transition moments; hence, they are referred to as spectroscopically orthogonal. Molecular complexes which are, for example, orthogonally spin-labeled with nitroxide(NO) and gadolinium(Gd) labels give access to three distinct DEER channels that are optimized to selectively probe NO-NO, NO-Gd, and Gd-Gd distances. Nevertheless, it has been previously recognized that crosstalk signals between individual DEER channels can occur, for example, when a Gd-Gd distance appears in a DEER channel optimized to detect NO-Gd distances. This is caused by residual spectral overlap between NO and Gd spins which, therefore, cannot be considered as perfectly orthogonal. Here, we present a systematic study on how to identify and suppress crosstalk signals that can appear in DEER experiments using mixtures of NO-NO, NO-Gd, and Gd-Gd molecular rulers characterized by distinct, nonoverlapping distance distributions. This study will help to correctly assign the distance peaks in homo- and heterocomplexes of biomolecules carrying not perfectly orthogonal spin labels.
Highlights
1.1 Double electron–electron resonance (DEER)Double electron–electron resonance (DEER, known as pulsed electron double resonance or PELDOR) is an electron paramagnetic resonance (EPR) pulsed dipolar spectroscopy (PDS) technique introduced by Milov et al (1981, 1984) and further developed by Spiess and Jeschke (Martin et al, 1998; Pannier et al, 2000) that probes the r−3-dependent dipolar coupling interaction between adjacent unpaired electron spins
If we consider that the NO spins of the NO–Gd ruler are observed and the Gd spins are partially excited by the pump pulse, we suggest that the absence of this crosstalk signal is due to the fact that the NO spins of the NO–Gd ruler have a shorterphase memory time Tm than those in the NO–NO ruler at 50 K (Tm ≈ 2 μs versus 4.6 μs; see Fig. S2 and Table S4; part B), which strongly decreases their contribution in the observer echo for the detected 2 μs time trace
In this work we thoroughly investigated the appearance of crosstalk signals between the three possible DEER channels at Q-band frequencies with mixtures of NO–NO, NO– Gd, and Gd–Gd rulers with nonoverlapping distance distributions
Summary
Double electron–electron resonance (DEER, known as pulsed electron double resonance or PELDOR) is an electron paramagnetic resonance (EPR) pulsed dipolar spectroscopy (PDS) technique introduced by Milov et al (1981, 1984) and further developed by Spiess and Jeschke (Martin et al, 1998; Pannier et al, 2000) that probes the r−3-dependent dipolar coupling interaction between adjacent unpaired electron spins. DEER is an established technique in structural biology (Jeschke, 2012, 2018), complementary to X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and cryoelectron microscopy. From this perspective, it is seen as being among the most promising methods for in-cell studies (Plitzko et al, 2017). The primary DEER trace contains an intermolecular background function that needs to be fitted and separated from the desired intramolecular dipolar signal
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