Allosteric and binding properties of Asp1-Glu382 truncated recombinant human serum albumin--an optical and NMR spectroscopic investigation.

Human serum albumin (HSA) is known for its exceptional ligand-binding capacity; indeed, its modular domain organization provides a variety of ligand-binding sites. Its flexible modular structure involves more than the immediate vicinity of the binding site(s), affecting the ligand-binding properties of the whole protein. Here, biochemical characterization by (1)H-NMR relaxometry and optical spectroscopy of a truncated form of HSA (tHSA) encompassing domains I and II (Asp1-Glu382) is reported. Removal of the C-terminal domain III results in a number of contacts that involve domain I (containing the heme site) and domain II (containing the warfarin site) being lost; however, the allosteric linkage between heme and warfarin sites is maintained. tHSA shows a nuclear magnetic relaxation dispersion profile similar to that of HSA, and displays increased affinity for ibuprofen, warfarin, and heme, suggesting that the fold is preserved. Moreover, the allosteric properties that make HSA a peculiar monomeric protein and account for the regulation of ligand-binding modes by heterotropic interactions are maintained after removal of domain III. Therefore, tHSA is a valuable model with which to investigate allosteric properties of HSA, allowing independent analysis of the linkages between different drug-binding sites.