Partly folded states of members of the lysozyme/lactalbumin superfamily: a comparative study by circular dichroism spectroscopy and limited proteolysis

Abstract

The partly folded states of protein members of the lysozyme (LYS)/alpha-lactalbumin (LA) superfamily have been analyzed by circular dichroism (CD) measurements and limited proteolysis experiments. Hen, horse, dog, and pigeon LYSs and bovine LA were used in the present study. These are related proteins of 123- to 129-amino-acid residues with similar three-dimensional structures but low similarity in amino acid sequences. Moreover, notable differences among them reside in their calcium-binding properties and capability to adopt partly folded states or molten globules in acid solution (A-state) or on depletion of calcium at neutral pH (apo-state). Far- and near-UV CD measurements revealed that although the structures of hen and dog LYS are rather stable in acid at pH 2.0 or at neutral pH in the absence of calcium, conformational transitions to various extents occur with all other LYS/LA proteins herewith investigated. The most significant perturbation of tertiary structure in acid was observed with bovine LA and LYS from horse milk and pigeon egg-white. Pepsin and proteinase K were used as proteolytic probes, because these proteases show broad substrate specificity, and therefore, their sites of proteolysis are dictated not by the specific amino acid sequence of the protein substrate but by its overall structure and dynamics. Although hen LYS at pH 2.0 was fully resistant to proteolysis by pepsin, the other members of the LYS/LA superfamily were cleaved at different rates at few sites of the polypeptide chain and thus producing rather large protein fragments. The apo-form of bovine LA, horse LYS, and pigeon LYS were attacked by proteinase K at pH 8.3, whereas dog and hen LYSs were resistant to proteolysis when reacted under identical experimental conditions. Briefly, it has been found that the proteolysis data correlate well with the extent of conformational transitions inferred from CD spectra and with existing structural informations regarding the proteins herewith investigated, mainly derived from NMR and hydrogen exchange measurements. The sites of initial proteolytic cleavages in the LYS variants occur at the level of the beta-subdomain (approximately chain region 34-57), in analogy to those observed with bovine LA. Proteolysis data are in agreement with the current view that the molten globule of the LYS/LA proteins is characterized by a structured alpha-domain and a largely disrupted beta-subdomain. Our results underscore the utility of the limited proteolysis approach for analyzing structure and dynamics of proteins, even if adopting an ensemble of dynamic states as in the molten globule.

Fig. 1.

(Top) Schematic view of the native structures of bovine α-lactalbumin (LA) and hen egg-white lysozyme (LYS). The diagrams were drawn using the PDB files 1hfz and 1jpo for bovine LA and hen LYS, respectively, using the program PREPI (Molecular Simulations Inc.) on a Silicon Graphics Iris Indigo Workstation. The β-subdomain in both proteins is composed by the β-pleated sheets colored in red; the α-helices are colored in blue; and the disulfide bridges are represented by yellow sticks. In the case of bovine LA, the connectivities of the four disulfide bridges along the 123-residue chain of the protein are 6–120, 28–111, 61–77, and 73–91. In the LA structure, the calcium atom is shown by a solid sphere in green. (Bottom) Alignment of amino acid sequences of LYS from horse (McKenzie and Shaw 1985), dog (Grobler et al. 1994), pigeon (Menéndez-Arias et al. 1985), and hen egg-white (Canfield 1963) and bovine LA (Hurley and Schuler 1987). Dashes indicate deletions relative to the other sequences. Conserved residues are highlighted in orange and labeled by a asterisk.

Fig. 2.

Far-UV (A) and near-UV (B) circular dichroism (CD) spectra of bovine α-lactalbumin (LA) and hen lysozyme (LYS). Spectra were obtained at 20°–22°C in 0.01 M Tris/0.1 M KCl (pH 7.5), containing 5 mM CaCl₂ (continuous lines) or 1 mM EDTA (dashed lines) and in 0.01 M HCl/0.1 M KCl (pH 2.0; dotted lines). In the case of bovine LA, near-UV CD spectra were recorded also in Tris/EDTA buffer(pH 7.5) at 4° and 37°C (dashed lines). The far- and near-UV CD spectra of hen LYS at pH 7.5 in the presence of 1 mM EDTA (data not shown) were identical to those recorded in Tris buffer only.

