REVERSIBLE FOLDING OF ADA PROTEIN (O-6-METHYLGUANINE-DNA METHYLTRANSFERASE) OF ESCHERICHIA-COLI

The multifunctional 39 kDa Escherichia coli Ada protein (O-6-methylgua nine-DNA methyltransferase) (EC 2.1.1.63), product of the ada gene, is a monomeric globular polypeptide with two distinct alkylacceptor acti vities located in two domains. The two domains are of nearly equal siz e and are connected by a hinge region. The Ada protein accepts stoichi ometrically the alkyl group from O-6-alkylguanine in DNA at the Cys-32 1 residue and from alkyl phosphotriester at the Cys-69 residue. This p rotein functions in DNA repair by direct dealkylation of mutagenic O-6 -alkylguanine. The protein methylated at Cys-69 becomes a transcriptio nal activator of the genes in the ada regulon, including its own. Each of the two domains functions independently as an alkyl acceptor. The purified homogeneous protein is unstable at 37 degrees C and spontaneo usly loses about 30% of its secondary structure in less than 30 min co ncomitant with a complete loss of activity. However, sedimentation equ ilibrium studies indicated that the inactive protein remains in the mo nomeric form without aggregation. Furthermore, electrospray mass spect roscopic analysis indicated the absence of oxidation of the inactive p rotein, This temperature-dependent inactivation of the Ada protein is inhibited by DNA. In the presence of increasing concentrations of urea or guanidine, the protein gradually loses more than 80% of its struct ure. The two alkyl acceptor activities appear to be differentially sen sitive to unfolding and the phosphotriester methyltransferase activity is resistant to 7 M urea. The partial or complete unfolding induced b y urea or guanidine is completely reversed within seconds by removal o f the denaturant. The heat-coagulated protein can also be restored to full activity by cycling it through treatment with 8 M urea or 6 M gua nidine. These results suggest that the nascent or unfolded Ada polypep tide folds to a metastable form which is active and that the thermodyn amically stable structure is partially unfolded and inactive.