EPR-Based Structural Elucidation of Biomolecules
- © Olav Schiemann
- Prof. Dr. Olav Schiemann, has been Professor of Physical Chemistry at the University of Bonn since 2011. His main field of work is EPR spectroscopy with applications in structural biology. He studied chemistry at the University of Marburg and received his doctorate there in the group of C. Elschenbroich with a thesis on organometallic chemistry. After postdoctoral work with J. K. Barton (Caltech) focusing on electron transfer into DNA, he habilitated at the University of Frankfurt in the group of T. F. Prisner with a topic in EPR spectroscopy. From 2007 to 2014, he was first a lecturer, then reader, and finally professor at the Biomedical Sciences Research Center of the University of St. Andrews in Scotland.
- Dipl. Chem. Jean Jacques Jassoy The chemistry graduate has been working on his doctorate since 2014 in the working group of Prof. Schiemann at the University of Bonn. The focus of his work is the synthesis of trityl spin labels for EPR-based distance measurements. He studied chemistry at the University of Bonn and did his diploma thesis in the field of redox-active polymers for energy storage under Prof. Esser at the University of Bonn.
- Fig. 1: Selected spin label types used in EPR spectroscopy: a) established nitroxide radicals, b) novel nitroxide radicals, c) gadolinium (III) complexes, and d) trityl radicals.
- Fig. 2: The protein mutant CYP101 C58 is labeled with a trityl radical, injected into living frog oocytes and the distance distribution between the trityl and the native iron(III) is measured through Relaxation Induced Dipolar Modulation Enhancement (RIDME) EPR experiment (green: in-cell, blue: in-vitro).
The combination of electron paramagnetic resonance (EPR) spectroscopy and site-directed spin-labeling (SDSL) has been used for the structural and dynamic elucidation of biomolecules for 25 years. The application limits of the previously established organic nitroxide radical markers can be overcome by new spin labels thereby increasing the potential of the method to a considerable extent.
The structural elucidation of proteins, DNA and RNA oligomers is an important prerequisite for a deeper understanding of metabolic processes in living organisms. This understanding is in turn fundamental for the development of new drugs and the design of new biotechnological methods for industrial production processes. When considering this vast scope of applications, the identification of biomacromolecular structures progresses rather slowly. Modern modeling software (e.g. Rosetta) provides ever better predictions using the biomolecules’ sequence information but still requires a much broader experimental data set than available today in order to find reliable biomacromolecular folding patterns . Of the estimated ~500,000 protein variants of the human proteome, only about 8,000 have been solved so far (> 60% peptide chain coverage), many of them only fragmentarily [2,3]. For some proteins, the appropriate expression conditions have yet to be found; in other cases, the established structural analysis methods have reached their current limitations.
Established Structural Analysis Methods
While crystallographic attempts often fail due to the impediments of finding suitable crystallization conditions and are intrinsically restricted to solid state reporting, nuclear magnetic resonance (NMR) spectroscopy struggles with resolution losses for increasing protein sizes (> 100 kDa) . Additionally, NMR requires costly isotope labeling to suppress intense background signals as well as rather large sample amounts for in-cell measurements (4 nmol protein/cell) .
Pulsed EPR Experiments
Complementary to crystallography and NMR, the combination of SDSL and EPR-based distance measurements is an important supplement to biomacromolecular structural analysis [6,7].
It facilitates topology studies in large protein-protein , protein-RNA  and protein-substrate complexes , as well as the investigation of oligonucleotide dynamics , the counting of monomers in biomolecular complexes [12,13], the extraction of angular information  and the triangulation of paramagnetic metal centers in proteins . The distances are calculated from the dipole-dipole interaction between paramagnetic centers and, compared to NMR, longer distances of up to 15 nm can be measured  because the magnetic moment of unpaired electrons is about three orders of magnitude larger than that of atomic nuclei. At the same time, the sensitivity of EPR spectroscopy enables the determination of reliable intracellular structural data even from sample amounts close to their physiological concentrations (100 pmol/cell) . Also, the predominantly diamagnetic cell background is invisible to EPR measurements, possible interferences by trace amounts of paramagnetic metal ions and short-lived organic radicals can usually be identified and separated by means of their spectroscopic signature.
