Abstract
Encodable lanthanide binding tags (LBTs) have become an attractive tool in modern structural biology as they can be expressed as fusion proteins of targets of choice. Previously, we have demonstrated the feasibility of inserting encodable LBTs into loop positions of interleukin-1β (Barthelmes et al. in J Am Chem Soc 133:808–819, 2011). Here, we investigate the differences in fast dynamics of selected loop-LBT interleukin-1β constructs by measuring 15N nuclear spin relaxation experiments. We show that the loop-LBT does not significantly alter the dynamic motions of the host protein in the sub-τc-timescale and that the loop-LBT adopts a rigid conformation with significantly reduced dynamics compared to the terminally attached encodable LBT leading to increased paramagnetic alignment strength. We further analyze residual dipolar couplings (RDCs) obtained by loop-LBTs and additional liquid crystalline media to assess the applicability of the loop-LBT approach for RDC-based methods to determine structure and dynamics of proteins, including supra-τc dynamics. Using orthogonalized linear combinations (OLCs) of RDCs and Saupe matrices, we show that the combined use of encodable LBTs and external alignment media yields up to five linear independent alignments.
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Dominic Barthelmes and Katja Barthelmes have contributed equally to this work.
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Barthelmes, D., Barthelmes, K., Schnorr, K. et al. Conformational dynamics and alignment properties of loop lanthanide-binding-tags (LBTs) studied in interleukin-1β. J Biomol NMR 68, 187–194 (2017). https://doi.org/10.1007/s10858-017-0118-5
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DOI: https://doi.org/10.1007/s10858-017-0118-5