Stabilities of uncomplemented and complemented M15 β-galactosidase (Escherichia coli) and the relationship to α-complementation

M15 β-galactosidase (Escherichia coli) is a mutant form of β-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 β-galactosidases decreased starting at 42°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 α-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 (10× less Mg2+ was needed for complemented protein). α-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 α-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 β-galactosidase, but high concentrations destabilized it. All concentrations destabilized complemented M15 β-galactosidase. α-Complementation was enhanced by 2-mercaptoethanol. Thus, there is a correlation between stability of the uncomplemented protein and α-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. Key words: β-galactosidase, α-complementation, stability.