The paramagnetic centers (spin labels, fig. 1) required for EPR distance measurements can either be intrinsical components of the biomolecules, e.g. paramagnetic metal co-factors, or can be introduced into the biomolecule structures via SDSL. In the latter case, the spin labels are bioconjugated either during  or after the biosynthesis of the macromolecule; the post-synthetical approach is most common and often uses mild “click” reactions for the chemo-selective attachment of spin labels to the correspondingly modified biomolecule [6,17,19]. In principle, these methods allow for the labeling of any accessible site in the biomolecule for both, proteins and oligonucleotides  whereby it has to be ensured that the incorporated spin labels do not lead to structural nor functional changes in the biomolecule. This can be checked by doing functional assays. The hitherto established nitroxide radical labels need low temperature EPR distance measurements in order to decelerate their electron spin relaxation and are easily reduced to EPR-inactive amines during in-cell measurements. Accordingly, the search for alternatives is in full swing and involves the testing of modified nitroxide labels , paramagnetic gadolinium complexes  and stable trityl radicals. Trityl spin labels show a high redox persistency within living cells as well as a considerably long relaxation time which allows for pulsed EPR measurements at room temperature . They also feature a narrow linewidth of the EPR signal, which benefits the signal-to-noise ratio (SNR) and the use of time-saving EPR pulse techniques.
Intracellular Distance Measurements
Recently, scientists achieved the organic synthesis of new trityl spin labels with diverse bioconjugation capabilities (disulphide bridges, thioether bridges, copper (I) catalyzed alkyne-azide cycloaddition, Sonogashira coupling) and subsequently performed the first intracellular EPR distance measurement between a spin label and a native paramagnetic metal center of a protein (fig. 2) . First, a mutant of the protein cytochrome P450 CYP101 (pseudomonas putida) was expressed, which contained only one accessible surface cysteine at position C58. A buffer solution of this protein was incubated with one of the new trityl labels during four hours at room temperature in order to achieve bioconjugation via a Michael addition type thiol-ene reaction. After a purification step, 253 pmol of the labelled protein were each injected into several living frog oocytes (xenopus laevis) using a micro-injector, and 20 of these pinhead sized cells were placed into an EPR sample tube. The distance between the spin label and the native ferric co-factor of the protein was measured with a pulsed EPR experiment by means of the Relaxation Induced Dipolar Modulation Enhancement (RIDME) pulse sequence at 25 K in the Q-band. The low temperature accounts for the relaxation properties of the low-spin iron (III) center.
The evaluation of the measurement yields a trityl-iron distance of 3.60 nm and is consistent with both the in-vitro reference measurement on the isolated protein and with the associated in-silico prediction. The additionally observed low range distances can presumably be attributed to contact interactions between the label and the protein surface; these interactions induce label conformers with shorter trityl-iron distances and are also typical for nitroxides and other label types .
Summary and Outlook
The combination of SDSL and EPR spectroscopy allows for nanometric distance measurements without the disadvantage of size restrictions and thus represents an important complementary method for the structural analysis of biomacromolecules. Compared to crystallography and NMR, in vitro EPR distance measurements provide structural data of hard to crystallize and large biomolecular complexes while at the same time using smaller sample amounts.The here described study represents an important step towards the investigation of such complexes within their natural environment and demonstrates the first intracellular application of trityl spin labels. Trityl radicals give rise to the hope for EPR distance measurements on biomolecules at room temperature in living cells. Future work could significantly contribute to the elucidation of the intracellular interactome and study the influence of molecular surroundings on biomolecules within cellular compartments.
We thank the Deutsche Forschungsgemeinschaft (DFG) for their funding under the Priority Program SPP1601 and the Collaborative Research Center SFB813.
