RESIDUE-SPECIFIC BIOINCORPORATION OF NONNATURAL, BIOLOGICALLY-ACTIVE AMINO-ACIDS INTO PROTEINS AS POSSIBLE DRUG CARRIERS - STRUCTURE AND STABILITY OF THE PER-THIAPROLINE MUTANT OF ANNEXIN-V

Residue-specific bioincorporation of 1,3-thiazolidine-4-carboxylic aci d [thiaproline, Pro(S)], a nonnatural amino acid analog of proline, in to human recombinant annexin V was achieved with a proline-auxotrophic Escherichia coil strain by fermentation procedures in minimal medium, Quantitative replacement of proline with thiaproline was confirmed by mass-spectrometric, amino acid, and x-ray crystallographic analyses, The wild-type protein and its per-Pro(S) mutant were found to crystall ize isomorphously and to show identical three-dimensional structures i n crystals, In solution the dichroic properties of the wild-type and p er-Pro(S) protein confirmed nearly identical overall folds. From therm al denaturation experiments, however, a reduced T-m (-4.5 K) value was determined whereas the van't Hoff enthalpy and entropy were not signi ficantly affected, Therefore, protein mutants containing bioactive ami no acid analogs like thiaproline at multiple sites would be expected t o fully retain their functional properties, including immunogenicity, and thus could serve as promising vehicles for targeted drug delivery.