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Stabilization of myoglobin by multiple alanine substitutions in helical positions
Author(s) -
Lin Laura,
Pinker Rachel J.,
Phillips George N.,
Kallenbach Neville R.
Publication year - 1994
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 - 10.1002/pro.5560030909
Subject(s) - myoglobin , chemistry , urea , crystallography , alanine , equilibrium unfolding , denaturation (fissile materials) , protein structure , mutant , sequence (biology) , biophysics , biochemistry , circular dichroism , amino acid , biology , gene , nuclear chemistry
We have carried out a series of multiple Xaa → Ala changes at nonadjacent surface positions in the sequence of sperm whale myoglobin. Although the corresponding single substitutions do not increase the thermal stability of the protein, multiple substitutions enhance the stability of the resulting myoglobins. The effect observed is an increase in the observed T m (midpoint unfolding temperature) relative to that predicted from assuming additivity of the free energy changes corresponding to single mutations. The stabilization occurs in the presence of urea, as measured by the dependence of the unfolding temperature on urea concentration. The sites that have been altered occur in different helices and are not close in sequence or in the native structure of myoglobin. The observed effect is consistent with a role of multiple alanines in residual interactions in the unfolded state of the mutant proteins.