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

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.

[1]  W. Anderson,et al.  Ca2+-induced hydrophobic site on calmodulin: application for purification of calmodulin by phenyl-Sepharose affinity chromatography. , 1982, Biochemical and biophysical research communications.

[2]  Charles E. Bugg,et al.  Three-dimensional structure of calmodulin , 1985, Nature.

[3]  I. Ota,et al.  Calcium affects the spontaneous degradation of aspartyl/asparaginyl residues in calmodulin. , 1989, Biochemistry.

[4]  L. Kay,et al.  A novel approach for sequential assignment of 1H, 13C, and 15N spectra of proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin. , 1990, Biochemistry.

[5]  L. Kay,et al.  A novel approach for sequential assignment of proton, carbon-13, and nitrogen-15 spectra of larger proteins: heteronuclear triple-resonance three-dimensional NMR spectroscopy. Application to calmodulin , 1990 .

[6]  G. Roberts,et al.  NMR of macromolecules : a practical approach , 1993 .

[7]  D. Aswad,et al.  In vitro aging of calmodulin generates isoaspartate at multiple Asn–Gly and Asp–Gly sites in calcium‐binding domains II, III, and IV , 1993, Protein science : a publication of the Protein Society.

[8]  Eva Thulin,et al.  Calcium-induced structural changes and domain autonomy in calmodulin , 1995, Nature Structural Biology.

[9]  Mitsuhiko Ikura,et al.  Calcium-induced conformational transition revealed by the solution structure of apo calmodulin , 1995, Nature Structural Biology.

[10]  J. Cavanagh.  Protein NMR Spectroscopy: Principles and Practice , 1995 .

[11]  A. Bax,et al.  Rotational dynamics of calcium-free calmodulin studied by 15N-NMR relaxation measurements. , 1995, European journal of biochemistry.

[12]  D. Aswad,et al.  Deamidation and isoaspartate formation in peptides and proteins , 1995 .

[13]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[14]  Ad Bax,et al.  Solution structure of calcium-free calmodulin , 1995, Nature Structural Biology.

[15]  Eric Oldfield,et al.  1H, 13C and 15N chemical shift referencing in biomolecular NMR , 1995, Journal of biomolecular NMR.

[16]  L. Steinrauf.  Structures of monoclinic lysozyme iodide at 1.6 A and of triclinic lysozyme nitrate at 1.1 A. , 1998, Acta crystallographica. Section D, Biological crystallography.

[17]  S. Martin,et al.  Ca2+ coordination to backbone carbonyl oxygen atoms in calmodulin and other EF-hand proteins: 15N chemical shifts as probes for monitoring individual-site Ca2+ coordination. , 1998, Biochemistry.

[18]  Z. Dauter,et al.  ATOMIC RESOLUTION REFINEMENT OF TRICLINIC HEW LYSOZYME AT 295K , 1998 .

[19]  W Wang,et al.  Instability, stabilization, and formulation of liquid protein pharmaceuticals. , 1999, International journal of pharmaceutics.

[20]  R. Nussinov,et al.  Folding and binding cascades: Dynamic landscapes and population shifts , 2008, Protein science : a publication of the Protein Society.

[21]  S. Martin,et al.  Ligand binding and thermodynamic stability of a multidomain protein, calmodulin , 2000, Protein science : a publication of the Protein Society.

[22]  T. Prangé,et al.  Structural effects of monovalent anions on polymorphic lysozyme crystals. , 1998, Acta crystallographica. Section D, Biological crystallography.

[23]  Ad Bax,et al.  Solution structure of Ca2+–calmodulin reveals flexible hand-like properties of its domains , 2001, Nature Structural Biology.

[24]  N. Robinson,et al.  Deamidation of human proteins , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[25]  W. Delano.  The PyMOL Molecular Graphics System , 2002 .

[26]  A. Wand,et al.  Calcium-induced conformational switching of Paramecium calmodulin provides evidence for domain coupling. , 2002, Biochemistry.

[27]  P. Bayley,et al.  Thermal stability of calmodulin and mutants studied by (1)H-(15)N HSQC NMR measurements of selectively labeled [(15)N]Ile proteins. , 2002, Biochemistry.

[28]  T. Squier,et al.  Oxidation of Met144 and Met145 in calmodulin blocks calmodulin dependent activation of the plasma membrane Ca-ATPase. , 2003, Biochemistry.

[29]  N. C. Price,et al.  How to study proteins by circular dichroism. , 2005, Biochimica et biophysica acta.

[30]  H. Schägger.  Tricine–SDS-PAGE , 2006, Nature Protocols.

[31]  Ronald T Borchardt,et al.  Formulation considerations for proteins susceptible to asparagine deamidation and aspartate isomerization. , 2006, Journal of pharmaceutical sciences.

[32]  C. R. Middaugh,et al.  Effects of pH and polyanions on the thermal stability of fibroblast growth factor 20. , 2007, Molecular pharmaceutics.

[33]  M. Uesugi,et al.  [Discovering high-affinity ligands for proteins: SAR by NMR]. , 2007, Tanpakushitsu kakusan koso. Protein, nucleic acid, enzyme.

[34]  R. Muhandiram,et al.  Carboxyl pK(a) values, ion pairs, hydrogen bonding, and the pH-dependence of folding the hyperthermophile proteins Sac7d and Sso7d. , 2007, Journal of molecular biology.

[35]  J. Laurence,et al.  High-field solution NMR spectroscopy as a tool for assessing protein interactions with small molecule ligands. , 2008, Journal of Pharmacy and Science.

[36]  M. Ikura,et al.  NMR Investigation of Calmodulin , 2008 .

[37]  Hanns-Christian Mahler,et al.  Protein aggregation: pathways, induction factors and analysis. , 2009, Journal of pharmaceutical sciences.

[38]  Dong Long,et al.  Buffer interference with protein dynamics: a case study on human liver fatty acid binding protein. , 2009, Biophysical journal.

[39]  Brian M. Murphy,et al.  Stability of Protein Pharmaceuticals: An Update , 2010, Pharmaceutical Research.

[40]  M. A. Shea,et al.  Thermodynamics and conformational change governing domain-domain interactions of calmodulin. , 2009, Methods in enzymology.

[41]  Margaret S. Cheung,et al.  The Effect of Macromolecular Crowding, Ionic Strength and Calcium Binding on Calmodulin Dynamics , 2011, PLoS Comput. Biol..

[42]  Yatin R. Gokarn,et al.  Effective charge measurements reveal selective and preferential accumulation of anions, but not cations, at the protein surface in dilute salt solutions , 2011, Protein science : a publication of the Protein Society.

[43]  KAP LIM.  Locations of Bromide Ions in Tetragonal Lysozyme Crystals , .