Fig. 3.

Far-UV (A) and near-UV (B) circular dichroism (CD) spectra of pigeon (left), horse (middle), and dog (right) lysozyme (LYS). Spectra were obtained at 20°–22°C in 0.01 M Tris/0.1 M KCl (pH 7.5), containing 5 mM CaCl₂ (continuous lines), in 0.01 M HCl/0.1 M KCl (pH 2.0; dotted lines), and in 10 mM Tris/0.1 M KCl (pH 7.5) containing 1 mM EDTA (dashed lines).

Fig. 4.

SDS-PAGE of the proteolytic digestion of hen, horse, dog, and pigeon lysozyme (LYS) and bovine α-lactalbumin (LA) by pepsin at pH 2.0. The proteolysis of bovine LA and hen LYS (top) and of horse, pigeon, and dog LYS (bottom) was conducted at 20°–22°C (enzyme-to-substrate ration [E/S], 1:500 by weight). Samples were taken from the reaction mixtures at the indicated times and stopped by alkalinization. LA and LYS refer to protein samples dissolved in acid solution without pepsin. The time-course digestion of LA and LYS polypeptide chain by pepsin was followed by SDS-PAGE under reducing conditions using the Tricine buffer system (Schägger and von Jagow 1987); under these conditions inter- and intramolecular disulfide bridges are broken, and protein fragments migrate in the gel as individual polypeptide chains. Numbers in the right part of the Coomassie-stained gel refer to the identity of the protein fragments produced during proteolysis.

Fig. 5.

SDS-PAGE of the proteolytic digestion of hen, horse, dog, and pigeon lysozyme (LYS) and bovine α-lactalbumin (LA) by proteinase K (at pH 8.3) in the presence of EDTA. The proteolysis of bovine LA and hen LYS (top) and of horse, pigeon, and dog LYS (bottom) was conducted at 20°–22°C (enzyme-to-substrate ration [E/S], 1:500 by weight). Samples were taken from the reaction mixtures at the indicated times, mixed with aqueous TFA in order to stop the proteolysis, and then mixed with the SDS-PAGE sample buffer (Schägger and von Jagow 1987). LA and LYS refer to protein samples incubated under identical solvent conditions without added protease. Numbers in the right part of the Coomassie-stained gel refer to the identity of the protein fragments produced during proteolysis.

Fig. 6.

(Top) Amino acid sequences of the polypeptide chain regions of members of the lysozyme (LYS)/α-lactalbumin (LA) superfamily encompassing the β-subdomain. The proteins were subjected to proteolysis in their partly folded states produced in acid or on depletion of the protein-bound calcium by EDTA at neutral pH (see text). Only the amino acid sequences of the chain region where initial proteolytic cleavages occur are shown. Closed and open arrowheads indicate sites of proteolysis by pepsin and proteinase K, respectively, and the bicolored arrowhead indicates cleavage by both pepsin and proteinase K. (Bottom) Secondary structure elements in the native structure of bovine LA (Pike et al. 1996). The four α-helices (H1 to H4) of the 123-residue chain are indicated by major boxes, and below them, the corresponding chain segments are given. The short 3₁₀-helices (h1b, 18–20; h2, 77–80; h3c, 115–118) are also shown by small boxes. The β-strands (S1, 41–44; S2, 47–50; S3, 55–56) of the β-subdomain are also shown by small boxes. (See Fig. 1 ▶ for a three-dimensional structure of bovine LA; note that in the computer-generated figure of α-LA, the short S3 β-sheet is not shown.) At variance from LA derived from other sources, the chain segment encompassing helix H4 (residues 105–110) in the crystal structure of bovine LA exhibits a variety of distinct conformers, including a distorted α-helical conformation (Pike et al. 1996).