Maintenance and thermal stabilization of NADH dehydrogenase-2 conformation upon elimination of its C-terminal region

VILLEGAS, J. M.; TORRES-BUGEAU, C. M.; CHEHÍN, R.; BURGOS, M. I.; FIDELIO, G. D.; RINTOUL, M. R.; RAPISARDA, V. A.

San Javier, Tucumán

Congreso; SAB 2012: XLI Reunión Anual de la Sociedad Argentina de Biofísica; 2012

Respiratory NADH dehydrogenase-2 (NDH-2) of Escherichia coli is a peripheral membrane-bound flavoprotein, belonging to a group of enzymes with scarce structural information. By eliminating the C-terminal region of NDH-2, a water soluble version with significant enzymatic activity was previously obtained. Here, structural features of the truncated and wild type NDH-2 were studied by different techniques, such as circular dichroism (CD), Fourier transform infrared spectroscopy (FTIR), fluorescence spectroscopy and limited proteolysis. Results showed that the elimination of C-terminal region did not affect NDH-2 globularity, since the overall structure of both proteins was similar at 30°C. Experimental data agree with the predicted NDH-2 structure (PDB:1OZK). Both enzymes suffered functional/conformational changes with increasing temperatures, including loss of enzymatic activity, alteration of tertiary structure, loss of some secondary structure elements and unfolding with aggregation. The midpoint transition temperatures detected were approximately 5?10°C higher for the truncated version than for NDH-2, indicating that the absence of C-terminal region stabilized the truncated protein conformation. However, truncation impaired enzymatic activity at low temperatures, probably due to the weak interaction of the mutant protein with FAD cofactor. A broad knowledge about NDH-2 enzymes could promote their potential applications in medical science and biotechnology, being the truncated protein a suitable tool for further structural characterizations.