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Monovalent cation binding to cubic insulin crystals.

O Gursky1, Y Li, J Badger

  • 1Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02254-9110.

Biophysical Journal
|March 1, 1992
PubMed
Summary

Two specific monovalent cation binding sites in cubic insulin were identified. These sites, crucial for crystal stability, are influenced by protein charge and pH, with specific ion sizes required.

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Area of Science:

  • Protein crystallography
  • Structural biology
  • Biochemistry

Background:

  • Insulin's crystal structure is vital for its function and pharmaceutical formulation.
  • Understanding cation interactions is key to stabilizing protein crystals.

Purpose of the Study:

  • To identify and characterize localized monovalent cation binding sites in cubic insulin crystals.
  • To investigate the influence of pH and ion type on these binding sites and crystal stability.

Main Methods:

  • Difference electron density mapping at 2.8 A resolution comparing sodium and thallium ion crystals.
  • Refinement of a 2 A resolution map of sodium-insulin crystals.
  • Crystallization of insulin with various monovalent cations (Li+, K+, NH4+, Rb+, Tl+) across a pH range (7-10).

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Main Results:

  • Two distinct monovalent cation binding sites were identified within the insulin crystal structure.
  • One site is in a buried cavity, stabilized by protein carbonyl dipoles.
  • The second site involves His B10 side chain conformations and is competitively occupied.
  • Cation occupancy is dependent on protein net charge and pH.
  • Monovalent cations with ionic radii <1.5 A are necessary for crystal stability.
  • Specific cations (Cs+, Mg++, Ca++, La+++) disrupted lattice order, while others (Li+, K+, NH4+, Rb+, Tl+) maintained diffraction.

Conclusions:

  • Localized cation binding sites play a critical role in stabilizing cubic insulin crystals.
  • Crystal stability is sensitive to pH, protein charge, and the specific type and size of monovalent cations used.
  • These findings have implications for insulin crystallization and formulation.