Gd3+ spin labeling for distance measurements by pulse EPR spectroscopy

Abstract

Methods for measuring nanometer scale distances between specific sites in biomolecules (proteins and nucleic acids) and their complexes are essential for describing and analyzing their structure and function. In the last decade pulse EPR techniques were proven very effective for measuring distances between two spin labels attached to a biomolecule. The most commonly used spin labels for such measurements are nitroxide stable radicals. Recently, a new family of spin labels, based on Gd(3+) chelates, has been introduced to overcome some of the limitations of using nitroxides, particularly at high magnetic fields, which are attractive due to the increased sensitivity they offer. The benefits that such S = 7/2 spin labels offer for frequencies of 30 GHz and higher, particularly at 95 GHz, include (1) high sensitivity, only ∼0.15 nmol of doubly labeled biomolecule is needed, (2) the lack of orientation selection, which allows straightforward data analysis. Gd(3+)-Gd(3+) DEER (double electron-electron resonance) distance measurements on labeled peptides, proteins and DNA have already been demonstrated and the results show that they are very promising in terms of sensitivity. In this Perspective we review these new developments. We briefly introduce the characteristics of the DEER experiment on a pair of S = 1/2 spins and characterize the EPR spectroscopic properties of Gd(3+) ions. We then introduce some of the tags employed to attach Gd(3+) to biomolecules and provide a few experimental examples of Gd(3+)-Gd(3+) DEER measurements. This is followed by a discussion of the parameters that affect the sensitivity of such DEER measurements. Since an important term in the spin Hamiltonian of Gd(3+) is the zero-field splitting (ZFS), its effect on the DEER modulation frequencies must be considered and this is discussed next. Finally, another recently reported approach for using Gd(3+) in distance measurements will be presented: the use of Gd(3+)-nitroxide pairs.

Encoded loop-lanthanide-binding tags for long-range distance measurements in proteins by NMR and EPR spectroscopy

“…-tag, which arises from the asymmetry of the LBT coordination sphere and is much larger than for Gd 3? -DOTA-tagged proteins (Goldfarb 2014). The width of the central transition (between the electron spin sublevels m s = -1/2 to m s = 1/2) defined at half height is *39 mT.…”

Gd(III)–Gd(III) distance measurements with chirp pump pulses

“…Besides possible distortions due to the duration of chirp pulses, the ultra-wideband frequency windows of the pump pulses may influence the obtained distance information. While orientation selection effects in Gd-Gd distance measurements are not significant [1], contributions of the ZFS may be altered [7,20]. For further reference, an overview over the subtle differences in distance distributions obtained with Gd-ruler 1 3 is provided in Fig.…”

“…EPR-based distance determination using Gd spin labels is becoming an important alternative to the common approach based on nitroxide spin labels [1]. The majority of experiments to date rely on the four-pulse DEER (double electronelectron resonance) sequence to access distance information between a pair of Gd(III) centers [2].…”

Averaging of nuclear modulation artefacts in RIDME experiments

“…[22,23] Most metal centres exhibit broad EPR spectra, which in com- 20 bination with the limited bandwidths of EPR microwave pulses give rise to low modulation depths in DEER experiments, thus decreasing the sensitivity. The two main approaches solving this problem are based on increasing the bandwidth for flipping the second spin in the dipolarly coupled spin pair.…”

“…[1,3,4,[7][8][9][10] However, a lot of effort has recently been invested to develop spin labels based 10 on metal complexes, as well as trilateration approaches to determine positions of native transition metal centres in metalloproteins. [6,[11][12][13][14][15][16][17][18][19][20][21] In this context metal ions, such as Fe(III), Cu(II), Co(II), Mn(II), or Gd(III), attract attention. Paramagnetic metal ion-based spin labels have spectroscopic properties 15 * Corresponding author.…”