NMR pseudocontact shifts in a symmetric protein homotrimer
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ARTICLE
NMR pseudocontact shifts in a symmetric protein homotrimer Thomas Müntener1 · Raphael Böhm1 · Kenneth Atz1 · Daniel Häussinger2 · Sebastian Hiller1 Received: 1 April 2020 / Accepted: 25 June 2020 © The Author(s) 2020
Abstract NMR pseudocontact shifts are a valuable tool for structural and functional studies of proteins. Protein multimers mediate key functional roles in biology, but methods for their study by pseudocontact shifts are so far not available. Paramagnetic tags attached to identical subunits in multimeric proteins cause a combined pseudocontact shift that cannot be described by the standard single-point model. Here, we report pseudocontact shifts generated simultaneously by three paramagnetic Tm-M7PyThiazole-DOTA tags to the trimeric molecular chaperone Skp and provide an approach for the analysis of this and related symmetric systems. The pseudocontact shifts were described by a “three-point” model, in which positions and parameters of the three paramagnetic tags were fitted. A good correlation between experimental data and predicted values was found, validating the approach. The study establishes that pseudocontact shifts can readily be applied to multimeric proteins, offering new perspectives for studies of large protein complexes by paramagnetic NMR spectroscopy. Keywords Solution NMR spectroscopy · Pseudocontact shift · Paramagnetism · Lanthanide chelating tag · Homotrimer · Chaperone Skp
Introduction Precise descriptions of protein structure and dynamics are key to understand biological functionality. NMR spectroscopy has proven a valuable source to gain such information under native or native-like conditions for highly relevant systems. Thereby, different geometrical restraints can be used to determine the structure of a protein. While residual dipolar couplings (RDCs) determine relative orientations of bond vectors (Chen and Tjandra 2012), nuclear Overhauser effects (NOEs) provide short-range distance information (Wagner and Wüthrich 1982; Williamson 2009). On the other hand, the two paramagnetic effects paramagnetic relaxation enhancement (PRE) and pseudocontact shift (PCS), which can be introduced into proteins by various Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10858-020-00329-7) contains supplementary material, which is available to authorized users. * Sebastian Hiller [email protected] 1
Biozentrum, University of Basel, Klingelbergstrasse 70, 4056 Basel, Switzerland
Department of Chemistry, University of Basel, St. Johanns‑Ring 19, 4056 Basel, Switzerland
2
lanthanide-chelating tags (LCTs), have proven to provide valuable distance- and orientation-dependent information over extremely long distances exceeding 100 Å (Joss and Häussinger 2019a; Nitsche and Otting 2017; Wang et al. 2007; Bertini et al. 2002a,b; Gochin and Roder 1995; Pearce et al. 2017). LCTs based on the DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) framework provide large PCSs and are usually attached to the protein surf
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