Mapping site-specific changes that affect stability of the N-terminal domain of calmodulin

Mol Pharm. 2012 Apr 2;9(4):734-43. doi: 10.1021/mp2004109. Epub 2012 Feb 15.

Abstract

Biophysical tools have been invaluable in formulating therapeutic proteins. These tools characterize protein stability rapidly in a variety of solution conditions, but in general, the techniques lack the ability to discern site-specific information to probe how solution environment acts to stabilize or destabilize the protein. NMR spectroscopy can provide site-specific information about subtle structural changes of a protein under different conditions, enabling one to assess the mechanism of protein stabilization. In this study, NMR was employed to detect structural perturbations at individual residues as a result of altering pH and ionic strength. The N-terminal domain of calmodulin (N-CaM) was used as a model system, and the ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) experiment was used to investigate effects of pH and ionic strength on individual residues. NMR analysis revealed that different solution conditions affect individual residues differently, even when the amino acid sequence and structure are highly similar. This study shows that addition of NMR to the formulation toolbox has the ability to extend understanding of the relationship between site-specific changes and overall protein stability.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Calmodulin / chemistry*
  • Magnetic Resonance Spectroscopy
  • Protein Stability
  • Protein Structure, Tertiary

Substances

  • Calmodulin