Effect of the extra N-terminal methionine residue on the stability and folding of recombinant alpha-lactalbumin expressed in Escherichia coli

The structure, stability, and unfolding-refolding kinetics of Escherichia c oli-expressed recombinant goat alpha-lactalbumin were studied by circular d ichroism spectroscopy, X-ray crystallography, and stopped-flow measurements , and the results were compared with those of the authentic protein prepare d from goat milk. The electric properties of the two proteins were also stu died by gel electrophoresis and ion-exchange chromatography. Although the o verall structures of the authentic and recombinant proteins are the same, t he extra methionine residue at the N terminus of the recombinant protein re markably affects the native-state stability and the electric properties. Th e native state of the recombinant protein was 3.5 kcal/mol less stable than the authentic protein, and the recombinant protein was more negatively cha rged than the authentic one. The recombinant protein unfolded 5.7 times fas ter than the authentic one, although there were no significant differences in the refolding rates of the two proteins. The destabilization of the reco mbinant protein tan be fully interpreted in terms of the increased unfoldin g rate of the protein, indicating that the N-terminal region remains unorga nized in the transition state of refolding,, and hence is not involved in t he folding initiation site of the protein. A comparison of the X-ray struct ures of recombinant alpha-lactalbumin determined here with that of the auth entic protein shows that the structural differences between the proteins ar e confined to the N-terminal region. Theoretical considerations for the dif ferences in the conformational and solvation free energies between the prot eins show that the destabilization of the recombinant protein is primarily due to excess conformational entropy of the N-terminal methionine residue i n the unfolded state, and also due to less exposure of hydrophobic surface on unfolding. The results suggest that when the N-terminal region of a prot ein has a rigid structure, expression of the protein by E. coli, which adds the extra methionine residue, destabilizes the native state through a conf ormational entropy effect. It also shows that differences in the electrosta tic interactions of the N-terminal amino group with the side-chain atoms of Thr38, Asp37, and Asp83 bring about a difference in the pK(a) value of the N-terminal amino group between the proteins, resulting in a greater negati ve net charge of the recombinant protein at neutral pH. (C) 1999 Academic P ress.