Stability and a-complementation of M15 B-galactosidase from escherichia coli
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AbstractM15 B-Galactosidase from Escherichia coli is an inactive variant of Bgalactosidase with a deletion of residues 11-41. The protein can become active by the addition of a peptide derived from the N-terminal end of the wild type protein, by a process termed a-complementation. A fusion protein system was developed which allowed for the production and purification of a-peptide. This significantly decreased the time required to obtain pure peptide. The peptide produced in this way was also found to complement to a higher activity than the chemically cleaved (by cyanogen bromide) peptide from wild type B-galactosidase. Factors which affected the a-complementation of M15 B-galactosidase were investigated for their effects on activation (a-complementation). It was found that NaCl and B-mercaptoethanol increased the activation. Mg2+ increased the activity at low concentration but inhibited activation at high concentrations. The equilibrium between monomers and dimers was investigated. From experiments done at a series of protein concentrations, the Kd for monomer-dimer equilibrium was found to be 2.78 ± 0.76 x 10-7 M. The concentration of the protein was important in the equilibrium. Mg2+, NaCl and B-mercaptoethanol were shown to lower the Kd. The pH also affected the monomer-dimer equilibrium. The stability of M15 B-galactosidase was examined by thermal denaturation. The uncomplemented protein was found to have a lower temperature of activity loss than wild type protein. The complemented protein was more stable to heat than the uncomplemented M15 B-galactosidase if Mg2+ was present. The complemented enzyme lost activity non-cooperatively. The conformational stability of the M15 B-galactosidase protein was examined by urea denaturation. Unfolding intermediates of wild type Bgalactosidase were stabilized by Mg2+ and NaCl. For the M15 Bgalactosidase, NaCl stabilizes the intermediate structures but there was minimal stabilization by Mg2+. Complementation of the M15 B-galactosidase did not appear to increase the AG(H2O). The binding of a-peptide to the protein was found to be a 1:1 ratio of peptide per monomer ofM15 B-galactosidase with both dimers and tetramers. It was also shown that the peptide derived from the fusion protein formed distinct states of tetramers. The a-complementation process did not alter the binding of substrate to enzyme. The catalytic activity was, however, altered.
Bibliography: p. 198-205.