Randomization of the Entire Active-site Helix α1 of the Thiol-disulfide Oxidoreductase DsbA from Escherichia coli

Abstract

DsbA from Escherichia coli is the most oxidizing member of the thiol-disulfide oxidoreductase family (E o ‫؍‬ ؊122 mV) and is required for efficient disulfide bond formation in the periplasm. The reactivity of the catalytic disulfide bond (Cys 30 -Pro 31 -His 32 -Cys 33 ) is primarily due to an extremely low pK a value (3.4) of Cys 30 , which is stabilized by the partial positive dipole charge of the active-site helix ␣1 (residues 30 -37). We have randomized all non-cysteine residues of helix ␣1 (residues 31, 32, and 34 -37) and found that two-thirds of the resulting variants complement DsbA deficiency in a dsbA deletion strain. Sequencing of 98 variants revealed a large number of non-conservative replacements in active variants, even at well conserved positions. This indicates that tertiary structure context strongly determines ␣-helical secondary structure formation of the randomized sequence. A subset of active and inactive variants was further characterized. All these variants were more reducing than wild type DsbA, but the redox potentials of active variants did not drop below ؊210 mV. All inactive variants had redox potentials lower than ؊210 mV, although some of the inactive proteins were still re-oxidized by DsbB. This demonstrates that efficient oxidation of substrate polypeptides is the crucial property of DsbA in vivo.

Thioredoxin‐like protein TlpA from Bradyrhizobium japonicum is a reductant for the copper metallochaperone ScoI

“…Redox equilibria were established between ScoI and different reference proteins having a thioredoxin fold, a single active‐site disulfide bond made up of the sequence motif CXXC, and a known redox potential. As reference proteins, we have chosen the periplasmic dithiol oxidase DsbA ( E o ′ −122 mV) [21,23] and a previously characterized thioredoxin variant, Trx DsbA ( E o −204 mV) [20], in which the dipeptide sequence between the active‐site cysteines was replaced by that of DsbA (Pro‐His). Reduced ScoI (ScoI red ) was then incubated at pH 7.0 and 25 °C with oxidized DsbA (DsbA ox ), or oxidized ScoI (ScoI ox ) with reduced Trx DsbA , and the reaction products were separated and quantified by RP‐HPLC after attainment of equilibrium.…”

“…(3)). Redox potentials of the reference proteins DsbA and Trx DsbA (−122 mV and −204 mV, respectively) had been established previously [19–21].…”

“…Redox potentials of the reference proteins DsbA and Trx DsbA (À122 mV and À204 mV, respectively) had been established previously [19][20][21].…”

Conserved Role of the Linker α-Helix of the Bacterial Disulfide Isomerase DsbC in the Avoidance of Misoxidation by DsbB

“…For the purification of DsbA, E. coli HK317 (15) carrying pCH3, a pBAD18 derivative encoding dsbA (15), was grown in NZ medium (NZ amine, 10 g/liter; yeast extract, 5 g/liter; NaCl, 10 g/liter) with 200 g/ml ampicillin, induced with 0.2% w/v arabinose when the A 600 reached 0.05, and the cells were harvested at A 600 ϭ 1.3. DsbA was purified from the periplasmic extract by a HiTrap Q FF anion exchange column (Amersham Biosciences), followed by hydrophobic chromatography on a HiTrap Phenyl HP column (Amersham Biosciences) as described (16).…”

Gas-phase acidities of cysteine-polyglycine peptides: The effect of the cysteine position

“…Interestingly, an amino acid residue located at different positions in a folded protein often exhibits different degrees of acidity or basicity. For example, an acidic residue, such as cysteine or aspartic acid, located at or near the N-terminus of a helix is often more acidic than that at or near the C-terminus [5,[7][8][9][10][11]. A wealth of studies on the acid-base properties of helical peptides have been carried out in condensed phase, in particular in aqueous solutions [11][12][13].…”