Protein Conformation, Dynamics and Function: A Correlation with Shape and Size of Macromolecules

Abstract

Macromolecular crowding, characterized by high concentrations of macromolecules within cellular environments, significantly influences protein dynamics and kinetics, affecting processes such as folding, stability, conformational transitions, and interactions. While factors such as pH and temperature have been extensively studied in relation to protein behaviour, the specific influence of macromolecules on protein folding pathways remains relatively underexplored. In our study, we investigated the model protein Cellular Retinoic Acid Binding Protein I (CRABP I) in the presence of α-, β-, and γ-cyclodextrins using circular dichroism (CD) and fluorescence spectroscopy at 25°C. Our findings demonstrate that the concentration and size of macromolecules have a pronounced impact on protein conformations, either promoting more compact structures or mitigating the effects of crowding. Additionally, we studied the effect of cyclodextrins on the reversibility of unfolded CRABP I using CD and differential scanning calorimeter (DSC), observing that cyclodextrins maximize the reversibility of the protein's unfolded state. This suggests that cyclodextrins can stabilize or facilitate refolding of the protein when it is prone to unfolding or aggregation. These results underscore the importance of understanding macromolecular crowding in the regulation of protein folding in vivo.