Label-free visualization of unfolding and crosslinking mediated protein aggregation in nonenzymatically glycated proteins

• Published in: Analyst (London) Vol. 149; no. 15; pp. 429 - 44
• Main Authors: Mukunda, Darshan Chikkanayakanahalli, Basha, Shaik, D'Souza, Meagan Gail, Chandra, Subhash, Ameera, K, Stanley, Weena, Mazumder, Nirmal, Mahato, Krishna Kishore
• Format: Journal Article
• Language: English
• Published: London Royal Society of Chemistry 22.07.2024
• Subjects: Crosslinking; Fluorescence; Imaging; Labels; Proteins; Spectroscopic analysis
• ISSN: 0003-2654; 1364-5528; 1364-5528
• DOI: 10.1039/d4an00358f

Nonenzymatic glycation (NEG) unfolds and crosslinks proteins, resulting in aggregation. Label-free evaluation of such structural changes, without disturbing molecular integrity, would be beneficial for understanding the fundamental mechanisms of protein aggregation. The current study demonstrates the assessment of NEG-induced protein aggregation by combining autofluorescence (AF) spectroscopy and imaging. The methylglyoxal (MG) induced protein unfolding and the formation of cross-linking advanced glycation end-products (AGEs) leading to aggregation were evaluated using deep-UV-induced-autofluorescence (dUV-AF) spectroscopy in proteins with distinct structural characteristics. Since the AGEs formed on proteins are fluorescent, the study demonstrated the possibility of autofluorescence imaging of NEG-induced protein aggregates. Autofluorescence spectroscopy can potentially reveal molecular alterations such as protein unfolding and cross-linking. In contrast, AGE-based autofluorescence imaging offers a means to visually explore the structural arrangement of aggregates, regardless of whether they are amyloid or non-amyloid in nature. Probing the nonenzymatic glycation induced protein aggregation by autofluorescence spectroscopy and microscopy.