02710nas a2200241   4500008004100000022001400041245011900055210006900174260010300243300001400346490000700360520177700367653003802144653001402182653001902196653001102215100002302226700002202249700002402271700002502295700001902320856012902339       2013                        eng d  a0300-908400aHuman mitochondrial NDUFS3 protein bearing leigh syndrome mutation is more prone to aggregation than its wild-type0 aHuman mitochondrial NDUFS3 protein bearing leigh syndrome mutati  a23 RUE LINOIS, 75724 PARIS, FRANCEbELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIERcDEC  a2392-24030 v953 a<p>NDUFS3 is an integral subunit of the Q module of the mitochondrial respiratory Complex-I. The combined mutation (T145I + R199W) in the subunit is reported to cause optic atrophy and Leigh syndrome accompanied by severe Complex-I deficiency. In the present study, we have cloned and overexpressed the human NDUFS3 subunit and its double mutant in a soluble form in Escherichia coli. The wild-type (w-t) and mutant proteins were purified to homogeneity through a serial two-step chromatographic purification procedure of anion exchange followed by size exclusion chromatography. The integrity and purity of the purified proteins was confirmed by Western blot analysis and MALDI-TOF/TOF. The conformational transitions of the purified subunits were studied through steady state as well as time resolved fluorescence and CD spectroscopy under various denaturing conditions. The mutant protein showed altered polarity around tryptophan residues, changed quenching parameters and also noticeably altered secondary and tertiary structure compared to the w-t protein. Mutant also exhibited a higher tendency than the w-t protein for aggregation which was examined using fluorescent (Thioflavin-T) and spectroscopic (Congo red) dye binding techniques. The pH stability of the w-t and mutant proteins varied at extreme acidic pH and the molten globule like structure of w-t at pH1 was absent in case of the mutant protein. Both the w-t and mutant proteins showed multi-step thermal and Gdn-HCI induced unfolding. Thus, the results provide insight into the alterations of NDUFS3 protein structure caused by the mutations, affecting the overall integrity of the protein and finally leading to disruption of Complex-I assembly. (C) 2013 Elsevier Masson SAS. All rights reserved.</p>10aAggregation Fluorescence lifetime10aComplex-I10aLeigh syndrome10aNDUFS31 aJaokar, Tulika, M.1 aPatil, Deepak, P.1 aShouche, Yogesh, S.1 aGaikwad, Sushama, M.1 aSuresh, C., G.  uhttps://library.ncl.res.in/content/human-mitochondrial-ndufs3-protein-bearing-leigh-syndrome-mutation-more-prone-aggregation