Thermal modulation of intrinsically disordered and globular proteins: A multi-modal spectroscopic and microscopic study

Understanding the thermal responsiveness of milk proteins is crucial for predicting their functional behavior during processing. In the current study, the temperature-dependent behavior of β-casein (β-CN) and β-lactoglobulin (β-Lg) was systematically examined at 50, 60, and 70 °C using complementary spectroscopic and microscopic techniques. Intrinsic fluorescence, ultraviolet–visible absorbance, 8-anilino-1-naphthalenesulfonic acid (ANS), Thioflavin T (ThT), and Congo red assays revealed distinct thermal response patterns. β-CN exhibited a biphasic response, with increased fluorescence intensity and probe accessibility at moderate temperatures followed by reduced signals at 70 °C, consistent with temperature-dependent changes in residue exposure and aggregate compaction that limit dye accessibility. In contrast, β-Lg showed progressive increases in intrinsic fluorescence, hydrophobic exposure, and dye binding across the investigated temperature range, reflecting continuous conformational loosening and enhanced intermolecular association. Fourier transform infrared spectroscopy showed only modest changes in the amide I–III regions for both proteins, including slight band shifts, reduced intensity, and broadening, indicative of subtle, local conformational rearrangements and increased structural heterogeneity rather than quantitative or definitive secondary-structure transitions. X-ray powder diffraction patterns displayed broad halos with minor intensity variations, suggesting limited changes in local packing without the emergence of long-range ordered structures. Scanning electron microscopy, fluorescence microscopy, atomic force microscopy, and dynamic light scattering consistently demonstrated temperature-dependent increases in aggregate size, surface roughness, and morphological heterogeneity, providing morphological support for aggregation behavior consistent with amyloid-like or β-sheet–enriched features. Overall, these findings delineate distinct thermal aggregation pathways in intrinsically disordered and globular milk proteins while avoiding definitive structural assignments.