Cyclodextrin: A spectroscopic and in-silico investigation as a size-dependent cytoplasmic protein stabilizer

Understanding the effect of the surrounding environment of the cytoplasm on protein stability gives insight into developing successful drug-delivery vehicles and biopharmaceutical formulations. Here we investigate the interaction of a small, monomeric cytoplasmic protein, Cellular retinoic acid binding protein I (CRABPI), with natural polysaccharides, cyclodextrins (α-, β-, and γ-CD) of different sizes. The characteristic toroidal shape with a hydrophobic cavity allows interaction with the protein surface through host-guest interactions. Using different multi-dimensional approaches, spectroscopic analyses, and in silico studies gives an impactful observation on structural and conformational stability. The output highlights that γ-cyclodextrin has the highest binding affinity and stabilization effect. Enhancement of fluorescence intensity, lifetime, and secondary contents of protein with a decrease in structural fluctuations in in silico study strongly supports the outcome. β-CD shows moderate stabilizing behaviour, while α-CD engages primarily in superficial hydrogen bonding with minimal impact on protein conformation. It is significant to highlight that the large cavity size of γ-CD encapsulates the surface amino acids of CRABP I, supporting localized structural rigidification without disturbing the global fold of the protein. These results establish a clear structure-function relationship among the CDs and highlight their potential as targeted molecular stabilizers. Our results underscore the importance of γ-cyclodextrin as a promising molecular chaperone in protein delivery and stabilization strategies, thereby extending cyclodextrin applications beyond drug encapsulation towards potential roles in structural stabilization of cytoplasmic proteins (like CRABPI).