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Crystallographic characterization of two novel crystal forms of human insulin induced by chaotropic agents and a shift in pH

Norrman, Mathias LU and Schluckebier, Gerd (2007) In BMC Structural Biology 7.
Abstract
Background: Insulin is a therapeutic protein that is widely used for the treatment of diabetes. Its biological function was discovered more than 80 years ago and it has since then been characterized extensively. Crystallization of the insulin molecule has always been a key activity since the protein is often administered by subcutaneous injections of crystalline insulin formulations. Over the years, insulin has been crystallized and characterized in a number of crystal systems. Results: Interestingly, we have now discovered two new crystal forms of human insulin. The crystals were obtained when the two chaotropic agents, urea and thiocyanate were present in the crystallization experiments, and their structures were determined by X-ray... (More)
Background: Insulin is a therapeutic protein that is widely used for the treatment of diabetes. Its biological function was discovered more than 80 years ago and it has since then been characterized extensively. Crystallization of the insulin molecule has always been a key activity since the protein is often administered by subcutaneous injections of crystalline insulin formulations. Over the years, insulin has been crystallized and characterized in a number of crystal systems. Results: Interestingly, we have now discovered two new crystal forms of human insulin. The crystals were obtained when the two chaotropic agents, urea and thiocyanate were present in the crystallization experiments, and their structures were determined by X-ray crystallography. The crystals belong to the orthorhombic and monoclinic crystal systems, with space groups C222(I) and C2 respectively. The orthorhombic crystals were obtained at pH 6.5 and contained three insulin hexamers in R-6 conformation in the asymmetric unit whilst the monoclinic C2 crystals were obtained at pH 7.0 and contained one R6 hexamer in the asymmetric unit. Common for the two new crystals is a hexamer-hexamer interaction that has not been found in any of the previous crystal forms of insulin. The contacts involve a tight glutamate-glutamate interaction with a distance of 2.3 angstrom between groups. The short distance suggests a low barrier hydrogen bond. In addition, two tyrosine-tyrosine interactions occupying a known phenol binding pocket contribute to the stabilization of the contacts. Within the crystals, distinct binding sites for urea were found, adding further to the discussion on the role of urea in protein denaturation. Conclusion: The change in space group from C222(I) to C2 was primarily caused by an increase in pH. The fewer number of hexamer-hexamer interactions comprising the short hydrogen bond in the C2 space group suggest that pH is the driving force. In addition, the distance between the two glutamates increases from 2.32 angstrom in the C222(I) crystals to 2.4 angstrom in the C2 crystals. However, in both cases the low barrier hydrogen bond and the tyrosine-tyrosine interaction should contribute to the stability of the crystals which is crucial when used in pharmaceutical formulations. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
BMC Structural Biology
volume
7
publisher
BioMed Central
external identifiers
  • wos:000253865400001
  • scopus:39049124128
ISSN
1471-2237
DOI
10.1186/1472-6807-7-83
language
English
LU publication?
yes
id
7869d6e9-3d77-46c2-92c6-4c752bdcb7d9 (old id 1407425)
date added to LUP
2009-06-02 10:49:06
date last changed
2017-09-24 04:32:40
@article{7869d6e9-3d77-46c2-92c6-4c752bdcb7d9,
  abstract     = {Background: Insulin is a therapeutic protein that is widely used for the treatment of diabetes. Its biological function was discovered more than 80 years ago and it has since then been characterized extensively. Crystallization of the insulin molecule has always been a key activity since the protein is often administered by subcutaneous injections of crystalline insulin formulations. Over the years, insulin has been crystallized and characterized in a number of crystal systems. Results: Interestingly, we have now discovered two new crystal forms of human insulin. The crystals were obtained when the two chaotropic agents, urea and thiocyanate were present in the crystallization experiments, and their structures were determined by X-ray crystallography. The crystals belong to the orthorhombic and monoclinic crystal systems, with space groups C222(I) and C2 respectively. The orthorhombic crystals were obtained at pH 6.5 and contained three insulin hexamers in R-6 conformation in the asymmetric unit whilst the monoclinic C2 crystals were obtained at pH 7.0 and contained one R6 hexamer in the asymmetric unit. Common for the two new crystals is a hexamer-hexamer interaction that has not been found in any of the previous crystal forms of insulin. The contacts involve a tight glutamate-glutamate interaction with a distance of 2.3 angstrom between groups. The short distance suggests a low barrier hydrogen bond. In addition, two tyrosine-tyrosine interactions occupying a known phenol binding pocket contribute to the stabilization of the contacts. Within the crystals, distinct binding sites for urea were found, adding further to the discussion on the role of urea in protein denaturation. Conclusion: The change in space group from C222(I) to C2 was primarily caused by an increase in pH. The fewer number of hexamer-hexamer interactions comprising the short hydrogen bond in the C2 space group suggest that pH is the driving force. In addition, the distance between the two glutamates increases from 2.32 angstrom in the C222(I) crystals to 2.4 angstrom in the C2 crystals. However, in both cases the low barrier hydrogen bond and the tyrosine-tyrosine interaction should contribute to the stability of the crystals which is crucial when used in pharmaceutical formulations.},
  author       = {Norrman, Mathias and Schluckebier, Gerd},
  issn         = {1471-2237},
  language     = {eng},
  publisher    = {BioMed Central},
  series       = {BMC Structural Biology},
  title        = {Crystallographic characterization of two novel crystal forms of human insulin induced by chaotropic agents and a shift in pH},
  url          = {http://dx.doi.org/10.1186/1472-6807-7-83},
  volume       = {7},
  year         = {2007},
}