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Backbone dynamics of the cytotoxic Ribonuclease α-sarcin by 15N NMR relaxation methods

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Abstract

The cytotoxic ribonuclease α-sarcin is a 150-residue protein that inactivates ribosomes by selectively cleaving a single phosphodiester bond in a strictly conserved rRNA loop. In order to gain insights on the molecular basis of its highly specific activity, we have previously determined its solution structure and studied its electrostatics properties. Here, we complement those studies by analysing the backbone dynamics of α-sarcin through measurement of longitudinal relaxation rates R1, off resonance rotating frame relaxation rates R1ρ, and the 15N1HNOE of the backbone amide 15N nuclei at two different magnetic field strengths (11.7 and 17.6 T). The two sets of relaxation parameters have been analysed in terms of the reduced spectral density mapping formalism, as well as by the model-free approach. α-Sarcin behaves as an axial symmetric rotor of the prolate type (D∥/D⊥=1.16 ± 0.02) which tumbles with a correlation time τm of 7.54 ± 0.02 ns. The rotational diffusion properties have been also independently evaluated by hydrodynamic calculations and are in good agreement with the experimental results. The analysis of the internal dynamics reveals that α-sarcin is composed of a rigid hydrophobic core and some exposed segments which undergo fast (ps to ns) internal motions. Slower motions in the μs to ms time scale are less abundant and in some cases can be assigned to specific motional processes. All dynamic data are discussed in relation to the role of some particular residues of α-sarcin in the process of recognition of its ribosomal target.

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Author notes

  1. José Manuel Pérez-Cañadillas

    Present address: Laboratory of Molecular Biology, Medical Research Council, Hills Road, CB2 2QH, Cambridge, U.K

Authors and Affiliations

  1. Instituto de Química Física `Rocasolano', Consejo Superior de Investigaciones Científicas, Serrano 119, 28006, Madrid, Spain

    José Manuel Pérez-Cañadillas, Ramón Campos-Olivas, Jorge Santoro, Manuel Rico & Marta Bruix

  2. Bijvoet Center for Biomolecular Research, Utrecht University, Padualaan 8, 3584, Utrecht, The Netherlands

    Marc Guenneugues

  3. ENTOMED S.A., rue Tobias Stimner, 67400, Illkirch, France

    Marc Guenneugues

  4. Structural and Computational Biology Program, Centro Nacional de Investigaciones Oncoloógicas, Melchor Fernández Almagro 3, 28029, Madrid, Spain

    Jorge Santoro

  5. Departamento de Bioquímica y Biología Molecular I, Facultad de Química, Universidad Complutense, 28040, Madrid, Spain

    Alvaro Martínez del Pozo & José G. Gavilanes

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  1. José Manuel Pérez-Cañadillas
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Correspondence to Marta Bruix.

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Pérez-Cañadillas, J.M., Guenneugues, M., Campos-Olivas, R. et al. Backbone dynamics of the cytotoxic Ribonuclease α-sarcin by 15N NMR relaxation methods. J Biomol NMR 24, 301–316 (2002). https://doi.org/10.1023/A:1021698308683

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