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

Pulsed EPR dipolar spectroscopy (PDS) offers several methods for measuring dipolar coupling and thus the distance between electron-spin centers. To date, PDS measurements to metal centers were limited to ions that adhere to the high-field approximation. Here, the PDS methodology is extended to cases where the high-field approximation breaks down on the example of the high-spin Fe³⁺/nitroxide spin-pair. First, the theory developed by Maryasov et al. (Appl. Magn. Reson. 2006 , 30, 683–702) was adapted to derive equations for the dipolar coupling constant, which revealed that the dipolar spectrum does not only depend on the length and orientation of the interspin distance vector with respect to the applied magnetic field but also on its orientation to the effective g-tensor of the Fe³⁺ ion. Then, it is shown on a model system and a heme protein that a PDS method called relaxation-induced dipolar modulation enhancement (RIDME) is well-suited to measuring such spectra and that the experimentally obtained dipolar spectra are in full agreement with the derived equations. Finally, a RIDME data analysis procedure was developed, which facilitates the determination of distance and angular distributions from the RIDME data. Thus, this study enables the application of PDS to for example, the highly relevant class of high-spin Fe³⁺ heme proteins.     

In Situ Labeling and Distance Measurements of Membrane Proteins in E. coli Using Finland and OX063 Trityl Labels

In situ investigation of membrane proteins is a challenging task. Previously we demonstrated that nitroxide labels combined with pulsed ESR spectroscopy is a promising tool for this purpose. However, the nitroxide labels suffer from poor stability, high background labeling, and low sensitivity. Here we show that Finland (FTAM) and OX063 based labels enable labeling of the cobalamin transporter BtuB and BamA, the central component of the β-barrel assembly machinery (BAM) complex, in E coli. Compared to the methanethiosulfonate spin label (MTSL), trityl labels eliminated the background signals and enabled specific in situ labeling of the proteins with high efficiency. The OX063 labels show a long phase memory time (T_M) of ≈5 μs. All the trityls enabled distance measurements between BtuB and an orthogonally labeled substrate with high selectivity and sensitivity down to a few μm concentration. Our data corroborate the BtuB and BamA conformations in the cellular environment of E. coli.     

Dead-time free measurement of dipole-dipole interactions between electron spins

A four-pulse version of the pulse double electron-electron resonance (DEER) experiment is presented, which is designed for the determination of interradical distances on a nanoscopic length-scale. With the new pulse sequence electron-electron couplings can be studied without dead-time artifacts, so that even broad distributions of electron-electron distances can be characterized. A version of the experiment that uses a pulse train in the detection period exhibits improved signal-to-noise ratio. Tests on two nitroxide biradicals with known length indicate that the accessible range of distances extends from about 1.5 to 8 nm. The four-pulse DEER spectra of an ionic spin probe in an ionomer exhibit features due to probe molecules situated both on the same and on different ion clusters. The former feature provides information on the cluster size and is inaccessible with previous methods.     

Addendum to the paper "Dead-time free measurement of dipole-dipole interactions between electron spins" by M. Pannier, S. Veit, A. Godt, G. Jeschke, and H.W. Spiess [J. Magn. Reson. 142 (2000) 331-340

The development of four-pulse DEER as described, which has been published in the Journal of Magnetic Resonance more than 10 years ago. The corresponding paper is an example where a slight advance, such as adding a refocusing pulse, which in retrospect looks so simple, can have a remarkable impact on an entire field of science. In our case it offered a simple way to exact measurements of distances between defined species in the nanometer range. The current applications are mainly in determining structures of proteins and nucleic acids.     

Synthesis of μ2-Oxo-Bridged Iron(III) Tetraphenylporphyrin–Spacer–Nitroxide Dimers and their Structural and Dynamics Characterization by using EPR and MD Simulations

Iron(III) porphyrins have the propensity to form μ₂-oxo-dimers, the structures of which resemble two wheels on an axle. Whereas their crystal structure is known, their solution structure and internal dynamics is not. In the present work, the structure and dynamics of such dimers were studied by means of electron paramagnetic resonance (EPR) spectroscopy and quantum chemistry based molecular dynamics (MD) simulations by using the semiempirical tight-binding method (GFN-xTB). To enable EPR investigation of the dimers, a nitroxide was attached to each of the tetraphenylporphyrin cores through a linear and a bent linker. The inter-nitroxide distance distributions within the dimers were determined by continuous-wave (cw)-EPR and pulsed electron–electron double resonance (PELDOR or DEER) experiments and, with the help of MD, interpreted in terms of the rotation of the porphyrin planes with respect to each other around the Fe–O–Fe axis. It was found that such rotation is restricted to the four registers defined by the phenyl substituents. Within the registers, the rotation angle swings between 30° and 60° in the proximal and between 125° and 145° in the distal register. With EPR, all four angles were found to be equally populated, whereas the 30° and 145° angles are strongly favored to the expense of the 60° and 125° angles in the MD simulation. In either case, the internal dynamics of these dimers thus resemble the motion of a step motor.     

Accurate long-range distance measurements in a doubly spin-labeled protein by a four-pulse, double electron-electron resonance method

Distance determination in disordered systems by a four-pulse double electron-electron resonance method (DEER or PELDOR) is becoming increasingly popular because long distances (several nanometers) and their distributions can be measured. From the distance distributions eventual heterogeneities and dynamics can be deduced. To make full use of the method, typical distance distributions for structurally well-defined systems are needed. Here, the structurally well-characterized protein azurin is investigated by attaching two (1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate spin labels (MTSL) by site-directed mutagenesis. Mutations at the surface sites of the protein Q12, K27, and N42 are combined in the double mutants Q12C/K27C and K27C/N42C. A distance of 4.3 nm is found for Q12C/K27C and 4.6 nm for K27C/N42C. For Q12C/K27C the width of the distribution (0.24 nm) is smaller than for the K27C/N42C mutant (0.36 nm). The shapes of the distributions are close to Gaussian. These distance distributions agree well with those derived from a model to determine the maximally accessible conformational space of the spin-label linker. Additionally, the expected distribution for the shorter distance variant Q12C/N42C was modeled. The width is larger than the calculated one for Q12C/K27C by 21%, revealing the effect of the different orientation and shorter distance. The widths and the shapes of the distributions are suited as a reference for two unperturbed MTSL labels at structurally well-defined sites.