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Structural Analysis of Murine Voltage Dependent Anion Channel (VDAC) 1
Author(s) -
Ujwal Rachna,
Ping Peipei,
Zhang Jun,
Mercado Gabriel,
Vondriska Thomas M.,
Kaback H. Ronald,
Abramson Jeff
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.5.lb17-d
Subject(s) - voltage dependent anion channel , vdac1 , bacterial outer membrane , porin , circular dichroism , biochemistry , protein secondary structure , biophysics , protein folding , inclusion bodies , chemistry , biology , microbiology and biotechnology , recombinant dna , escherichia coli , gene
VDAC is a mammalian integral protein expressed in the outer mitochondrial membrane. It has a bi‐functional role in mitochondrial biology: under physiological conditions, it transports nucleotides and small molecules across the outer membrane and under stress, it acts as a non‐selective pore in mediating mitochondrial permeability transition, which leads to cell death. VDAC is thought to be structurally similar to its bacterial homolog, porin; however little is known regarding the structure of VDAC. Because of its critical role in mitochondrial function, we sought to characterize VDAC structure as a first step to understanding its in vivo regulation and interaction with other proteins. N‐terminal His‐tagged murine VDAC1 cDNA was cloned and expressed in E. coli. Bacteria cultures were lysed via sonication and inclusion bodies isolated. After solubilization, the His tagged‐protein was affinity‐purified. Subsequent refolding of VDAC protein was facilitated with 2% lauryldimethylamine‐oxide. The integrity of the secondary structure of this purified and refolded VDAC protein was evaluated using circular dichroism (CD). CD spectra scanning wavelengths in the far‐UV range (λ=200–260nm) indicate that the purified VDAC exhibits classical beta‐barrel secondary structure. Thermal denaturation CD studies demonstrated irreversible unfolding of recombinant VDAC, indicating that the observed secondary structure was the non‐random, legitimate folding state of the molecule. Taken with ongoing crystallization trials to reveal the 3D structure of murine VDAC, these studies have fundamental implications for our understanding of mitochondrial biology and cell death.