Human α-fetoprotein as a Zn2+-binding protein. Tight cation binding is not accompanied by global changes in protein structure and stability

AbstractThe binding of zinc to human α-fetoprotein (AFP) isolated from human umbilical cord serum was studied by fluorimetric Zn2+-titration. We found that the total number of strong binding sites for zinc on this protein was 5: AFP has one very strong (dissociation constant, Kd<10−8 M) and at least four lower affinity zinc binding sites (Kd<10−5 M). Fourier transform infrared (FTIR) analysis revealed that aspartate and histidine residues could be involved in the strong coordination of zinc. Intriguingly, binding of zinc to the protein does not induce structural changes that can be detected by circular dichroism, FTIR, intrinsic fluorescence or (1,1′)-bi-(4-anilino)naphthalene-5,5′-disulfonic acid (bis-ANS) binding. Finally, scanning microcalorimetry measurements showed that stability of the protein is also unaffected by zinc binding in spite of the strength of the coordination. Such strong interactions without major structural consequences are highly unusual, and AFP may therefore be the first characterized representative of a new class of ligand-binding proteins

Structural Features of Amyloid Fibrils Formed from the Full-Length and Truncated Forms of Beta-2-Microglobulin Probed by Fluorescent Dye Thioflavin T

The persistence of high concentrations of beta-2-microglobulin (&beta;2M) in the blood of patients with acute renal failure leads to the development of the dialysis-related amyloidosis. This disease manifests in the deposition of amyloid fibrils formed from the various forms of &beta;2M in the tissues and biological fluids of patients. In this paper, the amyloid fibrils formed from the full-length &beta;2M (&beta;2m) and its variants that lack the 6 and 10 N-terminal amino acids of the protein polypeptide chain (&Delta;N6&beta;2m and &Delta;N10&beta;2m, respectively) were probed by using the fluorescent dye thioflavin T (ThT). For this aim, the tested solutions were prepared via the equilibrium microdialysis approach. Spectroscopic analysis of the obtained samples allowed us to detect one binding mode (type) of ThT interaction with all the studied variants of &beta;2M amyloid fibrils with affinity ~104 M&minus;1. This interaction can be explained by the dye molecules incorporation into the grooves that were formed by the amino acids side chains of amyloid protofibrils along the long axis of the fibrils. The decrease in the affinity and stoichiometry of the dye interaction with &beta;2M fibrils, as well as in the fluorescence quantum yield and lifetime of the bound dye upon the shortening of the protein amino acid sequence were shown. The observed differences in the ThT-&beta;2M fibrils binding parameters and characteristics of the bound dye allowed to prove not only the difference of the &Delta;N10&beta;2m fibrils from other &beta;2M fibrils (that can be detected visually, for example, by transmission electron microscopy (TEM), but also the differences between &beta;2m and &Delta;N6&beta;2m fibrils (that can not be unequivocally confirmed by other approaches). These results prove an essential role of N-terminal amino acids of the protein in the formation of the &beta;2M amyloid fibrils. Information about amyloidogenic protein sequences can be claimed in the development of ways to inhibit &beta;2M fibrillogenesis for the treatment of dialysis-related amyloidosis