Effect of molecular crowding on the conformation and thermal stability of _beta-Gal.

NOLAN MV; PERILLO MA

Villa Carlos Paz

Congreso; XLII Reunión Anual de la Sociedad Argentina de Biofísica; 2013

Sociedad Argentina de Biofísica

In previous works we studied the effect of molecular crowding on beta galactosidase (b-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 that opportunity we also found, as a first approach to the study of the enzyme conformation in crowded systems that b-Gal fluorescence spectrum suffered a red shift when the crowding agent concentration was increased from 0 to 35 % W/V. In the present work, studies on the conformation and thermal stability were carried out using both infrared (IR) and fluorescence spectroscopy were done to investigate possible changes on the enzyme structure due to overcrowding using polyethylene glycol molecular weight 6000 (PEG6000) was used as crowding agent. The b-Gal IR spectrum showed an important diminution in the main b-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. On the other hand, the effect of crowded systems on b-Gal thermal stability was noticeable, prevented the typical protein aggregation that occurs in proteins denatured by temperature and denaturation occurred in a less cooperative way. Taken together our results showed that in crowded conditions b-Gal is thermodynamically trapped in a conformation more open, flexible and stable than in dilute solutions.