title: Maintenance and thermal stabilization of NADH dehydrogenase-2 conformation upon elimination of its C-terminal region.
abstract: Development of an artificial enzyme with activity and structure comparable to that of natural enzymes is an important goal in biological chemistry. 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, NDH-2 structural features were established, in comparison to those of the truncated version. Far-UV circular dichroism, Fourier transform infrared spectroscopy and limited proteolysis analysis showed that the overall structure of both proteins was similar at 30 °C. Experimental data agree with the predicted NDH-2 structure (PDB: 1OZK). The absence of C-terminal region stabilized in ∼5-10 °C 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.
authors.affiliation: Instituto Superior de Investigaciones Biológicas (Consejo Nacional de Investigaciones Científicas y Técnicas-Universidad Nacional de Tucumán), and Instituto de Química Biológica Dr Bernabé Bloj (Universidad Nacional de Tucumán), Chacabuco 461, San Miguel de Tucumán T4000ILI, Argentina.
authors.forename: Josefina María
authors.initials: JM
authors.lastname: Villegas
authors.forename: Clarisa María
authors.initials: CM
authors.lastname: Torres-Bugeau
authors.forename: Rosana
authors.initials: R
authors.lastname: Chehín
authors.forename: Martha Inés
authors.initials: MI
authors.lastname: Burgos
authors.forename: Gerardo Daniel
authors.initials: GD
authors.lastname: Fidelio
authors.forename: María Regina
authors.initials: MR
authors.lastname: Rintoul
authors.forename: Viviana Andrea
authors.initials: VA
authors.lastname: Rapisarda
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000737:chemistry / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry; D007700:Kinetics; D009245:NADH Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017550:Spectroscopy, Fourier Transform Infrared; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; C088908:NADH dehydrogenase II; D009245:NADH Dehydrogenase