Advertisement

Journal of Biomolecular NMR

, Volume 40, Issue 1, pp 55–64 | Cite as

Structure of human insulin monomer in water/acetonitrile solution

  • Wojciech Bocian
  • Jerzy Sitkowski
  • Elżbieta Bednarek
  • Anna Tarnowska
  • Robert Kawęcki
  • Lech Kozerski
Article

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).

Keywords

Insulin monomer MD in explicit solvent NMR structure Water/acetonitrile solvent 

Notes

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.

Supplementary material

10858_2007_9206_MOESM1_ESM.pdf (4.9 mb)
(PDF 5042 kb)

References

  1. Antałek B, Windig W (1996) Generalized rank annihilation method applied to a single multicomponent pulsed gradient spin echo NMR data set. J Am Chem Soc 118:10331–10332CrossRefGoogle Scholar
  2. Bodenhausen G, Kogler H, Ernst RR (1984) Selection of coherence-transfer pathways in NMR pulse experiments. J Magn Reson 58:370–388Google Scholar
  3. Bodenhausen G, Reuben DJ (1980) Structural studies on a-conotoxin SI. Chem Phys Lett 69:185–189CrossRefADSGoogle Scholar
  4. Braunschweiler L, Ernst RR (1983) Coherence transfer by isotropic mixing: Application to proton correlation spectroscopy. J Magn Reson 53:521–528Google Scholar
  5. Brems DN, Alter LA, Beckage MJ, Chance RE, DiMarchi RD, Green LK, Long HB, Pekar AH, Shields JE, Frank BH (1992) Altering the association properties of insulin by amino acid replacement Protein Eng 5:527–533CrossRefGoogle Scholar
  6. Buck M. (1998) Trifluoroethanol and colleagues: cosolvents come of age. Recent studies with peptides and proteins. Q Rev Biophys 31:297–355CrossRefGoogle Scholar
  7. Cahill GF Jr (1971) The Banting Memorial Lecture 1971. Physiology of insulin in man. Diabetes 20:785–799Google Scholar
  8. Case DA, Darden TA, Cheatham III TE, Simmerling CL, Wang J, Duke RE, Luo R, Merz KM, Pearlman DA, Crowley M, Walker RC, Zhang W, Wang B, Hayik S, Roitberg A, Seabra G, Wong KF, Paesani F, Wu X, Brozell S, Tsui V, Gohlke H, Yang L, Tan C, Mongan J, Hornak V, Cui G, Beroza P, Mathews DH, Schafmeister C, Ross WS, Kollman PA (2006) AMBER 9. San Francisco, University of CaliforniaGoogle Scholar
  9. Chiti F, Stefani M, Taddei N, Ramponi G, Dobson CM (2003) Rationalization of the effects of mutations on peptide and protein aggregation rates. Nature 424:805–808CrossRefADSGoogle Scholar
  10. Ciszak E, Beals JM, Frank BH, Baker JC, Carter ND, Smith GD (1995) Role of C-terminal B-chain residues in insulin assembly: the structure of hexameric LysB28ProB29-human insulin. Structure 3:615–622CrossRefGoogle Scholar
  11. Cringus D, Yeremenko S, Pshenichnikov MS, Wiersma DA (2004) Hydrogen bonding and vibrational energy relaxation in water–acetonitrile mixtures. J. Phys. Chem B 108:10376–10387CrossRefGoogle Scholar
  12. Danielsson J, Jarvet J, Damberg P, Gräslund A (2002) Translational diffusion measured by PFG-NMR on full length and fragments of the Alzheimer Aβ(1-40) peptide. Determination of hydrodynamic radii of random coil peptides of varying length. Mag Reson Chem 40:S89–S97CrossRefGoogle Scholar
  13. Darrington RT, Anderson BD (1995) Effects of insulin concentration and self-association on the partitioning of its A-21 cyclic anhydride intermediate to desamido insulin and covalent dimer. Pharm Res 12:1077–1084CrossRefGoogle Scholar
  14. Goddard TD, Kneller DN (2004) SPARKY 3. San Francisco, University of CaliforniaGoogle Scholar
  15. Griesinger C, Otting G, Wüthrich K, Ernst RR (1988) Clean TOCSY for proton spin system identification in macromolecules. J Am Chem Soc 110:7870–7872CrossRefGoogle Scholar
  16. Griesinger C, Søerensen OW, Ernst RR (1985) Two-dimensional correlation of connected NMR transitions. J Am Chem Soc 107:6394–6396CrossRefGoogle Scholar
