Mutational analysis of protein splicing, cleavage, and self-association reactions mediated by the naturally split Ssp DnaE intein.

The ability to separately purify the naturally split Synechocystis sp. PCC6803 (Ssp) DnaE intein domains has allowed detailed examination of both universal and Ssp DnaE intein-specific steps in the protein splicing pathway. By engineering substitutions at both the +1 and penultimate intein positions, we have further characterized intein reaction kinetics in this system. Replacement of the crucial +1Cys with serine decreased N-terminal cleavage and trans-splicing rates; however, this substitution did not prevent splicing or the ability of ZnCl2 to inhibit it. Substitution of the penultimate intein residue (alanine) with a typically conserved histidine did not increase the rate or extent of trans-splicing or cleavage under typical assay conditions. Despite the observation that this histidine aids in asparagine cyclization for other inteins, it did not encourage C-terminal cleavage for the Ssp DnaE intein or uncouple it from N-terminal cleavage. Both the +1Ser and Ala to His mutants were insensitive to ZnCl2 during trans-cleavage experiments, uncoupling a previously linked inhibition in asparagine cyclization from an inhibition in trans-thioesterification detected for the wild-type intein.

Zinc ion effects on individual Ssp DnaE intein splicing steps: regulating pathway progression.

Use of the naturally split, self-splicing Synechocystis sp. PCC6803 DnaE intein permits separate purification of the N- and C-terminal intein domains. Otherwise spontaneous intein-mediated reactions can therefore be controlled in vitro, allowing detailed study of intein kinetics. Incubation of the Ssp DnaE intein with ZnCl(2) inhibited trans splicing, hydrolysis-mediated N-terminal trans cleavage, and C-terminal trans cleavage reactions. Maximum inhibition of the splicing reaction was achieved at equal molar concentrations of ZnCl(2) and intein domains, suggesting a 1:1 metal ion:intein binding stoichiometry. Mutation of the (+)1 cysteine residue to valine (C(+)1V) alleviated the inhibitory effects of ZnCl(2). Valine substitution in the absence of ZnCl(2) blocked trans splicing and decreased C-terminal cleavage kinetics in a manner similar to that of the native (+)1 cysteine in the presence of ZnCl(2). These data are consistent with Zn(2+)-mediated inhibition of the Ssp DnaE intein via chelation of the (+)1 cysteine residue. N-Terminal trans cleavage can occur via both spontaneous hydrolysis and nucleophilic (e.g., DTT) attack. Comparative examination of N-terminal cleavage rates using amino acid substitution (C(+)1V) and Zn(2+)-mediated inhibition permitted the maximum contribution of hydrolysis to overall N-terminal cleavage kinetics to be determined. Stable intermediates consisting of the associated intein domains were detected by PAGE and provided evidence of a rapid C-terminal cleavage step. Acute control of the C-terminal reaction was achieved by the rapid reversal of Zn(2+)-mediated inhibition by EDTA. By inhibiting both the splicing pathway and spontaneous hydrolysis with Zn(2+), reactants can be diverted from the trans splicing to the trans cleavage pathway where DTT and EDTA can regulate N- and C-terminal cleavage, respectively.

Human p53 phosphorylation mimic, S392E, increases nonspecific DNA affinity and thermal stability.

DNA binding is crucial to the protective role of the tumor suppressor protein p53, a nuclear phosphoprotein and transcription factor. The mutant human p53 protein S392E is a phosphorylation mimic that has been previously demonstrated to represent an "activated" form of p53 in both in vivo and in vitro assays [Hupp and Lane (1995) J. Biol. Chem. 270, 18165; Hao et al. (1996) J. Biol. Chem. 271, 29380]. Herein, we describe an analysis of structural and functional differences between this mutant and the wild-type protein. Structurally, the S392E protein exhibits increased thermal stability compared to wild-type p53, as monitored by circular dichroism and conformational antibody Ab1620 reactivity. These structural effects include alterations to the core DNA binding domain, remote in sequence space from the site of mutation. Functionally, the S392E mutation does not increase p53 binding to its 20 bp consensus DNA sequence in the absence of nonspecific DNA additives. In contrast, affinity of S392E for a 20 bp nonspecific DNA sequence is enhanced. Embedding 20 bp consensus DNA in the context of longer DNA sequences does not substantially alter S392E affinity, whereas wild-type affinity for these DNAs decreases with increased proportion of nonspecific DNA. These differences may account for the S392E "activated" phenotype and illuminate the role of this modified p53 in vivo.