Revealing Advanced Glycation End Products Associated Structural Changes in Serum Albumin.

Structural alterations in proteins have a significant impact on their function and body physiology. Glycation via nonenzymatic forms of cross-linking leads to proteins' conformational changes, the macromolecule being recognized as a stable fibrillary structure, oligomerization, and becoming advanced glycation end products (AGEs). Protein that undergoes glycation-related modifications, namely, β-sheet enriched structural changes, are recognized as amyloid. In the current study, we characterized a single protein modified in vitro under physiological conditions to represent a protein glycation model. The glycation altered the helical conformation of serum albumin (SA) and promoted the formation of a β-sheet enriched with amyloid fibrils detected at multidimensional levels. The nanoscale resolution by spectroscopy in the presence of thioflavin-T (ThT) and 8-anilinonaphthalene-1-sulfonic acid (8-ANS) showed binding of the fibrils formed in the presence of glucose (GLU) and the carbonyl metabolites methylglyoxal (MGO) and glycolaldehyde (GAD). In the presence of MGO and GAD, the SA becomes insoluble aggregates, demonstrated by TEM microscopy and dynamic light scattering (DLS). The protein oligomerization was visualized when separated via SDS gel electrophoresis and mass photometry (MP) assays. Following the glycation, eventually, the material polymerized and became stiffer. The level of stiffness was analyzed by a rheometer that revealed a quick alteration under MGO and GAD. This is the first study to combine multiple spectroscopy assays, imaging, and rheology measurements of SA and to demonstrate a resolution on a nanoscale structural toward better resolution of the conformational changes of glycated SA, oligomerization, and protein aggregations under physiological conditions.