Determination of Phosphorylation and Deprotonation Induced Higher Order Structural Transitions in α_s-Caseins

One extremely sensitive and highly successful application of Raman spectroscopy is the structural characterization of proteins. Understanding higher order structure and its effect on protein stability is essential not only for biopharmaceutical and food manufacturing but also for the understanding of diseases that result from the misfolding of proteins including diabetes type II, Alzheimer's, and Parkinson's disease. Due to the large amount of structural information available in Raman spectra, even small alterations in protein conformations including increased exposure of binding regions or changes in geometry of secondary structural elements can be identified. In this study, we demonstrate the unique sensitivity of Raman spectroscopy to subtle structural transitions in an intrinsically open, flexible protein, α_s-casein, in response to phosphorylation and deprotonation. Through the application of 2D correlation analysis two separate transition phases have been identified from pH 6-9 and pH 10-12 for both phosphorylated and dephosphorylated α_s-casein. However, the actual structural changes observed in each pH range differed considerably between the phosphorylated and dephosphorylated α_s-casein. Furthermore, the presence of the phosphorylated serine residues is demonstrated to have a shielding effect during deprotonation of the protein.