An FTIR study of the complex melting behavior of α-lactalbumin

We have studied the thermal denaturation of holo α-lactalbumin (α-LA) using FTIR spectroscopy by monitoring the absorbance of protein bands in the secondary structure sensitive amide I region. In agreement with results obtained by other techniques, a major cooperative melting transition is observed between 60 and 80°C. This main transition is found to involve predominantly the unfolding of helical structures. Addition of excess Ca²⁺ ions raises the T_m of the cooperative melting transition but does not change the protein substructures involved in the transition. In addition to the main transition, there are at least two other distinct melting processes. One of them is gradual, is independent of excess Ca²⁺, and occurs throughout the measured temperature range below the major cooperative transition. This melting process involves denaturation of α-LA's β-hairpin. The other melting process is affected by the addition of excess Ca²⁺ and involves the denaturation of helical structures. In the absence of excess calcium, this transition, occurring between 45 and 60°C, strongly overlaps with the major cooperative melting. Our results indicate that the β-hairpin of α-LA is not directly stabilized by the bound calcium ion present in the holo protein and melts independently from the rest of the protein.