α-Lactalbumin, Amazing Calcium-Binding Protein

Abstract

α-Lactalbumin (α-LA) is a small (Mr 14,200), acidic (pI 4-5), Ca²⁺-binding protein. α-LA is a regulatory component of lactose synthase enzyme system functioning in the lactating mammary gland. The protein possesses a single strong Ca²⁺-binding site, which can also bind Mg²⁺, Mn²⁺, Na⁺, K⁺, and some other metal cations. It contains several distinct Zn²⁺-binding sites. Physical properties of α-LA strongly depend on the occupation of its metal binding sites by metal ions. In the absence of bound metal ions, α-LA is in the molten globule-like state. The binding of metal ions, and especially of Ca²⁺, increases stability of α-LA against the action of heat, various denaturing agents and proteases, while the binding of Zn²⁺ to the Ca²⁺-loaded protein decreases its stability and causes its aggregation. At pH 2, the protein is in the classical molten globule state. α-LA can associate with membranes at neutral or slightly acidic pH at physiological temperatures. Depending on external conditions, α-LA can form amyloid fibrils, amorphous aggregates, nanoparticles, and nanotubes. Some of these aggregated states of α-LA can be used in practical applications such as drug delivery to tissues and organs. α-LA and some of its fragments possess bactericidal and antiviral activities. Complexes of partially unfolded α-LA with oleic acid are cytotoxic to various tumor and bacterial cells. α-LA in the cytotoxic complexes plays a role of a delivery carrier of cytotoxic fatty acid molecules into tumor and bacterial cells across the cell membrane. Perhaps in the future the complexes of α-LA with oleic acid will be used for development of new anti-cancer drugs.

Keywords: amyloid fibrils; cytotoxicity; folding; liprotides; metal binding; molten globule; nanoparticles; nanotubes; structure; unfolding; α-lactalbumin.

Figure 1

X-ray structure of Ca²⁺-bound human α-lactalbumin (PDB ID 1a4v). α-Helices are shown in blue; β-structure is shown in green; S-S bridges are shown in yellow. Gray sphere represents bound calcium ion, and amino acid residues taking part in its coordination are represented by stick model.

Figure 2

X-ray structure of the primary and secondary Ca^2+-binding sites in human α-lactalbumin (PDB ID: 1a4v). Gray spheres represent calcium ions bound in the strong primary (Ca₁)- and the weak secondary (Ca₂)-binding sites.

Figure 3

Heat sorption curves for apo-(1 mM EGTA) and Ca²⁺-loaded (1 mM CaCl₂) bovine α-lactalbumin derived from differential scanning calorimetry measurements. 10 mM HEPES, pH 8.

Scheme 1

The equilibrium scheme of the binding of one metal ion to the protein molecule with a single binding site, taking into consideration equilibria between the native (P, PMe) and thermally changed (P*, P*Me) states of the protein.

Scheme 2

The equilibrium scheme of the competitive binding of Ca²⁺ and Mg²⁺ ions to a protein molecule with a single binding site, taking into consideration equilibria between the native (PCa, PMg) and thermally changed (P*Ca, P*Mg) states of the protein.

Figure 4

Three-dimensional phase diagram for bovine α-lactalbumin in free calcium (pCa = −log(Ca²⁺))—temperature coordinates in the presence of competing Mg²⁺ ions. Normalized contributions of various protein states (equilibrium Scheme 2): PCa state—green; PMg state—light blue; P*Ca state—yellow; P*Mg state—gray; P state—dark blue; P* state—red.

Scheme 3

The scheme of a transition of a protein from an unfolded to the native state with the participation of an intermediate state.

Figure 5

Temperature dependence of specific heat capacity of human α-lactalbumin (α-LA) in the complex with poly-L-Lys (n = 119), estimated from scanning microcalorimetry data (pH 7.8, 10 mM HEPES-KOH). Concentration of calcium ions was controlled by addition of either 1 mM CaCl₂ (Ca²⁺) or 1 mM EDTA (apo). Concentration of α-LA was 1 mg/mL. Poly-L-Lys was added in equimolar concentration.

Figure 6

Viability of human larynx carcinoma cells in vitro plotted vs. concentration of oleic acid (OA) within the sample. The protein-OA complexes were prepared at 45 °C under alkaline Ca²⁺-free conditions (pH 12.0, 1 mM EDTA). bLG-OA-45 (bovine β-lactoglobulin, open circles), pPA-OA-45 (pike parvalbumin, open triangles), bLA-OA-45 (bovine α-LA, open diamonds), human α-lactalbumin made lethal to tumor cells (HAMLET) (solid squares), OA (oleic acid, solid diamonds).