Strategies to identify and suppress crosstalk signals in double electron–electron resonance (DEER) experiments with gadolinium<sup>III</sup> and nitroxide spin-labeled compounds

Abstract

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.

Gd3+–Trityl–Nitroxide Triple Labeling and Distance Measurements in the Heterooligomeric Cobalamin Transport Complex in the Native Lipid Bilayers

“…The amplitude of the Gd 3+ –Gd 3+ crosstalk signal is decreased (DEERNet suggested a total absence; see Figure b vs d). This might be due to the presence of additional Gd 3+ (BtuF) in the sample, which reduces the relative ratio between different spin pairs . The Gd 3+ –NO (BtuF–T-CNCbl) PELDOR revealed a somewhat longer distance (Figure c, also shown in Figure e), and a partial contribution (crosstalk) from it might account for the broadening of Gd 3+ –trityl distances.…”

“…For the Gd 3+ –NO PELDOR, it was shown that an additional crosstalk signal corresponding to the Gd 3+ –Gd 3+ distance could appear due to the (suboptimal) coexcitation of underlying Gd 3+ spins by the NO pump pulse. , Their nutation experiments showed that, even at ∼290 MHz lower from the spectral maximum, pulses mostly excited the M S = −1/2 to M S = +1/2 transition. Pumping trityl could enhance this crosstalk signal due to the smaller frequency offset (Figure d).…”

“…This might be due to the presence of additional Gd 3+ (BtuF) in the sample, which reduces the relative ratio between different spin pairs. 47 The Gd 3+ −NO (BtuF−T-CNCbl) PELDOR revealed a somewhat longer distance (Figure 6c, also shown in Figure 4e), and a partial contribution (crosstalk) from it might account for the broadening of Gd 3+ −trityl distances. Importantly, no crosstalk signals (from Gd 3+ −Gd 3+ ) were evident for the Gd 3+ −NO data under the experimental conditions.…”

Milliwatt three- and four-pulse double electron electron resonance for protein structure determination

“…S1 (ESI†) for the DEER setups and Table S3 (ESI†) for the experimental parameters) on a dinitroxide ruler (NO–NO ruler; see Materials and Methods) with 2.0 nm interspin distance that is soluble in aqueous solvents. 54 The MS-3 resonator required an incident power of 45 W at the resonator to perform the experiment. In contrast, the microhelix required only 43 mW of incident power.…”

“…The structural formula is presented in Fig. 9(a), and the synthesis is described in Teucher et al 54 The ruler consists of two nitroxides that are held by a stiff rod-like spacer at a distance of 2.0 nm. 64 A stock solution of the NO–NO ruler was prepared in D 2 O using 50% v/v deuterated glycerol to ensure a glass-like freezing at a 1.4 mM final molecule concentration.…”