Affiliation of both authors:
Universität Bonn, Institut für Physikalische und Theoretische Chemie, AG EPR-Spektroskopie, Bonn, Germany
Prof. Dr. Olav Schiemann
Institut für Physikalische und Theoretische Chemie
Rheinische Friedrich-Wilhelms-Universität Bonn
 P.-S. Huang, S. E. Boyken, D. Baker, Nature 2016, 537, 320. DOI: 10.1038/nature19946
 M. S. Baker, S. B. Ahn, A. Mohamedali, M. T. Islam, D. Cantor, P. D. Verhaert, S. Fanayan, S. Sharma, E. C. Nice, M. Connor, S. Ranganathan, Nat. Commun. 2016, 8, 1. DOI: 10.1038/ncomms14271
 www.rcsb.org, H.M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H. Weissig, I. N. Shindyalov, P. E. Bourne, Nucleic Acids Res. 2000, 28, 235. PMCID: PMC102472
 A. Viegas, T. Viennet, M. Etzkorn, Biol. Chem. 2016, 397, 1335. DOI: 10.1515/hsz-2016-0224
 C. Altenbach, T. Marti, H. G. Khorana, W. L. Hubbell, Science 1990, 248, 1088. DOI: 10.1126/science.2160734
 J. P. Klare, H. J. Steinhoff Struct. Bond. 2014, 152, 205. DOI: 10.1007/430_2012_88
 C. Pilotas, R. Ward, E. Branigan, A. Rasmussen, G. Hagelueken, H. Huang, S. S. Black, I.R. Booth, O. Schiemann, J. H. Naismith, Proc. Natl. Acad. Sci. USA 2012, 109, 15983. DOI: 10.1073/pnas.1202286109
 O. Duss, E. Michel, M. Yulikov, M. Schubert, G. Jeschke, F. H.-T. Allain, Nature 2014, 509, 588. DOI: 10.1038/nature13271
 M. Herget, C. Baldauf, C. Schölz, D. Parcej, K.-H. Wiesmüller, R. Tampé, R. Abele, E. Bordignon, Proc. Natl. Acad. Sci. USA 2011, 108, 1349. DOI: 10.1073/pnas.1012355108
 A. Marko, V. P. Deysenkov, D. Margraf, P. Cekan, O. Schiemann, S. Th. Sigurdsson, T.F. Prisner, J. Am. Chem. Soc. 2011, 133, 13375. DOI: 10.1021/ja201244u
 B. E. Bode, D. Margraf, J. Plackmeyer, G. Dürner, T. F. Prisner, O. Schiemann, J. Am. Chem. Soc. 2007, 129, 6736. DOI: 10.1021/ja065787t
 J. E. Banham, C. R. Timmel, R. J. M. Abbott, S. M. Lea, G. Jeschke, Angew. Chem. Int. Ed. 2006, 45, 1058. DOI: 10.1002/anie.200503720
 V. P. Denysenkov, T. F. Prisner, J. Stubbe, M. Bennati, Proc. Natl. Acad. Sci. USA 2006, 103, 13386. DOI: 10.1073/pnas.0605851103
 D. Abdullin, N. Florin, G. Hagelueken, O. Schiemann, Angew. Chem. Int. Ed. 2015, 54, 1827. DOI: 10.1002/anie.201410396
 R. Ward, A. Bowman, E. Sozudogru, H. El-Mkami, T. Owen-Huhes, D. G. Norman, J. Magn. Res. 2010, 207,164. DOI: 10.1016/j.jmr.2010.08.002
 M. J. Schmidt, J. Borbas, M. Drescher, D. Summerer, J. Am. Chem. Soc. 2014, 136, 1238. DOI: 10.1021/ja411535q
 E. H. Abdelkader, A. Feintuch, X. Yao, L. A. Adams, L. Aurelio, B. Graham, D. Godlfarb, G. Otting, Chem. Commun. 2015, 51, 15898. DOI: 10.1039/c5cc07121f
 Structure and Bonding 152, Structural Information from Spin-Labels and Intrinsic Paramagnetic Centres in the Biosciences (Ed.:C. R. Timmel, J. R. Harmer), Springer-Verlag, Berlin Heidelberg 2014. DOI: 10.1007/978-3-642-39125-5
 V. Meyer, M. A. Swanson, L. J. Clouston, P. J. Boratynski, R. A. Stein, H. S. Mchaourab, A. Rajca, S. S. Eaton, G. R. Eaton, Biophys. J. 2015, 108, 1213. DOI: 10.1016/j.bpj.2015.01.015
 G. Yu. Shevelev, O. A. Krumkacheva, A. A. Lomzov, A. A. Kuzhelev, O. Yu. Rogozhnikova, D. V. Trukhin, T. I. Troitskaya, V. M. Tormyshev, M. V. Fedin, D. V. Pyshnyi, E.G .Bagryanskaya, J. Am. Chem. Soc. 2014, 136, 9874. DOI: 10.1021/ja505122n
 J. J. Jassoy, A. Berndhäuser, F. Duthie, S. P. Kühn, G. Hagelueken, O. Schiemann, Angew. Chem. Int. Ed. 2017, 56, 177. DOI: 10.1002/anie.201609085
 D. Abdullin, G. Hagelueken, O. Schiemann, Phys. Chem. Chem. Phys. 2016, 18, 10428. DOI: 10.1039/C6CP01307D