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Ligand binding and thermodynamic stability of a multidomain protein, calmodulin
Author(s) -
Masino Laura,
Martin Stephen R.,
Bayley Peter M.
Publication year - 2000
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.9.8.1519
Subject(s) - chemistry , denaturation (fissile materials) , crystallography , chemical stability , protein folding , ionic strength , ionic bonding , calmodulin , ligand (biochemistry) , protein domain , biophysics , ion , biochemistry , biology , enzyme , receptor , aqueous solution , nuclear chemistry , organic chemistry , gene
Chemical and thermal denaturation of calmodulin has been monitored spectroscopically to determine the stability for the intact protein and its two isolated domains as a function of binding of Ca 2+ or Mg 2+ . The reversible urea unfolding of either isolated apo‐domain follows a two‐state mechanism with relatively low δG° 20 values of ˜2.7 (N‐domain) and ˜1.9 kcal/mol (C‐domain). The apo‐C‐domain is significantly unfolded at normal temperatures (20‐25 °C!. The greater affinity of the C‐domain for Ca 2+ causes it to be more stable than the N‐domain at [Ca 2+ ] ≤0.3 mM. By contrast, Mg 2+ causes a greater stabilization of the N‐ rather than the C‐domain, consistent with measured Mg 2+ affinities. For the intact protein (+Ca 2+ ), the bimodal denaturation profiles can be analyzed to give two δ G ° 20 values, which differ significantly from those of the isolated domains, with one domain being less stable and one domain more stable. The observed stability of the domains is strongly dependent on solution conditions such as ionic strength, as well as specific effects due to metal ion binding. In the intact protein, different folding intermediates are observed, depending on the ionic composition. The results illustrate that a protein of low intrinsic stability is liable to major perturbation of its unfolding properties by environmental conditions and liganding processes and, by extension, mutation. Hence, the observed stability of an isolated domain may differ significantly from the stability of the same structure in a multidomain protein. These results address questions involved in manipulating the stability of a protein or its domains by site directed mutagenesis and protein engineering.