All of the individual carboxyl groups (the side-chain carboxyl groups of Asp and Glu, and the C-terminal alpha-carboxyl group) in Escherichia coli ribonuclease HI, which is an enzyme that cleaves the RNA strand of a RNA/DNA hybrid, were pH-titrated, and their ionization constants (pKa) were determined from an analysis of the pH-dependent chemical shifts of the carboxyl carbon resonances obtained from 1H-13C heteronuclear two-dimensional NMR. The pKa values in the enzyme varied widely among individual residues, for example, in the unusual pKa values for two important catalytic residues, Asp10 (pKa 6.1) and Asp70 (pKa 2.6). Moreover, remarkable two-step titrations were observed for these carboxylates. The binding of Mg2+ ion to the enzyme, which is the cofactor necessary for catalytic activity, caused no significant change in the pKa values of the carboxyl groups, except for that of Asp10. The variations of the pKas that were dependent on the microenvironment in the protein were theoretically reproduced to compare with the experimental results by a numerical calculation, using a continuum electrostatic model. Most of the significant pKa decreases were brought about through strong electrostatic interactions with the neighboring basic amino acids, Arg or Lys. The pKa shifts and the two-step titrations of Asp10 and -70, which are close to each other, were interpreted to be due to the neighboring effect of two functional groups, as observed in the interacting titratable groups of a dicarboxyl compound or in the active site carboxylates of lysozyme and aspartic protease. The role of Asp10 in the catalytic action is either to be the proton donor to the RNA moiety or the binding partner of the Mg2+ ion cofactor. Asp70, on the other hand, is considered to be the proton acceptor from a water molecule.

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Supplementary data are available at Bioinformatics online.

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Protein aggregation is a common phenomenon. The preparation of highly concentrated protein samples, typically required for biophysical measurements, often involves a time consuming and tedious testing of solvent conditions for improving protein solubility. Here, in a systematic analysis, we have determined the increase in solubility upon the addition of SEP-tags (solubility enhancement peptide tags) containing, one, three, and five lysines or arginines (or six arginines) to either the N or C terminus of our low solubility model protein, bovine pancreatic trypsin inhibitor variant, BPTI-22 (a BPTI variant containing 22 alanines). As anticipated, the BPTI-22 solubility increased in direct relation to the number of charged residues contained in the SEP-tag, and without altering either the activity or the structure of the protein. The largest solubility increases were of 4.2-, 4.8-, and 6.2-folds produced by the addition, at the C terminus, of five lysine (BPTI-22-C5K), five and six arginine residues (BPTI-22-C5R and BPTI-22-C6R), respectively. The increased solubility of the tagged BPTI-22 yielded higher quality NMR spectra (hetero single quantum correlation HSQC spectra; with respect of the signal-to-noise and line shapes) in a much shorter time than for the untagged BPTI-22. Furthermore, tagged samples remained soluble for over ten days, as observed by their HSQC spectra. We believe that lysine- and arginine-based SEP-tags may provide an effective and versatile method for enhancing protein solubility.