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Evidence of oligomerization of bovine insulin in solution given by NMR

  • S. V. Efimov
  • Yu. O. Zgadzay
  • N. B. Tarasova
  • V. V. Klochkov
Original Article

Abstract

The protein hormone insulin exists in several forms in nature, and a large number of modified sequences are used in pharmacy. They differ by physicochemical properties and efficiency of biological action. Pancreatic bovine insulin was studied in an acidic solution by nuclear magnetic resonance spectroscopy. \(^{1}\)H and \(^{13}\)C NMR signal assignment of backbone and side chains was made by analysis of a set of 2D spectra obtained on a sample with natural isotope abundance. The presence of certain secondary structure elements was revealed on a qualitative level based on nuclear Overhauser effect spectroscopy, which are similar to those observed in the crystal structure. The C-terminus of the B-chain possessed a remarkable flexibility. The molecule was shown to exist in exchange with oligomers based on its self-diffusion coefficient and correlation time measurements performed at different concentrations. Certain signals in the NOESY and HSQC spectra are consistent with the presence of minor conformers; this is an obstacle in simulating the molecular structure under the conditions used in the experiment.

Keywords

Bovine insulin Secondary structure NMR NOESY DOSY Oligomerization 

Notes

Acknowledgements

This work was funded by the subsidy of the Russian Government to support the Program of Competitive Growth of Kazan Federal University among world’s leading academic centers and by the subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities (Center of Shared Facilities; VVK acknowledges the project no. 3.5283.2017/6.7).

