Effect of molecular crowding on the conformation of β-Gal: An FT-IR and analytical ultracentrifugation study.

VERÓNICA M. NOLAN; PEDRO D. CLOP; MARIA A. PERILLO

Santiago del Estero

Congreso; XLIV Reunión Anual SAB 2015; 2015

Sociedad Aregentina de Biof{isica

In previous works we have studied the effect of molecular crowding on beta galactosidase (β-Gal) enzymatic activity. The results showed that Vmax was just slightly affected, while the affinity of the enzyme (KM) suffered a significant decrease at growing molecular crowding levels. In the present work we explored the hypothesis that catalytic effects were related to the enzyme conformation change induced by crowded systems. Thus, the effect of molecular crowding on the conformation of β-Gal was studied using FT-IR and analytical centrifugation techniques. As in previous works, polyethylene glycol molecular weight 6000 (PEG6000) in a range from 0 to 35 % W/V was used as crowding agent. The β-Gal FT-IR spectrum showed an important diminution in the main β-structure band (at around 1620 cm-1) when the molecular crowding agent concentration was increased up to 35 % W/V. At the same time, typical bands corresponding to disordered structures appeared. The effect of crowding on β-Gal thermal stability was noticeable and denaturation occurred in a less cooperative manner. Additionaly, it was found that PEG6000 prevented the typical protein aggregation that occurs in thermal denatured proteins. Through sedimentation velocity approaches, hydrodynamic information about the size and shape of the protein could be obtained which also supported a more opened protein conformation in the presence of PEG.