Residue-specific bioincorporation of non-natural, biologically active amino acids into proteins as possible drug carriers: structure and stability of the per-thiaproline mutant of annexin V

Abstract

Residue-specific bioincorporation of 1,3-thiazolidine-4-carboxylic acid [thiaproline, Pro(S)], a non-natural amino acid analog of proline, into human recombinant annexin V was achieved with a proline-auxotrophic Escherichia coli 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 crystallize isomorphously and to show identical three-dimensional structures in crystals. In solution the dichroic properties of the wild-type and per-Pro(S) protein confirmed nearly identical overall folds. From thermal denaturation experiments, however, a reduced Tm (-4.5 K) value was determined whereas the van't Hoff enthalpy and entropy were not significantly affected. Therefore, protein mutants containing bioactive amino acid analogs like thiaproline at multiple sites would be expected to fully retain their functional properties, including immunogenicity, and thus could serve as promising vehicles for targeted drug delivery.

Scheme 1

The structures of proline (Left) and its non-natural analog thiaproline (Right). The values for thiaproline were taken from the Cambridge database for small molecules, the data for proline are from ref. .

Figure 1

Ribbon plot of human recombinant annexin V (top view) with difference densities at Cγ for all proline residues successfully substituted with thiaprolines (contouring level is 1.5 σ).

Figure 2

Electrospray mass spectra of wt and per-Pro(S) annexin V. The deconvoluted spectra of separate measurements were superimposed at the same mass scale.

Figure 3

Electron density difference between -CH₂- and sulfur at the site of replacement (Cγ) shown by the difference Fourier map (F_Pro(S)–F_Pro) contoured at 2.8 σ for the residue Pro-87.

Figure 4

CD spectra of wt and per-Pro(S) annexin V in PBS containing 10% glycerol at 20°C.

Figure 5

Thermal denaturation of wt (•) and per-Pro(S) annexin V (○) in PBS containing 10% glycerol. Fractions of denatured protein are calculated from CD data monitored at 222 nm.

Figure 6

Fluorescence emission spectra for native (solid line) and per-Pro(S) annexin V (dashed line) at 20°C in PBS containing 10% glycerol; samples were excitated at 280 nm.