References

  1. Bocian W, Borowicz P, Mikołajczyk L, Sitkowski J, Tarnowska A, Bednarek E (2008) T. Gła̧bski, B. Tejchman-Małecka, M. Bogiel, L. Kozerski (2008) NMR Structure of Biosynthetic Engineered Human Insulin Monomer B31\(\tt ^{Lys}\)-B32\(\tt ^{Arg}\) in Water/Acetonitrile Solution. Comparison with the Solution Structure of Native Human Insulin Monomer. Biopolymers 89:320.  https://doi.org/10.1002/bip.21018 CrossRefGoogle Scholar
  2. Boelens R, Ganadu M, Verheyden P, Kaptein R (1990) Two-dimensional NMR studies on des-pentapeptide-insulin. Proton resonance assignments and secondary structure analysis. Eur J Biochem 191:147.  https://doi.org/10.1111/j.1432-1033.1990.tb19104.x CrossRefPubMedGoogle Scholar
  3. Brange J, Andersen L, Laursen E, Meyn G, Rasmussen E (1997) Toward understanding insulin fibrillation. J Pharm Sci 86:517.  https://doi.org/10.1021/js960297s CrossRefPubMedGoogle Scholar
  4. Brange J, Havelund S, Hougaard P (1992) Chemical stability of insulin. 2. Formation of higher molecular weight transformation products during sorage of pharmaceutical preparations. Pharm Res 9:727.  https://doi.org/10.1023/A:1015835017916 CrossRefPubMedGoogle Scholar
  5. Classification and diagnosis of diabetes: standards of medical care in diabetes—2018 (2018). Diabetes Care 41:S13.  https://doi.org/10.2337/dc18-S002
  6. Delaglio F, Grzesiek S, Vuister G, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277.  https://doi.org/10.1007/BF00197809 CrossRefPubMedGoogle Scholar
  7. Dupradeau FY, Richard T, Le Flem G, Oulyadi H, Prigent Y, Monti JP (2002) A new B-chain mutant of insulin: comparison with the insulin crystal structure and role of sulfonate groups in the B-chain structure. J Pept Res 60:56CrossRefPubMedGoogle Scholar
  8. Ernst R, Bodenhausen G, Wokaun A (1987) Principles of nuclear magnetic resonance in one and two dimensions. Clarendon Press, OxfordGoogle Scholar
  9. Goddard T, Kneller D (2017) Sparky 3. http://www.cgl.ucsf.edu/home/sparky/
  10. Hawkins B, Cross K, Craik D (1994) A \(^{1}\)H-NMR determination of the solution structure of the A-chain of insulin: comparison with the crystal structure and an examination of the role of solvent. Biochim Biophys Acta.  https://doi.org/10.1016/0167-4838(94)90182-1 Google Scholar
  11. Hawkins B, Cross K, Craik D (1995) Solution structure of the B-chain of insulin as determined by \(^{1}\)H NMR spectroscopy. Int J Peptide Protein Res. 46:424CrossRefPubMedGoogle Scholar
  12. Hua Q, Weiss M (1991) Comparative 2D NMR studies of human insulin and despentapeptide insulin: sequential resonance assignment and implications for protein dynamics and receptor recognition. Biochemistry 30:5505.  https://doi.org/10.1021/bi00236a025 CrossRefPubMedGoogle Scholar
  13. Kadima W (1999) Role of metal ions in the T- to R-allosteric transition in the insulin hexamer. Biochemistry 38:13443.  https://doi.org/10.1021/bi9903188 CrossRefPubMedGoogle Scholar
  14. Kay L (1997) NMR methods for the study of protein structure and dynamics. Biochem Cell Biol. 75:1.  https://doi.org/10.1139/o97-023 CrossRefPubMedGoogle Scholar
  15. Keller D, Clausen R, Josefsen K, Led J (2001) Flexibility and bioactivity of insulin: an NMR investigation of the solution structure and folding of an unusually flexible human insulin mutant with increased biological activity. Biochemistry 40:10732.  https://doi.org/10.1021/bi0108150 CrossRefPubMedGoogle Scholar
  16. Khodov I, Alper G, Mamardashvili G, Mamardashvili N (2015) Hybrid multi-porphyrin supramolecular assemblies: synthesis and structure elucidation by 2D DOSY NMR studies. J Mol Struct 1099:174.  https://doi.org/10.1016/j.molstruc.2015.06.062 CrossRefGoogle Scholar
  17. Kilo C, Mezitis N, Jain R, Mersey J, McGill J, Raskin P (2003) Starting patients with type 2 diabetes on insulin therapy using once-daily injections of biphasic insulin aspart 70/30, biphasic human insulin 70/30, or NPH insulin in combination with metformin. J Diabetes Complic 17:307.  https://doi.org/10.1016/S1056-8727(03)00076-X CrossRefGoogle Scholar
  18. Kline A, Justice R Jr (1990) Complete sequence-specific \(^{1}\)H NMR assignments for human insulin. Biochemistry 29:2906.  https://doi.org/10.1021/bi00464a003 CrossRefPubMedGoogle Scholar
  19. Kosinová L, Veverka V, Novotná P, Collinsová M, Urbanová M, Moody N, Turkenburg J, Jiráček J, Brzozowski A , L. Z̧áková (2014) Insight into the sructural and biological relevance of the T/R transition of the N-terminus of the B-Chain in human insulin. Biochemistry 53:3392.  https://doi.org/10.1021/bi500073z CrossRefPubMedPubMedCentralGoogle Scholar
  20. Krüger P, Straßburger W, Wollmer A, van Gunsteren W, Dodson G (1987) The simulated dynamics of the insulin monomer and their relationship to the molecule’s structure. Eur Biophys. J 14:449.  https://doi.org/10.1007/BF00293254 CrossRefPubMedGoogle Scholar
  21. De Leeuw I, Vague P, Selam JL, Skeie S, Lang H, Draeger E, Elte J (2005) Insulin detemir used in basal-bolus therapy in people with type 1 diabetes is associated with a lower risk of nocturnal hypoglycaemia and less weight gain over 12 months in comparison to NPH insulin. Diabetes Obes Metab 7:73.  https://doi.org/10.1111/j.1463-1326.2004.00363.x CrossRefGoogle Scholar
  22. Leszek J, Trypka E, Tarasov V, Ashraf G, Aliev G (2017) Type 3 Diabetes Mellitus: a novel implication of Alzheimers disease. Curr Top Med Chem 17:1331.  https://doi.org/10.2174/1568026617666170103163403 CrossRefPubMedGoogle Scholar
  23. Van Lokeren L, Cartuyvels E, Absillis G, Willem R, Parac-Vogt T (2008) Phosphoesterase activity of polyoxomolybdates: diffusion ordered NMR spectroscopy as a tool for obtaining insights into the reactivity of polyoxometalate clusters. Chem Commun.  https://doi.org/10.1039/B802671H Google Scholar
  24. Lycknert K, Rundlöf T, Widmalm G (2002) Solution structure of a type 1 H antigen trisaccharide at a micellar surface: NMR Relaxation and Molecular Dynamics Simulation Studies. J Phys Chem B 106:5275.  https://doi.org/10.1021/jp0136462 CrossRefGoogle Scholar
  25. Mayer J, Faming Z, DiMarchi R (2007) Insulin structure and function. Pept Sci 88:687.  https://doi.org/10.1002/bip.20734 CrossRefGoogle Scholar
  26. Menting J, Yang Y, Chan S, Phillips N, Smith B, Whittaker J, Wickramasinghe N, Whittaker L, Pandyarajan V, Wan ZL, Yadav S, Carroll J, Strokes N, Roberts C, Ismail-Beigi F, Milewski W, Steiner D, Chauhan V, Ward C, Weiss M, Lawrence M (2014) Protective hinge in insulin opens to enable its receptor engagement. Proc. Natl Acad Sci USA 111:E3395.  https://doi.org/10.1073/pnas.1412897111 CrossRefPubMedPubMedCentralGoogle Scholar
  27. Mishra N, Joshi K, Verma S (2013) Inhibition of human and bovine insulin fibril formation by designed peptide conjugates. Mol Pharm 10:3903.  https://doi.org/10.1021/mp400364w CrossRefPubMedGoogle Scholar
  28. Mohan V (2002) Which insulin to use? Human or animal? Curr Sci 83:1544Google Scholar
  29. Neuhaus D, Williamson M (2000) The nuclear overhauser effect in structural and conformational analysis, 2nd edn. Wiley, New YorkGoogle Scholar
  30. Nielsen L, Frokjaer S, Brange J, Uversky V, Fink A (2001) Probing the mechanism of insulin fibril formation with insulin mutants. Biochemistry 40:8397.  https://doi.org/10.1021/bi0105983
  31. Olsen H, Ludvigsen S, Kaarsholm N (1996) Solution structure of an engineered insulin monomer at neutral pH. Biochemistry 35:8836.  https://doi.org/10.1021/bi960292+ CrossRefPubMedGoogle Scholar
  32. O’Donoghue S, Chang X, Abseher R, Nilges M, Led J (2000) Unraveling the symmetry ambiguity in a hexamer: calculation of the R\(_{6}\) human insulin structure. J Biomol NMR 16:93.  https://doi.org/10.1023/A:1008323819099 CrossRefPubMedGoogle Scholar
