title: Semisynthesis of human thymidine monophosphate kinase.
abstract: Protein semisynthesis based on native chemical ligation has become a major protein engineering tool that allows manipulation of domains of proteins of all sizes. It helps to overcome limitations in chemical protein synthesis set by the inherent size limits of solid phase peptide synthesis. Here we present a semisynthesis approach that provides access to N-terminally-modified variants of human thymidine monophosphate kinase (TMPK). This enzyme is intimately involved in activating nucleoside-based drugs directed against viral infections such as HIV and against certain types of cancers. The option to chemically synthesize and manipulate the first 30 amino acids of this enzyme via protein semisynthesis allows direct substitution of vital amino acids in the P-loop of this enzyme for probing the mechanism of phosphate transfer and direct observation of substrate or inhibitor binding. Efficient native chemical ligation of two N-terminal segments, one comprising the wild type sequence and one containing a small fluorescent probe, provides milligram amounts of two semisynthetic TMPK variants. An efficient folding procedure in the presence of substrate nucleotides provides access to active semisynthetic TMPK variants.
authors.affiliation: Center of Integrated Protein Science Munich and Department of Chemistry, Technische Universität München, Lichtenbergstr. 4, 85747 Garching b. München, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Gogolin
authors.forename: Ralf
authors.initials: R
authors.lastname: Seidel
authors.forename: Martin
authors.initials: M
authors.lastname: Engelhard
authors.forename: Roger S
authors.initials: RS
authors.lastname: Goody
authors.forename: Christian F W
authors.initials: CF
authors.lastname: Becker
mesh_terms: D019943:Amino Acid Substitution; D006801:Humans; D009703:Nucleoside-Phosphate Kinase / Q000138:chemical synthesis* / Q000737:chemistry; D009711:Nucleotides / Q000737:chemistry; D017510:Protein Folding; D017434:Protein Structure, Tertiary
chemical_list: D009711:Nucleotides; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase