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Bridge over troubled proline: assignment of intrinsically disordered proteins using (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H experiments concomitantly with HNCO and i(HCA)CO(CA)NH

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Abstract

NMR spectroscopy is by far the most versatile and information rich technique to study intrinsically disordered proteins (IDPs). While NMR is able to offer residue level information on structure and dynamics, assignment of chemical shift resonances in IDPs is not a straightforward process. Consequently, numerous pulse sequences and assignment protocols have been developed during past several years, targeted especially for the assignment of IDPs, including experiments that employ HN, Hα or 13C detection combined with two to six indirectly detected dimensions. Here we propose two new HN-detection based pulse sequences, (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H, that provide correlations with 1HN(i − 1), 13C′(i − 1) and 15N(i), and 1HN(i + 1), 13C′(i) and 15N(i) frequencies, respectively. Most importantly, they offer sequential links across the proline bridges and enable filling the single proline gaps during the assignment. We show that the novel experiments can efficiently complement the information available from existing HNCO and intraresidual i(HCA)CO(CA)NH pulse sequences and their concomitant usage enabled >95 % assignment of backbone resonances in cytoplasmic tail of adenosine receptor A2A in comparison to 73 % complete assignment using the HNCO/i(HCA)CO(CA)NH data alone.

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Acknowledgments

This work was financially supported by the Grants 259447 (to P. P.) and 132138 (to V. P. J.) from the Academy of Finland, and by the IRG 249081 from FP7 Marie Curie European Reintegration Grant and Biocenter Oulu (to V. P. J.). Biocenter Finland and Biocentrum Helsinki are acknowledged for the infrastructure support.

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Authors and Affiliations

  1. Program in Structural Biology and Biophysics, Institute of Biotechnology/NMR Laboratory, University of Helsinki, P.O. Box 65, 00014, Helsinki, Finland

    Maarit Hellman & Perttu Permi

  2. Department of Biochemistry, University of Oulu, P.O. Box 3000, 90014, Oulu, Finland

    Henni Piirainen & Veli-Pekka Jaakola

  3. Biocenter Oulu, University of Oulu, P.O. Box 5000, 90014, Oulu, Finland

    Henni Piirainen & Veli-Pekka Jaakola

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  1. Maarit Hellman
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  2. Henni Piirainen
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  3. Veli-Pekka Jaakola
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  4. Perttu Permi
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Correspondence to Perttu Permi.

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Hellman, M., Piirainen, H., Jaakola, VP. et al. Bridge over troubled proline: assignment of intrinsically disordered proteins using (HCA)CON(CAN)H and (HCA)N(CA)CO(N)H experiments concomitantly with HNCO and i(HCA)CO(CA)NH. J Biomol NMR 58, 49–60 (2014). https://doi.org/10.1007/s10858-013-9804-0

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