  33. Palmieri L, Fávero-Retto M, Lourenço D, Lima L (2013) A T\(_{3}\)R\(_{3}\) hexamer of the human insulin variant B28Asp. Biophys Chem 173–174:1.  https://doi.org/10.1016/j.bpc.2013.01.003 CrossRefPubMedGoogle Scholar
  34. Patil S, Keire D, Chen K (2017) Comparison of NMR and dynamic light scattering for measuring diffusion coefficients of formulated insulin: implications for particle size distribution measurements in drug products. AAPS J. 19:1760.  https://doi.org/10.1208/s12248-017-0127-z CrossRefPubMedGoogle Scholar
  35. Polshakov V, Frenkiel T, Westley B, Chadwick M, May F, Carr M, Feeney J (1995) NMR-based structural studies of the pNR-2/pS2 single domain trefoil peptide: similarities to porcine spasmolytic peptide and evidence for a monomeric structure. Eur J Biochem 233:847.  https://doi.org/10.1111/j.1432-1033.1995.847_3.x CrossRefPubMedGoogle Scholar
  36. Roy M, Lee RK, Kaarsholm N, Thøgersen H, Brange J, Dunn M (1990) Sequence-specific \(^{1}\)H-NMR assignments for the aromatic region of several biologically active, monomeric insulins including native human insulin. Biochim Biophys Acta 1053:63.  https://doi.org/10.1016/0167-4889(90)90027-B CrossRefPubMedGoogle Scholar
  37. Ryan G, Jobe L, Martin R (2005) Pramlintide intreatment of type 1 and type 2 diabetes mellitus. Clinical Therapeutics 27:1500.  https://doi.org/10.1016/j.clinthera.2005.10.009 CrossRefPubMedGoogle Scholar
  38. Seigler D, Olsson G, Agramonte R, Lohman V, Ashby M, Reeves M, Skyler J (1991) Pharmacokinetics of long-acting (ultralente) insulin preparations, Diabetes. Nutr Metab 4:267Google Scholar
  39. Smith G, Pangborn W, Blessing R (2005) The structure of T\(_{6}\) bovine insulin. Acta Crystallogr D. D61:1476.  https://doi.org/10.1107/S0907444905025771 CrossRefGoogle Scholar
  40. Usachev K, Efimov S, Kolosova O, Klochkova E, Aganov A, Klochkov V (2015) Antimicrobial peptide protegrin-3 adopt an antiparallel dimer in the presence of DPC micelles: a high-resolution NMR study. J Biomol NMR 62:71.  https://doi.org/10.1007/s10858-015-9920-0 CrossRefPubMedGoogle Scholar
  41. Viollet B, Guigas B, Garcia N, Leclerc J, Foretz M, Andreelli F (2012) Cellular and molecular mechanisms of metformin: an overview. Clin Sci 122:253.  https://doi.org/10.1042/CS20110386 CrossRefPubMedPubMedCentralGoogle Scholar
  42. Weiss M (2013) Design of ultra-stable insulin analogues for the developing world. J Health Spec 1:59.  https://doi.org/10.4103/1658-600X.114683 CrossRefGoogle Scholar
  43. Whittingham J, Scott D, Chance K, Wilson A, Finch J, Brange J, Guy G (2002) Dodson, Insulin at pH 2: structural analysis of the conditions promoting insulin fibre formation. J Mol Biol 318:479.  https://doi.org/10.1016/S0022-2836(02)00021-9 CrossRefPubMedGoogle Scholar
  44. Williamson M, Waltho J (1992) Peptide structure from NMR. Chem Soc Rev 21:227.  https://doi.org/10.1039/CS9922100227 CrossRefGoogle Scholar
  45. Yang Y, Petkova A, Huang K, Xu B, Hua QX, Ye IJ, Chu YC, Hu SQ, Phillips N, Whittaker J, Ismail-Beigi F, Mackin R, Katsoyannis P, Tycko R, Weiss M (2010) An Achilles’ heel in an amyloidogenic protein and its repair. J.Biol Chem 285:10806.  https://doi.org/10.1074/jbc.M109.067850 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© European Biophysical Societies' Association 2018

Authors and Affiliations

  • S. V. Efimov
    • 1
  • Yu. O. Zgadzay
    • 1
  • N. B. Tarasova
    • 2
  • V. V. Klochkov
    • 1
  1. 1.Laboratory of NMR spectroscopy, Institute of PhysicsKazan Federal UniversityKazanRussia
  2. 2.Laboratory of Molecular Biology, Kazan Institute of Biochemistry and BiophysicsFRC Kazan Scientific Center of RASKazanRussia

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