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