  17. Griesinger C, Sørensen OW, Ernst RR (1987) Practical aspects of the E.COSY technique. Measurement of scalar spin-spin coupling constants in peptides. J Magn Reson 75:474–492Google Scholar
  18. Güntert P, Mumenthaler C, Wüthrich K (1997) Torsion angle dynamics for NMR structure calculation with the new program DYANA. J Mol Biol 273:283–298CrossRefGoogle Scholar
  19. Hua QX, Gozani SN, Chance RE, Hoffmann JA, Frank BH, Weiss MA (1995) Structure of a protein in a kinetic trap. Nat Struct Biol 2:129–138CrossRefGoogle Scholar
  20. Jeener J, Meier BH, Bachmann P, Ernst RR (1979) Investigation of Exchange Processes by 2-Dimensional NMR-Spectroscopy. J Chem Phys 71:4546–4553CrossRefADSGoogle Scholar
  21. Jimenez JL, Nettleton EJ, Bouchard M, Robinson CV, Dobson CM, Saibil HR (2002) The protofilament structure of insulin amyloid fibrils. Proc Natl Acad Sci USA 99:9196–9201CrossRefADSGoogle Scholar
  22. Johnson CS (1999) Diffusion ordered NMR spectroscopy: principles and applications. Prog Nucl Magn Reson 34:203CrossRefADSGoogle Scholar
  23. Jørgensen AM, Kristensen SM, Led JJ, Balschmidt P (1992) Three-dimensional solution structure of an insulin dimer. A study of the B9(Asp) mutant of human insulin using nuclear magnetic resonance, distance geometry and restrained molecular dynamics. J Mol Biol 227:1146–1163CrossRefGoogle Scholar
  24. Jørgensen AM, Olsen HB, Balschmidt P, Led JJ (1996) Solution structure of the superactive monomeric des-[Phe(B25)] human insulin mutant: elucidation of the structural basis for the monomerization of des-[Phe(B25)] insulin and the dimerization of native insulin. J Mol Biol 257:684–699CrossRefGoogle Scholar
  25. Kadima W, Øgendal L, Bauer R, Kaarsholm N, Brodersen K, Hansen JF, Porting P (1993) The influence of ionic strength and pH on the aggregation properties of zinc-free insulin studied by static and dynamic laser light scattering. Biopolymers 33:1643–1657CrossRefGoogle Scholar
  26. Kadima W, Roy M, Lee RW, Kaarsholm NC, Dunn MF (1992) Studies of the association and conformational properties of metal-free insulin in alkaline sodium chloride solutions by one- and two-dimensional 1H NMR. J Biol Chem 267:8963–8970Google Scholar
  27. Keller D, Clausen R, Josefsen K, Led JJ (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 40:10732–10740CrossRefGoogle Scholar
  28. Kline AD, Justice RM Jr (1990) Complete sequence-specific 1H NMR assignments for human insulin. Biochemistry 29:2906–2913CrossRefGoogle Scholar
  29. Kwon YM, Baudys M, Knutson K, Kim SW (2001) In situ study of insulin aggregation induced by water-organic solvent interface. Pharm Res 18:1754–1759CrossRefGoogle Scholar
  30. Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8:477–486CrossRefGoogle Scholar
  31. Le Roith D, Zick Y (2001) Recent advances in our understanding of insulin action and insulin resistance. Diabetes Care 24:588–597CrossRefGoogle Scholar
  32. Lin M, Larive CK (1995) Detection of insulin aggregates with pulsed-field gradient nuclear magnetic resonance spectroscopy. Anal Biochem 229:214–220CrossRefGoogle Scholar
  33. Lougheed WD, Albisser AM, Martindale HM, Chow JC, Clement JR (1983) Physical stability of insulin formulations. Diabetes 32:424–432CrossRefGoogle Scholar
  34. Ludvigsen S, Roy M, Thogersen H, Kaarsholm NC (1994) High-resolution structure of an engineered biologically potent insulin monomer, B16 Tyr-->His, as determined by nuclear magnetic resonance spectroscopy. Biochemistry 33:7998–8006CrossRefGoogle Scholar
  35. Luo RZ, Beniac DR, Fernandes A, Yip CC, Ottensmeyer FP (1999) Quaternary structure of the insulin-insulin receptor complex. Science 285:1077–1080CrossRefGoogle Scholar
  36. Nettleton EJ, Tito P, Sunde M, Bouchard M, Dobson CM, Robinson CV (2000) Characterization of the oligomeric states of insulin in self-assembly and amyloid fibril formation by mass spectrometry. Biophys J 79:1053–1065CrossRefGoogle Scholar
  37. Neuhaus D, Williamson MP (1995) Nuclear overhauser effect in conformational and structural analysis. A guide for Chemists. Wiley-VCH, NYGoogle Scholar
  38. Olsen HB, Ludvigsen S, Kaarsholm NC (1996) Solution structure of an engineered insulin monomer at neutral pH. Biochemistry 35:8836–8845CrossRefGoogle Scholar
  39. Onufriev A, Bashford D, Case DA (2000) Modification of the Generalized Born Model Suitable for Macromolecules. J Phys Chem B 104:3712–3720CrossRefGoogle Scholar
  40. Piantini U, Sorensen OW, Ernst RR (1982) Multiple quantum filters for elucidating NMR coupling networks. J Am Chem Soc 104:6800–6801CrossRefGoogle Scholar
  41. Pocker Y, Biswas SB (1981) Self-association of insulin and the role of hydrophobic bonding: a thermodynamic model of insulin dimerization. Biochemistry 20:4354–4361CrossRefGoogle Scholar
  42. Price WS, Tsuchiya F, Suzuki C, Arata Y (1999) Characterization of the solution properties of Pichia Farinosa killer toxin using PGSE NMR diffusion measurements. J Biomol NMR 13:113–117CrossRefGoogle Scholar
  43. Rodger A, Norden B (1997) Circular dichroism and linear dichroism. Oxford University PressGoogle Scholar
  44. Smith GD, Pangborn WA, Blessing RH (2003) The structure of T6 human insulin at 1.0 A resolution. Acta Crystallogr D Biol Crystallogr 59:474–482CrossRefGoogle Scholar
  45. States DJ, Haberkorn RA, Ruben DJ (1982) A two-dimensional nuclear overhauser experiment with pure absorption phase in four quadrants. J Magn Reson 48:286–292Google Scholar
  46. Summers MF, Marzilli LG, Bax A (1986) Complete proton and carbon-13 assignments of coenzyme B12 through the use of new two-dimensional NMR experiments. J Am Chem Soc 108:4285–4294CrossRefGoogle Scholar
  47. Uversky VN, Garriques LN, Millett IS, Frokjaer S, Brange J, Doniach S, Fink AL (2003) Prediction of the association state of insulin using spectral parameters. J Pharm Sci 92:847–858CrossRefGoogle Scholar
  48. Vis H, Heinemann U, Dobson CM, Robinson CV (1998) Detection of a monomeric intermediate associated with dimerization of protein HU by mass spectrometry. J Am Chem Soc 120:6427–6428CrossRefGoogle Scholar
  49. Wider G, Døtsch V, Wüthrich K (1994) Self-compensating pulsed magnetic-field gradients for short recovery times. J Magn Reson Series A 108:255–258CrossRefGoogle Scholar
  50. Windig W, Antałek B (1997) Direct exponential curve resolution algorithm (DECRA): A novel application of the generalized rank annihilation method for a single spectral mixture data set with exponentially decaying contribution profiles. Chemom Intell Lab Syst 37:241–254CrossRefGoogle Scholar
  51. Wishart DS, Bigam CG, Holm A, Hodges RS, Sykes BD (1995) 1H, 13C and 15N random coil NMR chemical shifts of the common amino acids. I. Investigations of nearest-neighbor effects. J Biomol NMR 5:67–81CrossRefGoogle Scholar
  52. Wishart DS, Sykes BD, Richards FM (1992) The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry 31:1647–1651CrossRefGoogle Scholar
  53. Wu DH, Chen AD, Johnson CS (1995) An improved diffusion-ordered spectroscopy experiment incorporating bipolar-gradient pulses. J Magn Reson Series A 115:260–264CrossRefGoogle Scholar
  54. Xia B, Tsui V, Case DA, Dyson HJ, Wright PE (2002) Comparison of protein solution structures refined by molecular dynamics simulation in vacuum, with a generalized Born model, and with explicit water. J Biomol NMR 22:317–331CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Wojciech Bocian
    • 1
    • 2
  • Jerzy Sitkowski
    • 1
    • 2
  • Elżbieta Bednarek
    • 1
  • Anna Tarnowska
    • 2
  • Robert Kawęcki
    • 2
  • Lech Kozerski
    • 1
    • 2
  1. 1.National Medicines InstituteWarsawPoland
  2. 2.Institute of Organic Chemistry Polish Academy of SciencesWarsawPoland

Personalised recommendations