Evaluation of the secondary structure of vaccinia-virus thymidine kinase by circular-dichroism spectroscopy of overlapping synthetic peptides.
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 alpha-helical content mainly showed CD spectra with increasing and decreasing Cotton effects typical of alpha-helices. This phenomenon was used to localize the helices on the sequence. In contrast, only single CD spectra with clear beta-sheet conformation, or CD spectra of mixed secondary-structure content were observed for beta-sheets. Therefore, the exact localization of beta-sheet-containing residues was deduced by comparison with isofunctional sequence-dissimilar proteins. We identified seven alpha-helices and six beta-sheet-containing regions, which we used for a secondary-structure model of the vaccinia-virus TK protein.
Behrends
H W
HW
Department of Pharmacy, Swiss Federal Institute of Technology, Zürich, Switzerland.
Beck-Sickinger
A G
AG
Folkers
G
G
D000595:Amino Acid Sequence; D002942:Circular Dichroism; D008969:Molecular Sequence Data; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry; D017433:Protein Structure, Secondary; D013937:Thymidine Kinase / Q000737:chemistry*; D014616:Vaccinia virus / Q000201:enzymology*
D010455:Peptides; D013937:Thymidine Kinase