Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 1998 Sep;75(3):1172–1179. doi: 10.1016/S0006-3495(98)74036-6

Structural and morphological characterization of ultralente insulin crystals by atomic force microscopy: evidence of hydrophobically driven assembly.

C M Yip 1, M R DeFelippis 1, B H Frank 1, M L Brader 1, M D Ward 1
PMCID: PMC1299792  PMID: 9726919

Abstract

Although x-ray crystal structures exist for many forms of insulin, the hormone involved in glucose metabolism and used in the treatment of diabetes, x-ray structural characterization of therapeutically important long-acting crystalline ultralente insulin forms has been elusive because of small crystal size and poor diffraction characteristics. We describe tapping-mode atomic force microscopy (TMAFM) studies, performed directly in crystallization liquor, of ultralente crystals prepared from bovine, human, and porcine insulins. Lattice images obtained from direct imaging of crystal planes are consistent with R3 space group symmetry for each insulin type, but the morphology of the human and porcine crystals observed by AFM differs substantially from that of the bovine insulin crystals. Human and porcine ultralente crystals exhibited large, molecularly flat (001) faces consisting of hexagonal arrays of close packed hexamers. In contrast, bovine ultralente crystals predominantly exhibited faces with cylindrical features assignable to close-packed stacks of insulin hexamers laying in-plane, consistent with the packing motif of the (010) and (011) planes. This behavior is attributed to a twofold increase in the hydrophobic character of the upper and lower surfaces of the donut-shaped insulin hexamer in bovine insulin compared to its human and porcine counterparts that results from minor sequence differences between these insulins. The increased hydrophobicity of these surfaces can promote hexamer-hexamer stacking in precrystalline aggregates or enhance attachment of single hexamers along the c axis at the crystal surface during crystal growth. Both events lead to enhanced growth of ¿hk0¿ planes instead of (001). The insulin hexamers on the (010) and (110) faces are exposed "edge-on" to the aqueous medium, such that solvent access to the center of the hexamer and to solvent channels is reduced compared to the (001) surface, consistent with the slower dissolution and reputed unique basal activity of bovine ultralente insulin. These observations demonstrate that subtle variations in amino acid sequence can dramatically affect the interfacial structure of crystalline proteins.

