Abstract
Paramagnetic relaxation enhancement (PRE) can be used to determine long-range distance restraints in biomolecules. The PREs are typically determined by analysis of intensity differences in HSQC experiments of paramagnetic and diamagnetic spin labels. However, this approach requires both isotope- and spin-labelling. Herein, we report a novel method to evaluate NOESY intensities in the presence of a paramagnetic moiety to determine PRE restraints. The advantage of our approach over HSQC-based approaches is the increased number of available signals without the need for isotope labelling. NOESY intensities affected by a paramagnetic center were evaluated during a structure calculation within the paramagnetic iterative relaxation matrix approach (P-IRMA). We applied P-IRMA to a 14-mer RNA with a known NMR solution structure, which allowed us to assess the quality of the PRE restraints. To this end, three different spin labels have been attached at different positions of the 14-mer to test the influence of flexibility on the structure calculation. Structural disturbances introduced by the spin label have been evaluated by chemical shift analysis. Furthermore, the impact of P-IRMA on the quality of the structure bundles were tested by intentionally leaving out available diamagnetic restraints. Our analyses show that P-IRMA is a powerful tool to refine RNA structures for systems that are insufficiently described by using only diamagnetic restraints.
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References
Azarkh M, Singh V, Okle O, Seemann IT, Dietrich DR, Hartig JS, Drescher M (2013) Site-directed spin-labeling of nucleotides and the use of in-cell EPR to determine long-range distances in a biologically relevant environment. Nat Protoc 8:131–147
Battiste JL, Wagner G (2000) Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data†. Biochemistry 39:5355–5365
Beswick V, Guerois R, Cordier-Ochsenbein F, Coïc Y-M, Huynh-Dinh T, Tostain J, Noël J-P, Sanson A, Neumann J-M (1998) Dodecylphosphocholine micelles as a membrane-like environment: new results from NMR relaxation and paramagnetic relaxation enhancement analysis. Eur Biophys J 28:48–58
Boelens R, Koning TMG, Kaptein R (1988) Determination of biomolecular structures from proton-proton NOE’s using a relaxation matrix approach. J Mol Struct 173:299–311
Boelens R, Koning TMG, van der Marel GA, van Boom JH, Kaptein R (1989) Iterative procedure for structure determination from proton-proton NOEs using a full relaxation matrix approach. Application to a DNA octamer. J Magn Reson 82:290–308
Brueschweiler R, Roux B, Blackledge M, Griesinger C, Karplus M, Ernst RR (1992) Influence of rapid intramolecular motion on NMR cross-relaxation rates. A molecular dynamics study of antamanide in solution. J Am Chem Soc 114:2289–2302
Brünger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS et al (1998) Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr D 54:905–921
Büttner L, Seikowski J, Wawrzyniak K, Ochmann A, Höbartner C (2013) Synthesis of spin-labeled riboswitch RNAs using convertible nucleosides and DNA-catalyzed RNA ligation. Bioorg Med Chem 21:6171–6180
Dedmon MM, Lindorff-Larsen K, Christodoulou J, Vendruscolo M, Dobson CM (2005) Mapping long-range interactions in α-synuclein using spin-label NMR and ensemble molecular dynamics simulations. J Am Chem Soc 127:476–477
Duchardt E, Schwalbe H (2005) Residue specific ribose and nucleobase dynamics of the cUUCGg RNA tetraloop motif by MNMR 13C relaxation. J Biomol NMR 32:295–308
Edwards TE, Sigurdsson ST (2007) Site-specific incorporation of nitroxide spin-labels into 2′-positions of nucleic acids. Nat Protoc 2:1954–1962
Fürtig B, Richter C, Bermel W, Schwalbe H (2004) New NMR experiments for RNA nucleobase resonance assignment and chemical shift analysis of an RNA UUCG tetraloop. J Biomol NMR 28:69–79
Grünewald C, Kwon T, Piton N, Förster U, Wachtveitl J, Engels JW (2008) RNA as scaffold for pyrene excited complexes. Bioorg Med Chem 16:19–26
Helmling C, Bessi I, Wacker A, Schnorr KA, Jonker HRA, Richter C, Wagner D, Kreibich M, Schwalbe H (2014) Noncovalent spin labeling of riboswitch rnas to obtain long-range structural NMR restraints. ACS Chem Biol 9:1330–1339
