Methanococcus vannielii selenium-binding protein (SeBP): chemical reactivity of recombinant SeBP produced in Escherichia coli

Abstract

A selenium-binding protein (SeBP) from Methanococcus vannielii was recently identified, and its gene was isolated and overexpressed in Escherichia coli [Self, W. T., Pierce, R. & Stadtman, T. C. (2004) IUBMB Life 56, 501-507]. SeBP and recombinant SeBP (rSeBP) migrated as approximately 42-kDa species on native gels and as approximately 33-kDa species on SDS gels. rSeBP consists of identical 8.8-kDa subunits, each containing a single cysteine residue. rSeBP isolated in the absence of reducing agents contained oxidized cysteine (89%) and very little bound selenium (0.05 eq or less per subunit). Complete reduction of the oxidized cysteine residues in rSeBP with Tris(2-carboxyethyl)phosphine required addition of a denaturant, such as 1 M guanidine-hydrochloride. With selenite as the selenium source and the isolated reduced protein as sole reductant, binding of one selenium per tetramer under anaerobic conditions required four cysteine thiol groups, one on each subunit. In the corresponding reaction, with reduced glutathione (GSH), equimolar amounts of selenodiglutathione (GSSeSG) and glutathione disulfide are formed from selenite and 4 GSH. At GSH-to-selenite ratios >4:1, conversion of GSSeSG to a perselenide derivative, GSSe(-), occurs. However, with the reduced rSeBP as sole electron donor in the reaction with selenite, further conversion of the R-SSeS-R product apparently did not occur. Prior alkylation of the cysteine thiol groups in reduced rSeBP prevented selenite reduction and selenium binding under comparable conditions.

Fig. 1.

Mobilities of native forms of SeBP from M. vannielii and rSeBP from E. coli. Both proteins were eluted from a rSeBP-specific antibody column (14). (A) Electrophoretic mobilities of SeBP and rSeBP on a native Tris-glycine gel. (B) MALDI-TOF spectra of SeBP from M. vannielii and rSeBP from E. coli.

Fig. 2.

Alkylation of rSeBP. Squares indicate solutions of reduced rSeBP in 50 mM Tricine·KOH/0.5 mM EDTA/1 M Gnd-HCl adjusted to the indicated pH values alkylated with DCIA; the fluorescence was measured as described in Materials and Methods. From the curve (—□—), pK_a of the cysteine residue in rSeBP under these denaturing conditions is ≈6.75. All measurements were made in triplicate, and the standard deviation of the difference is plotted. Circles indicate alkylation of rSeBP with bromo[1-¹⁴C]-acetate at the indicated pH values as described in Materials and Methods.

Fig. 3.

Thermal denaturation data using DSC and light-scattering detection. Left axis indicates units of heat capacity (Cp) for DSC measurements. Solid line is the initial scan from 25°C to 105°C using DSC detection. Dashed line represents the scan from 25°C to 105°C after allowing the original sample to cool slowly (10°C per hr) and keeping it at 25° for 16 hr. Right axis indicates A₃₆₀ light-scattering measurements. Filled circles (•) represent light-scattering data from 25°C to 105°C.

Fig. 4.

Protein structural changes induced by denaturation. (A) CD scans from 260 to 195 nm of 2 mg/ml monomer rSeBP. Fitting the signal with the convex constraint algorithm shows the sample to be ≈60% α-helical, 20% β-sheet, and 20% random coil. (B) Shown is the loss of secondary structure as the concentration of Gnd-HCl increases (0—6 M). Signals decrease from –17°C to –6°C at 220 nm with increasing concentrations of Gnd-HCl. (C) The protein sample after denaturation at the given Gnd-HCl concentration is dialyzed back to nondenaturing conditions for 16 hr. The increase in signal at 220 nm corresponds to lower Gnd-HCl concentrations.

Fig. 5.

Selenium content of rSeBP samples. Oxidized, reduced, and alkylated protein samples were incubated with selenite as described in Materials and Methods. For each condition, there is a control to which no selenite was added. The selenium content of selenite-treated samples is expressed as eq of selenium bound per monomer. In the reaction of reduced protein with selenite, four cysteine thiol groups are consumed: Two are converted to RS-Se-SR, and two are oxidized to RS-SR as in the reaction with GSH (Eq. 1), resulting in one selenium eq bound per tetramer or 0.25 eq per monomer. The selenium content of the enzyme samples not treated with selenite (controls) was <0.02 eq selenium per monomer.