Evaluation of the secondary structure of vaccinia-virus thymidine kinase by circular-dichroism spectroscopy of overlapping synthetic peptides

Abstract

A new approach is reported that includes multiple-peptide synthesis and CD spectroscopy of overlapping peptides to evaluate the secondary structure of the vaccinia-virus thymidine kinase (TK). We divided the sequence of the vaccinia-virus TK into 82 peptides of 15 residues that overlapped by 13 residues and covered the complete sequence of vaccinia-virus TK. All peptides were synthesized by solid-phase multiple-peptide synthesis by means of the Fmoc/tert-butyl strategy. Subsequently, the secondary structure of each peptide was studied by means of CD spectroscopy in a mixture of 30% trifluoroethanol and sodium phosphate, pH 7. Secondary-structure evaluation led to determination of a vaccinia-virus-TK secondary-structure pattern. Consecutive peptides with α-helical content mainly showed CD spectra with increasing and decreasing Cotton effects typical of α-helices. This phenomenon was used to localize the helices on the sequence. In contrast, only single CD spectra with clear β-sheet conformation, or CD spectra of mixed secondary-structure content were observed for β-sheets. Therefore, the exact localization of β-sheet-containing residues was deduced by comparison with isofunctional sequence-dissimilar proteins. We identified seven α-helices and six β-sheet-containing regions, which we used for a secondary-structure model of the vaccinia-virus TK protein.