Höbartner C, Sicoli G, Wachowius F, Gophane DB, Sigurdsson STh (2012) Synthesis and characterization of RNA containing a rigid and nonperturbing cytidine-derived spin label. J Org Chem 77:7749–7754
Iwahara J, Tang C, Marius Clore G (2007) Practical aspects of 1H transverse paramagnetic relaxation enhancement measurements on macromolecules. J Magn Reson 184:185–195
Kalk A, Berendsen HJC (1976) Proton magnetic relaxation and spin diffusion in proteins. J Magn Reson 1969(24):343–366
Kellner R, Mangels C, Schweimer K, Prasch SJ, Weiglmeier PR, Rösch P, Schwarzinger S (2009) SEMPRE: spectral editing mediated by paramagnetic relaxation enhancement. J Am Chem Soc 131:18016–18017
Linge JP, O’Donoghue SI, Nilges M (2001) Automated assignment of ambiguous nuclear overhauser effects with ARIA. Methods in Enzymology 339:71–90
Lipari G, Szabo A (1982) Model-free approach to the interpretation of nuclear magnetic resonance relaxation in macromolecules. 1. Theory and range of validity. J Am Chem Soc 104:4546–4559
Mackereth CD, Madl T, Bonnal S, Simon B, Zanier K, Gasch A, Rybin V, Valcárcel J, Sattler M (2011) Multi-domain conformational selection underlies pre-mRNA splicing regulation by U2AF. Nature 475:408–411
Macosko JC, Pio MS, Tinoco I, Shin Y-K (1999) A novel 5′ displacement spin-labeling technique for electron paramagnetic resonance spectroscopy of RNA. RNA 5:1158–1166
Martin NH, Floyd RM, Woodcock HL, Huffman S, Lee C-K (2008) Computation of through-space NMR shielding effects in aromatic ring π-stacked complexes. J Mol Graph Model 26:1125–1130
Nozinovic S, Fürtig B, Jonker HRA, Richter C, Schwalbe H (2010) High-resolution NMR structure of an RNA model system: the 14-mer cUUCGg tetraloop hairpin RNA. Nucleic Acids Res 38:683–694
Ramos A, Varani G (1998) A new method to detect long-range protein–RNA contacts: nMR detection of electron–proton relaxation induced by nitroxide spin-labeled RNA. J Am Chem Soc 120:10992–10993
Schiemann O, Piton N, Plackmeyer J, Bode BE, Prisner TF, Engels JW (2007) Spin labeling of oligonucleotides with the nitroxide TPA and use of PELDOR, a pulse EPR method, to measure intramolecular distances. Nat Protoc 2:904–923
Schnorr K, Gophane DB, Helmling C, Cetiner E, Pasemann K, Fürtig B, Wacker A, Qureshi NS, Gränz M, Barthelmes D et al (2017) Impact of spin label rigidity on extent and accuracy of distance information from PRE data. J Biomol NMR 68:53–63
Seven I, Weinrich T, Gränz M, Grünewald C, Brüß S, Krstić I, Prisner TF, Heckel A, Göbel MW (2014) Photolabile protecting groups for nitroxide spin labels. Eur J Org Chem 2014:4037–4043
Solomon I (1955) Relaxation processes in a system of two spins. Phys Rev 99:559–565
Stoll S, Schweiger A (2006) EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J Magn Reson 178:42–55
Unger SW, Lecomte JTJ, La Mar GN (1985) The utility of the nuclear overhauser effect for peak assignment and structure elucidation in paramagnetic proteins. J Magn Reson 64:521–526
van der Walt S, Colbert SC, Varoquaux G (2011) The numPy array: a structure for efficient numerical computation. Comput Sci Eng 13:22–30
Volkov AN, Worrall JAR, Holtzmann E, Ubbink M (2006) Solution structure and dynamics of the complex between cytochrome c and cytochrome c peroxidase determined by paramagnetic NMR. Proc Natl Acad Sci 103:18945–18950
Wunderlich CH, Huber RG, Spitzer R, Liedl KR, Kloiber K, Kreutz C (2013) A novel paramagnetic relaxation enhancement tag for nucleic acids: a tool to study structure and dynamics of RNA. ACS Chem Biol 8:2697–2706
Acknowledgements
E.C.C. gratefully acknowledges a MainCampus scholarship provided by the Stiftung Polytechnische Gesellschaft (Frankfurt). We would like to thank E. Stirnal, M. Gränz and E. Jaumann for help with experiments. The work was supported by DFG through CRC902 and by the state of Hesse through institutional funding to BMRZ.
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Cetiner, E.C., Jonker, H.R.A., Helmling, C. et al. Paramagnetic-iterative relaxation matrix approach: extracting PRE-restraints from NOESY spectra for 3D structure elucidation of biomolecules. J Biomol NMR 73, 699–712 (2019). https://doi.org/10.1007/s10858-019-00282-0
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DOI: https://doi.org/10.1007/s10858-019-00282-0