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

NOLAN MV; CLOP PD; PERILLO MA

Santiago del Estero

Congreso; XLIV Reunión Anual de la Sociedad Argentina de Biofísica (SAB); 2015

Sociedad Argentina de Biofísica

 In previous works we have studiedthe effect of molecular crowding on beta galactosidase (b-Gal) enzymatic activity. The results showed that Vmaxwas just slightly affected, while the affinity of the enzyme (KM)suffered a significant decrease at growing molecular crowding levels. In thepresent work we explored the hypothesis that catalytic effects were related to theenzyme conformation change induced by crowded systems. Thus, the effect ofmolecular crowding on the conformationof b-Gal was studied using FT-IR and analyticalcentrifugation techniques. As in previous works, polyethylene glycol molecularweight 6000 (PEG6000) in a range from 0 to 35 % W/V was used ascrowding agent. The b-Gal FT-IR spectrum showed an important diminution in themain b-structure band (at around 1620 cm-1) when themolecular crowding agent concentration was increased up to 35 % W/V. At thesame time, typical bands corresponding to disordered structures appeared. Theeffect of crowding on b-Gal thermal stability was noticeable and denaturationoccurred in a less cooperative manner. Additionaly, it was found that PEG6000prevented the typical protein aggregation that occurs in thermal denaturedproteins. Through sedimentation velocityapproaches, hydrodynamic information about the size and shape of the proteincould be obtained which also supported amore opened protein conformation in the presence of PEG. Acknowledgements:Workfinanced with grants from CONICET, Foncyt, SeCyT-UNC. MVN and MAP are career membersof CONICET.