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Novel medium for monomer proteins structure elucidation. Human insulin structure in water/ acetonitrile solvent. Search for nonamyloidogenic conditions

Lech Kozerski 1,2Wojciech Bocian 1,2Elżbieta Bednarek 2Robert Kawęcki 1Jerzy Sitkowski 1,2Anna Tarnowska 1

1. Polish Academy of Science, Institute of Organic Chemistry, Kasprzaka 44/52, Warszawa 01-224, Poland
2. National Medicines Institut (NIL), Chełmska 30/34, Warsaw 00-725, 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).
The nonamyloidogenic property of a protein depends essentially on intrinsic propensity of AA sequence to retain hydrophobic core on one hand and on its side chains character determining the solvation shell which should prevent extensive aggregation. The water/acetonitrile solvent allows to retain tertiary structure exactly matching the native structure of a monomer embeded in a crystal of hexamer and prevents the aggregation. It therefore potentially allows to study the effect of various external factors on misfolded conformation of a monomer in a critical stage leading to amyloid formation.

Acknowledgements: This work was supported by the network "Synthesis, structure and therapeutic properties of compounds and organic substances".


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Related papers

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