Full Text

The Full Text of this article is available as a PDF (866.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Baker E. N., Blundell T. L., Cutfield J. F., Cutfield S. M., Dodson E. J., Dodson G. G., Hodgkin D. M., Hubbard R. E., Isaacs N. W., Reynolds C. D. The structure of 2Zn pig insulin crystals at 1.5 A resolution. Philos Trans R Soc Lond B Biol Sci. 1988 Jul 6;319(1195):369–456. doi: 10.1098/rstb.1988.0058. [DOI] [PubMed] [Google Scholar]
  2. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F., Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. doi: 10.1016/s0022-2836(77)80200-3. [DOI] [PubMed] [Google Scholar]
  3. Berson S. A., Yalow R. S. Insulin in blood and insulin antibodies. Am J Med. 1966 May;40(5):676–690. doi: 10.1016/0002-9343(66)90148-3. [DOI] [PubMed] [Google Scholar]
  4. Carugo O., Argos P. Protein-protein crystal-packing contacts. Protein Sci. 1997 Oct;6(10):2261–2263. doi: 10.1002/pro.5560061021. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Derewenda U., Derewenda Z., Dodson E. J., Dodson G. G., Reynolds C. D., Smith G. D., Sparks C., Swenson D. Phenol stabilizes more helix in a new symmetrical zinc insulin hexamer. Nature. 1989 Apr 13;338(6216):594–596. doi: 10.1038/338594a0. [DOI] [PubMed] [Google Scholar]
  6. Durbin S. D., Feher G. Protein crystallization. Annu Rev Phys Chem. 1996;47:171–204. doi: 10.1146/annurev.physchem.47.1.171. [DOI] [PubMed] [Google Scholar]
  7. Galloway J. A., Chance R. E. Improving insulin therapy: achievements and challenges. Horm Metab Res. 1994 Dec;26(12):591–598. doi: 10.1055/s-2007-1001766. [DOI] [PubMed] [Google Scholar]
  8. Graham D. T., Pomeroy A. R. An in-vitro test for the duration of action of insulin suspensions. J Pharm Pharmacol. 1984 Jul;36(7):427–430. doi: 10.1111/j.2042-7158.1984.tb04418.x. [DOI] [PubMed] [Google Scholar]
  9. HALLAS-M|OLLER K., PETERSEN K., SCHLICHTKRULL J. Crystalline and amorphous insulin-zinc compounds with prolonged action. Science. 1952 Oct 10;116(3015):394–398. doi: 10.1126/science.116.3015.394. [DOI] [PubMed] [Google Scholar]
  10. Hillier A. C., Ward M. D. Atomic force microscopy of the electrochemical nucleation and growth of molecular crystals. Science. 1994 Mar 4;263(5151):1261–1264. doi: 10.1126/science.263.5151.1261. [DOI] [PubMed] [Google Scholar]
  11. Hollenberg M. D. Receptor triggering and receptor regulation: structure-activity relationships from the receptor's point of view. J Med Chem. 1990 May;33(5):1275–1281. doi: 10.1021/jm00167a001. [DOI] [PubMed] [Google Scholar]
  12. Kaarsholm N. C., Ko H. C., Dunn M. F. Comparison of solution structural flexibility and zinc binding domains for insulin, proinsulin, and miniproinsulin. Biochemistry. 1989 May 16;28(10):4427–4435. doi: 10.1021/bi00436a046. [DOI] [PubMed] [Google Scholar]
  13. Konnert J. H., D'Antonio P., Ward K. B. Observation of growth steps, spiral dislocations and molecular packing on the surface of lysozyme crystals with the atomic force microscope. Acta Crystallogr D Biol Crystallogr. 1994 Jul 1;50(Pt 4):603–613. doi: 10.1107/S0907444994001988. [DOI] [PubMed] [Google Scholar]
  14. Kuznetsov YuG, Malkin A. J., Land T. A., DeYoreo J. J., Barba A. P., Konnert J., McPherson A. Molecular resolution imaging of macromolecular crystals by atomic force microscopy. Biophys J. 1997 May;72(5):2357–2364. doi: 10.1016/S0006-3495(97)78880-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Land TA, Malkin AJ, Kuznetsov YG, McPherson A, De Yoreo JJ Mechanisms of protein crystal growth: An atomic force microscopy study of canavalin crystallization. Phys Rev Lett. 1995 Oct 2;75(14):2774–2777. doi: 10.1103/PhysRevLett.75.2774. [DOI] [PubMed] [Google Scholar]
  16. Lijnzaad P., Argos P. Hydrophobic patches on protein subunit interfaces: characteristics and prediction. Proteins. 1997 Jul;28(3):333–343. [PubMed] [Google Scholar]
  17. Lyon M. K., Marr K. M., Furcinitti P. S. Formation and characterization of two-dimensional crystals of photosystem II. J Struct Biol. 1993 Mar-Apr;110(2):133–140. doi: 10.1006/jsbi.1993.1014. [DOI] [PubMed] [Google Scholar]
  18. Malkin AJ, Land TA, Kuznetsov YG, McPherson A, DeYoreo JJ. Investigation of virus crystal growth mechanisms by in situ atomic force microscopy. Phys Rev Lett. 1995 Oct 2;75(14):2778–2781. doi: 10.1103/PhysRevLett.75.2778. [DOI] [PubMed] [Google Scholar]
  19. Melander W., Horváth C. Salt effect on hydrophobic interactions in precipitation and chromatography of proteins: an interpretation of the lyotropic series. Arch Biochem Biophys. 1977 Sep;183(1):200–215. doi: 10.1016/0003-9861(77)90434-9. [DOI] [PubMed] [Google Scholar]
  20. Müller D. J., Engel A., Carrascosa J. L., Vélez M. The bacteriophage phi29 head-tail connector imaged at high resolution with the atomic force microscope in buffer solution. EMBO J. 1997 May 15;16(10):2547–2553. doi: 10.1093/emboj/16.10.2547. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nicholls A., Sharp K. A., Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 1991;11(4):281–296. doi: 10.1002/prot.340110407. [DOI] [PubMed] [Google Scholar]
  22. Schabert F. A., Henn C., Engel A. Native Escherichia coli OmpF porin surfaces probed by atomic force microscopy. Science. 1995 Apr 7;268(5207):92–94. doi: 10.1126/science.7701347. [DOI] [PubMed] [Google Scholar]
  23. Smith G. D., Swenson D. C., Dodson E. J., Dodson G. G., Reynolds C. D. Structural stability in the 4-zinc human insulin hexamer. Proc Natl Acad Sci U S A. 1984 Nov;81(22):7093–7097. doi: 10.1073/pnas.81.22.7093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Walz T., Tittmann P., Fuchs K. H., Müller D. J., Smith B. L., Agre P., Gross H., Engel A. Surface topographies at subnanometer-resolution reveal asymmetry and sidedness of aquaporin-1. J Mol Biol. 1996 Dec 20;264(5):907–918. doi: 10.1006/jmbi.1996.0686. [DOI] [PubMed] [Google Scholar]
  25. Yip C. M., Brader M. L., DeFelippis M. R., Ward M. D. Atomic force microscopy of crystalline insulins: the influence of sequence variation on crystallization and interfacial structure. Biophys J. 1998 May;74(5):2199–2209. doi: 10.1016/S0006-3495(98)77929-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yip C. M., Ward M. D. Atomic force microscopy of insulin single crystals: direct visualization of molecules and crystal growth. Biophys J. 1996 Aug;71(2):1071–1078. doi: 10.1016/S0006-3495(96)79307-4. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES