Conformational and epitope mapping of herpes-simplex-virus type-1 thymidine kinase using synthetic peptide segments.
Adjacent peptide segments covering the complete sequence of herpes-simplex-virus type-1 thymidine kinase (HSV1-TK) of 376 amino acids were synthesized in order to experimentally verify the three-dimensional structure of the HSV1-TK active site, which was previously determined by molecular modeling. 26 peptides have been prepared by multiple solid-phase synthesis using the 9-fluorenylmethoxycarbonyl strategy. The purified peptides were linked covalently to bovine serum albumin. The peptide/ELISA of the synthesized bovine-serum-albumin conjugates using polyclonal rabbit anti(HSV1-TK)serum resulted in ten epitopes, which correlate excellently with the computer-proposed active site of HSV1-TK. CD spectra of the HSV1-TK peptides were recorded in trifluoroethanol/water (9:1 by vol.) An eigenvalue method based on CD spectra of 15 well known protein structures was used to calculate the relative percentage of secondary structures from the CD data. The computer model of the HSV1-TK showed full conformity with the folding pattern determined by CD of the synthetic peptide segments. Therefore, conformational peptide mapping with CD-based secondary structures combined with epitope mapping from the peptide/ELISA is an efficient and reliable method to support computer-aided protein design.
Zimmermann
N
N
Institut für Organische Chemie, Universität Tübingen, Federal Republic of Germany.
Beck-Sickinger
A G
AG
Folkers
G
G
Krickl
S
S
Müller
I
I
Jung
G
G
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry*; D010455:Peptides / Q000138:chemical synthesis; D011487:Protein Conformation; D018139:Simplexvirus / Q000201:enzymology*; D013937:Thymidine Kinase / Q000737:chemistry / Q000276:immunology*
D000596:Amino Acids; D000939:Epitopes; D010446:Peptide Fragments; D010455:Peptides; D013937:Thymidine Kinase