pH-induced structural transitions of caseins

Caseins are relatively small (molecular mass ∼20 kDa), unstructured milk proteins of which the main components are referred to as αs-, β- and κ-casein. All three components lack a compact folded conformation, which can be ascribed to a combination of their low overall hydrophobicity and high net charge. Structural transitions of the three caseins in response to variation of pH were investigated using fluorescence and circular dichroism (CD) spectroscopy. Tryptophan emission parameters (intensity and wavelength of emission maximum) and CD spectra showed that at neutral and alkaline pH the caseins exist predominantly in random coil conformation. As the solvent is made acidic the added protons compensate the negative charges on the caseins and reduce the repulsion between like charged residues, allowing the casein chains to fold. At the pI (pH 4–5), the net charge on the protein tends to zero and the protein should approach its maximally structured state. Below pI, the uncompensated charges and their interactions reappear, resulting in slackening of the compact structure and formation of a partially unfolded intermediate. These conclusions were borne out by the biphasic pH-dependence of the fluorescence emission parameters of Trp as well as of ANS incubated with the caseins. Measurement of the efficiency of energy transfer between Trp (donor) and ANS (acceptor) and of the CD spectra of caseins as functions of pH were also consistent with this scenario.