Exploring the chemistry behind protein-glycosaminoglycan conjugate: A steady-state and kinetic spectroscopy based approach.

Abstract

The impact of glycosaminoglycan (chondroitin sulphate, CS) on bone morphogenetic protein - 2 (BMP - 2) structure, stability (thermal and chemical), association kinetics and conformation was monitored by multiple spectroscopic techniques (UV-Visible, fluorescence and circular dichroism). The absorbance in peptide region and fluorescence intensity of BMP - 2 was quenched in presence of CS; thus, confirming the formation of a ground-state complex. As there was an increase in Stern-Volmer constant observed as a function of temperature, idea of dynamic quenching was established. However, the negligible changes in lifetime indicated static quenching; thus, making the process a combination of static-dynamic quenching. Basically, the protein - glycan interaction was driven by entropy of the system and mediated by hydrophobic interactions. Secondary structure (CD spectroscopy) of native protein was significantly affected (intensity became more negative) in presence of CS, thus, introducing more compactness in the protein. CS infused thermal and chemical stability into BMP - 2 via alteration in its conformation. The rate of association was inversely proportional to concentration of quencher (CS), which confirmed the correlation between large size (~ 5 times the size of protein) and structural complexity of CS with fewer binding sites present in BMP - 2. The rate of association in presence of urea, suggested a decrease in association rate as a function of urea concentration for 15μM CS. Experimental evidences suggested an interaction between protein and glycan mediated by hydrophobic interactions, which deciphers structural, thermal and chemical stability into protein.