Abstract
Two VDAC (voltage-dependent anion-selective channel) isoforms were purified from seed cotyledons of Phaseolus vulgaris by chromatofocusing chromatography. Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy was used to study the structural properties of the two isoforms reconstituted in a mixture of asolectin and 5% stigmasterol. The IR spectra of the two VDAC isoforms were highly similar indicating 50 to 53% anti-parallel beta-sheet. The orientation of the beta-strands relative to the barrel axis was calculated from the experimentally obtained dichroic ratios of the amide I beta-sheet component and the amide II band. Comparing the IR spectra of the reconstituted VDAC isoforms with the IR spectra of the bacterial porin OmpF, for which a high resolution structure is available, provided evidence for a general structural organization of the VDAC isoforms similar to that of bacterial porins. Hydrogen-deuterium exchange measurements indicated that the exchange of the amide protons occurs to a higher extent in the two VDAC isoforms than in the OmpF porin.
Highlights
Two voltage-dependent anion-selective channel (VDAC) isoforms were purified from seed cotyledons of Phaseolus vulgaris by chromatofocusing chromatography
It seems reasonable to assume that the multitude of functional properties that are attributed to VDAC could arise from the presence of different VDAC isoforms in mitochondria
Two VDAC isoforms were purified from kidney bean cotyledons in a single purification step using chromatofocusing chromatography as previously reported [22]
Summary
Bacterial porin was diluted in 2 ml of 1 mM Hepes, 0.1% (v/v) Genapol, pH 7.2, to a similar concentration as were the VDAC proteins and reconstitution was carried out as above described for VDAC. The protein spectra were recorded with parallel and perpendicular polarized light with respect to the plane of incidence, averaging 512 scans at a resolution of 2 cmϪ1. To determine the dichroic ratio of the different secondary structure components, the amide I band (1700 to 1600 cmϪ1) was decomposed by a least-square curve-fitting procedure using a Cauchy (Lorenzian/Gaussian) function as described previously [25]. The dichroic ratio of the lipid (CϭO), called below RATRiso, was obtained from the parallel and perpendicular polarized spectra. To account for varying absorbance intensities in sample and lipid spectra, a subtraction coefficient was used to cancel the lipid (CϭO) area integrated between 1705 and 1770 cmϪ1
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