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Structure of human insulin monomer in water/acetonitrile solution

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Abstract

Here we present evidence that in water/acetonitrile solvent detailed structural and dynamic information can be obtained for important proteins that are naturally present as oligomers under native conditions. An NMR-derived human insulin monomer structure in H2O/CD3CN, 65/35 vol%, pH 3.6 is presented and compared with the available X-ray structure of a monomer that forms part of a hexamer (Acta Crystallogr. 2003 Sec. D59, 474) and with NMR structures in water and organic cosolvent. Detailed analysis using PFGSE NMR, temperature-dependent NMR, dilution experiments and CSI proves that the structure is monomeric in the concentration and temperature ranges 0.1–3 mM and 10–30°C, respectively. The presence of long-range interstrand NOEs, as found in the crystal structure of the monomer, provides the evidence for conservation of the tertiary structure. Starting from structures calculated by the program CYANA, two different molecular dynamics simulated annealing refinement protocols were applied, either using the program AMBER in vacuum (AMBER_VC), or including a generalized Born solvent model (AMBER_GB).

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Acknowledgement

A. T. gratefully acknowledges the financial support during this work by means of Ministry of Science and Higher Education grant no. 0278/P01/2006/30.

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Authors and Affiliations

  1. National Medicines Institute, Chełmska 30/34, Warsaw, 00-725, Poland

    Wojciech Bocian, Jerzy Sitkowski, Elżbieta Bednarek & Lech Kozerski

  2. Institute of Organic Chemistry Polish Academy of Sciences, Kasprzaka 44, Warsaw, 01-224, Poland

    Wojciech Bocian, Jerzy Sitkowski, Anna Tarnowska, Robert Kawęcki & Lech Kozerski

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  1. Wojciech Bocian
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  2. Jerzy Sitkowski
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  3. Elżbieta Bednarek
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Correspondence to Lech Kozerski.

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Wojciech Bocian contributed equally to this work.

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Bocian, W., Sitkowski, J., Bednarek, E. et al. Structure of human insulin monomer in water/acetonitrile solution. J Biomol NMR 40, 55–64 (2008). https://doi.org/10.1007/s10858-007-9206-2

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