Molecular properties of purified human uncoupling protein 2 refolded from bacterial inclusion bodies.
One way to study low-abundance mammalian mitochondrial carriers is by ectopically expressing them as bacterial inclusion bodies. Problems encountered with this approach include protein refolding, homogeneity, and stability. In this study, we investigated protein refolding and homogeneity properties of inclusion body human uncoupling protein 2 (UCP2). N-methylanthraniloyl-tagged ATP (Mant-ATP) experiments indicated two independent inclusion body UCP2 binding sites with dissociation constants (Kd) of 0.3-0.5 and 23-92 microM. Dimethylanthranilate, the fluorescent tag without nucleotide, bound with a Kd of greater than 100 microM, suggesting that the low affinity site reflected binding of the tag. By direct titration, UCP2 bound [8-(14)C] ATP and [8-(14)C] ADP with Kds of 4-5 and 16-18 microM, respectively. Mg2+ (2 mM) reduced the apparent ATP affinity to 53 microM, an effect entirely explained by chelation of ATP; with Mg2+, Kd using calculated free ATP was 3 microM. A combination of gel filtration, Cu2+-phenanthroline cross-linking, and ultracentrifugation indicated that 75-80% of UCP2 was in a monodisperse, 197 kDa form while the remainder was aggregated. We conclude that (a) Mant-tagged nucleotides are useful fluorescent probes with isolated UCP2 when used with dimethylanthranilate controls; (b) UCP2 binds Mg2+-free nucleotides: the Kd for ATP is about 3-5 microM and for Mant-ATP it is about 10 times lower; and (c) in C12E9 detergent, the monodisperse protein may be in dimeric form.
Jekabsons
Mika B
MB
Medical Research Council, Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge CB2 2XY, United Kingdom. mjekabsons@buckinstitute.org
Echtay
Karim S
KS
Arechaga
Ignacio
I
Brand
Martin D
MD
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D001665:Binding Sites; D004926:Escherichia coli / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D002479:Inclusion Bodies / Q000378:metabolism*; D007473:Ion Channels; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008928:Mitochondria / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D017510:Protein Folding; D012044:Regression Analysis; D000071246:Uncoupling Protein 2; D062367:ortho-Aminobenzoates / Q000737:chemistry
D005456:Fluorescent Dyes; D007473:Ion Channels; D026901:Membrane Transport Proteins; D024101:Mitochondrial Proteins; C000604389:UCP2 protein, human; D000071246:Uncoupling Protein 2; D062367:ortho-Aminobenzoates; C045473:3'-O-(N-methylanthraniloyl) ATP; D000255:Adenosine Triphosphate