Stabilities of uncomplemented and complemented M15 beta-galactosidase (Escherichia coli) and the relationship to alpha-complementation.
M15 beta-galactosidase (Escherichia coli) is a mutant form of beta-galactosidase having residues 11-41 deleted. It is an inactive dimer but can be complemented to the active tetrameric form by the addition of a peptide containing the deleted residues. The activities of uncomplemented and complemented M15 beta-galactosidases decreased starting at 42 degrees C--uncomplemented over a narrow temperature range, complemented over a broad range. This is because uncomplemented protein is a simple dimer while complemented is a mix of interacting oligomers at high temperatures. The effects of added components on stability and alpha-complementation are best explained by binding effects on equilibria between native forms and forms susceptible to inactivation. Mg2+ stabilized complemented protein but destabilized uncomplemented protein (10x less Mg2+ was needed for complemented protein). Alpha-complementation increased somewhat at low Mg2+ but decreased at high Mg2+. These effects can be explained by differential Mg2+ binding to the native and susceptible forms. The enhancement of both stability and alpha-complementation by Na+ can be explained by preferential binding of Na+ to the native forms of both the uncomplemented and complemented proteins. Low 2-mercaptoethanol concentrations stabilized uncomplemented M15 beta-galactosidase, but high concentrations destabilized it. All concentrations destabilized complemented M15 beta-galactosidase. Alpha-complementation was enhanced by 2-mercaptoethanol. Thus, there is a correlation between stability of the uncomplemented protein and alpha-complementation at low 2-mercaptoethanol owing to interactions with native forms. The lack of correlation at higher 2-mercaptoethanol probably results from precipitation by 2-mercaptoethanol. In contrast to irreversible thermal inactivation, differences in reversible stability in urea were small. This suggests that quaternary structure and Mg2+ and Na+ sites are lost at low urea concentrations and are unimportant at the urea concentrations that result in reversible denaturation.
Gallagher
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Division of Biochemistry, Faculty of Science, University of Calgary, Canada.
Huber
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D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology*; D006361:Heating; D008274:Magnesium; D008623:Mercaptoethanol; D011487:Protein Conformation; D012965:Sodium Chloride; D013696:Temperature; D000519:alpha-Galactosidase / Q000737:chemistry; D001616:beta-Galactosidase / Q000737:chemistry*
D012965:Sodium Chloride; D008623:Mercaptoethanol; D000519:alpha-Galactosidase; D001616:beta-Galactosidase; D008274:Magnesium