Metal Centres in Pulsed Dipolar Spectroscopy – From Methodology to Application

Abstract

Knowledge of structure does not imply understanding of function of biological systems, but understanding of function is hardly possible without having some knowledge about the structure. Likewise, mechanistic models that rely on a dynamic view, i.e. structural transitions, are crucial for the design of biomimetic systems or for a rational approach to pharmacological intervention. In this framework, it is important to obtain structural data on biological systems with increasing size as well as complexity and to understand how well such data constrain these systems as well as models. In that respect, pulsed dipolar spectroscopy has become a valuable tool in structural biology for the measurement of distance distributions in the nanometre range without the need of longrange order within the biological samples, i.e. crystallization, irrespective of the size of the system. This is particularly important to access functional states. In the past few years new insight into protein function has been obtained by hybrid structural models that incorporate restraints from pulsed dipolar spectroscopy in combination with site-directed spin labelling cooperatively with restraints from other techniques. The work realized within this thesis pushes for increased sensitivity and reliable data analysis of pulse dipolar spectroscopy data at low hardware requirements. It contributes towards extending the arsenal of spectroscopically orthogonal spin probes that are important for site-directed spin labelling strategies, which in turn are crucial for applying Electron Paramagnetic Resonance (EPR) to heterologous complexes of biomacromolecules. In that context, the thesis embarked on a study of several metal ions (Gd(III), Mn(II), Cu(II), Fe(III), Co(II)) with respect to their suitability in pulsed dipolar spectroscopy. Complexes of Cu(II), Mn(II) as well as Gd(III) were found to be most promising, with the optimal choice depending on the problem at hand. The EPR properties, i.e. lineshape or electronic relaxation, of Mn(II) and Gd(III) are strongly influenced by the zero-field splitting (ZFS) interaction that in turn influences the performance of distance measurements on such compounds. Hence, the reliability of extracting broadly distributed ZFS parameters was studied for a variety of Gd(III) complexes and two different models present in literature. These ZFS data allowed for the development of a superposition model to predict the magnitude of the ZFS based on knowledge of Gd(III)-complex’s structure. This superposition model may be useful for designing new Gd(III) labels prior to synthesis efforts. Currently, the major technique for obtaining distance distributions between spin pairs is the double electron electron resonance (DEER) experiment, in combination with nitroxide-based spin labelling. The reliability of this approach and the resulting distance distributions may be compromised by orientation selection effects in orthogonal labelling strategies, in which one of the two nitroxides is replaced by a metal ion that typically exhibits much more strongly anisotropically-broadened EPR lines. Further, if both labels are metal ions low modulation depths are observed in conventional DEER experiments consequently limiting sensitivity. In addition, level mixing for high-spin pairs with insufficient difference in the resonance offset between the observed and pumped transitions may induce distortions to the distance distribution. To some extent, these complications can be addressed by the relaxation induced dipolar modulation enhancement (RIDME) experiment without the need for technically demanding EPR spectrometers. Relaxation-induced spin flips have virtual infinite bandwidth and spins distributed over the whole spectrum contribute to the dipolar signal. Therefore, the average resonance frequency difference in spin pairs contributing to the modulation is much larger than the dipoledipole coupling, which reduces distortions from level mixing. This also leads to reduced orientation selection compared to spin inversion induced by a pump pulse that only covers a small fraction of the EPR spectrum. Before commencing this thesis, RIDME at high field had shown potential for improving modulation depth as well as resolution of the distance distribution for Gd(III)-Gd(III) pairs. The technique had also been shown to provide information on metalloproteins with native paramagnetic metal ions. However, broad application of the RIDME technique in the framework of spin-labelled biomolecules is hindered by some difficulties: (i) for high-spin species, multiples of the dipolar frequency contribute to the dipolar signal, (ii) the technique is highly sensitive to nuclear modulation artefacts, (iii) no simple analytical expressions exist for intermolecular background decay and the signal decays faster than in the DEER experiment. Therefore the separation of the intermolecular background decay from the intramolecular modulation is more challenging, in particular for broad distance distributions, and the accessible distance range might be restricted. In this respect, the thesis commenced a thorough study of several aspects that influence data analysis, such as background correction, nuclear modulations and contributions of higher harmonics. An approach - based on a modified kernel in Tikhonov regularization - was implemented that can provide the anticipated distance distributions for high-spin RIDME data if the relative contributions of the different higher harmonics are known. For either high-spin metal centre, Mn(II) or Gd(III), a significant gain in modulation depth could be confirmed and the harmonic overtone coefficients occurred to be relatively stable for spin-spin distances > 3 nm, different pulse sequence parameters as well as measurement temperature, ligand environment and in spin-labelled proteins compared to model compounds. By processing simulated data sets under the assumptions of too large or too small content of harmonics in the data, it was found how distance distributions are influenced by errors in estimating this content and that a mismatch of about 20% appears to be within the uncertainties due to other approximations for Gd(III)-Gd(III) measurements in biomolecules. Some deviations were observed for short spin-spin distances as well as for shifting the detection position away from the maximum of the Gd(III) spectrum or towards the low-field part of the Mn(II) spectrum. These deviations shall be investigated in more detail in follow-up studies appended with newly – to be synthesised – model compounds in the short distance range. It was shown in previous work on Gd(III)-Gd(III) DEER measurements that using ultrawideband (UWB) excitation over a range of 2.5 GHz, polarization can be transferred from satellite transitions to the central observer transition and thereby increase sensitivity. Within this thesis, it was demonstrated that similar schemes can be used to enhance the sensitivity in Gd(III)-Gd(III) as well as Mn(II)-Mn(II) RIDME measurements. In addition, such pre-polarization schemes were found to be promising to reduce the intensity of artefact peaks by enhancing only echoes excited by all pulses in the RIDME sequence. Further, a new approach for suppression of nuclear modulation artefacts in RIDME was introduced. The approach avoids losses in signal-to-noise ratio that are present in the previously established approaches. Interesting trends were recognized in the experimental intermolecular RIDME background decay that are in agreement with approximate analytical equations computed under the assumption of instantaneous jumps of the non-resonant spins following an approach by Hu and Hartman. The observed proton-driven spectral diffusion processes in the RIDME background decay may allow designing new RIDME-based experimental schemes to characterize soft matter and biomacromolecules through the determination of the local proton distribution in the vicinity of the spin-labelled site. A comparison of RIDME and UWB-DEER measurements on molecular Cu(II)-rulers revealed that both techniques perform similarly depending on the available hardware. For both experimental schemes, a much larger modulation depth and thus higher sensitivity was obtained in comparison to a conventional DEER setup. Notably, the RIDME technique reduces orientation selectivity from the inverted species and appears to be beneficial for situations with limited power over a broad range. Such situations are currently still often encountered at high field, where nuclear modulations in RIDME experiments are conveniently suppressed. Eventually, paramagnetic metal-ion substitution of Mg(II) by Mn(II) was found to provide valuable means to follow metal binding in nucleotide binding domains of ATP-fuelled proteins as well as for accessing geometric assemblies through measurement of spin-spin distances. It was demonstrated how such data can be combined with solid-state NMR data in order to obtain deeper structural insight.