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

Mutating Aspartate in The Calcium-binding Site of α-lactalbumin: Effects on The Protein Stability and Cation Binding

The residue Asp87, which is in the calcium-binding loop of bovine α-lactalbumin (α-LA) and provides a side-chain carboxylate oxygen for ligand Ca(II) co-ordination, was substituted by either alanine or asparagine. The physical properties and calcium-binding affinities were monitored by intrinsic fluorescence and circular dichroism spectroscopy. D87A α-LA displayed a total loss of rigid tertiary structure, a dramatic loss in secondary structure and negligible calcium affinity [Anderson et al. (1997) Biochemistry, 36, 11648–11654]. On the contrary, D87N α-LA displayed native-like secondary structure with a somewhat de-stabilized tertiary structure. When the well-documented N-terminal methionine was enzymatically removed from D87N α-LA [Veprintsev et al. (1999) Proteins: Struct. Funct. Genet., 37, 65–72], the structure appeared to more closely resemble native α-LA. Remarkably, the thermal transition mid-temperature of apo-desMetD87N α-LA was ~31°C versus native apo- α-LA (~25°C), probably due to negative charge `compensation\u27 in the calcium co-ordination site. On the other hand, the transition mid-temperature of Ca(II)-bound desMetD87N α-LA was ~57°C versus native α-LA (~66°C), which was related to a decreased Ca(II) affinity (K = ~2.1×105 versus ~1.7×107/M at 40°C, respectively). These results reaffirm that alanine substitution in site specific mutagenesis is not always a prudent choice. Substitutions must be conservative with only minimal changes in functional groups and side-chain volume

Fine Tuning the N-terminus of a Calcium Binding Protein: α-lactalbumin

The effects of amino acid substitutions in the N-terminus of bovine recombinant α-lactalbumin (including enzymatic removal of the N-terminal methionine and deletion of Glu-1) were studied by intrinsic fluorescence, circular dichroism (CD), and differential scanning microcalorimetry (DSC). Wild-type recombinant α-lactalbumin has a lower thermostability and calcium affinity compared to the native protein, while the properties of wild-type protein with the N-terminal methionine enzymatically removed are similar to the native protein. Taken together, the fluorescence, CD, and DSC results show that recombinant wild type α-lactalbumin in the absence of calcium ion is in a type of molten globule state. The delta-E1 mutant, where the Glu1residue of the native sequence is genetically removed, leaving an N-terminal methionine in its place, shows almost one order of magnitude higher affinity for calcium and higher thermostability (both in the absence and presence of calcium) than the native protein isolated from milk. It was concluded that the N-terminus of the protein dramatically affects both stability and function as manifested in calcium affinity. Proteins 1999;37:65–72. © 1999 Wiley-Liss, Inc

Effects of Mutations in The Calcium-binding Sites of Recoverin on Its Calcium Affinity: Evidence for Successive Filling of The Calcium Binding Sites

A molecule of the photoreceptor Ca2+-binding protein recoverin contains four potential EF-hand Ca2+-binding sites, of which only two, the second and the third, are capable of binding calcium ions. We have studied the effects of substitutions in the second, third and fourth EF-hand sites of recoverin on its Ca2+-binding properties and some other characteristics, using intrinsic fluorescence, circular dichroism spectroscopy and differential scanning microcalorimetry. The interaction of the two operating binding sites of wild-type recoverin with calcium increases the protein\u27s thermal stability, but makes the environment around the tryptophan residues more flexible. The amino acid substitution in the EF-hand 3 (E121Q) totally abolishes the high calcium affinity of recoverin, while the mutation in the EF-hand 2 (E85Q) causes only a moderate decrease in calcium binding. Based on this evidence, we suggest that the binding of calcium ions to recoverin is a sequential process with the EF-hand 3 being filled first. Estimation of Ca2+-binding constants according to the sequential binding scheme gave the values 3.7 × 106 and 3.1 × 105 M–1 for third and second EF-hands, respectively. The substitutions in the EF-hand 2 or 3 (or in both the sites simultaneously) do not disturb significantly either tertiary or secondary structure of the apo-protein. Amino acid substitutions, which have been designed to restore the calcium affinity of the EF-hand 4 (G160D, K161E, K162N, D165G and K166Q), increase the calcium capacity and affinity of recoverin but also perturb the protein structure and decrease the thermostability of its apo-form

Point Amino Acid Substitutions in The Ca\u3csup\u3e2+\u3c/sup\u3e-binding Sites of Recoverin: III. A Mutant with The Fourth Reconstructed Ca\u3csup\u3e2+\u3c/sup\u3e-binding Site

Unlike wild type recoverin with only two (the second and the third) functioning Ca+2-binding sites out of four potential ones, the +EF4 mutant contains a third active Ca+2-binding site. This site was reconstructed from the fourth potential Ca+2-binding domain by the introduction of several amino acid substitutions in it by site-directed mutagenesis. The effect of these mutations in the fourth potential Ca+2-binding site of myristoylated recoverin on the structural features and conformational stability of the protein was studied by fluorimetry and circular dichroism. The apoform of the resulting mutant (free of Ca2+ ions) was shown to have a higher calcium capacity, significantly lower thermal stability, and noticeably different secondary and tertiary structures as compared with the apoform of wild-type recoverin

Recoverin Is a Zinc-binding Protein

Recoverin is an N-myristoylated 23 kDa calcium-binding protein from retina, which modulates the Ca2+-sensitive deactivation of rhodopsin via Ca2+-dependent inhibition of rhodopsin kinase. It was shown by intrinsic and bis-ANS probe fluorescence, circular dichroism, and differential scanning calorimetry that myristoylated recombinant recoverin interacts specifically with zinc ions. Similar to the calcium binding, the binding of zinc to Ca2+-loaded recoverin additionally increases its α-helical content, hydrophobic surface area, and environmental mobility/polarity of its tryptophan residues. In contrast to the calcium binding, the binding of zinc decreases thermal stability of the Ca2+-loaded protein. Zn2+-titration of recoverin, traced by bis-ANS fluorescence, reveals binding of a single Zn2+ ion per protein molecule. It was shown that the double-mutant E85Q/E121Q with inactivated Ca2+-binding EF-hands 2 and 3 (Alekseev, A. M.; Shulga-Morskoy, S. V.; Zinchenko, D. V.; Shulga-Morskaya, S. A.; Suchkov, D. V.; Vaganova, S. A.; Senin, I. I.; Zargarov, A. A.; Lipkin, V. M.; Akhtar, M.; Philippov, P. P. FEBS Lett.1998, 440, 116−118), which can be considered as an analogue of the apo-protein, binds Zn2+ ion as well. Apparent zinc equilibrium binding constants evaluated from spectrofluorimetric Zn2+-titrations of the protein are 1.4 × 105 M-1 (dissociation constant 7.1 μM) for Ca2+-loaded wild-type recoverin and 3.3 × 104 M-1 (dissociation constant 30 μM) for the E85Q/E121Q mutant (analogue of apo-recoverin). Study of the binding of wild-type recoverin to ROS membranes showed a zinc-dependent increase of its affinity for the membranes, without regard to calcium content, suggesting further solvation of a protein myristoyl group upon Zn2+ binding. Possible implications of these findings to the functioning of recoverin are discussed