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Novel medium for monomer proteins structure elucidation. Human insulin structure in water/ acetonitrile solvent. Search for nonamyloidogenic conditions
|Lech Kozerski 1,2, Wojciech Bocian 1,2, Elżbieta Bednarek 2, Robert Kawęcki 1, Jerzy Sitkowski 1,2, Anna Tarnowska 1|
1. Polish Academy of Science, Institute of Organic Chemistry, Kasprzaka 44/52, Warszawa 01-224, Poland
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. In particular, the detailed comparison of a structure in 20% acetic acid is presented and discussed. The 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 oC, 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).
Acknowledgements: This work was supported by the network "Synthesis, structure and therapeutic properties of compounds and organic substances".
Presentation: Oral at VI Multidyscyplinarna Konferencja Nauki o Leku, by Lech Kozerski
See On-line Journal of VI Multidyscyplinarna Konferencja Nauki o Leku
Submitted: 2008-02-06 10:31 Revised: 2009-06-07 00:48