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-HCl 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.