Dissimilarity in the reductive unfolding pathways of two ribonuclease homologues.

Using DTT(red) as the reducing agent, the kinetics of the reductive unfolding of onconase, a frog ribonuclease, has been examined. An intermediate containing three disulfides, Ir, that is formed rapidly in the reductive pathway, is more resistant to further reduction than the parent molecule, indicating that the remaining disulfides in onconase are less accessible to DTT(red). Disulfide-bond mapping of Ir indicated that it is a single species lacking the (30-75) disulfide bond.

Dissociation of the dimeric SecA ATPase during protein translocation across the bacterial membrane.

The ATPase SecA mediates post-translational translocation of precursor proteins through the SecYEG channel of the bacterial inner membrane. We show that SecA, up to now considered to be a stable dimer, is actually in equilibrium with a small fraction of monomers. In the presence of membranes containing acidic phospholipids or in certain detergents, SecA completely dissociates into monomers.

Role of individual disulfide bonds in hen lysozyme early folding steps.

To probe the role of individual disulfide bonds in the folding kinetics of hen lysozyme, the variants with two mutations, C30A,C115A, C64A,C80A, and C76A,C94A, were constructed. The corresponding proteins, each lacking one disulfide bond, were produced in Escherichia coli as inclusion bodies and solubilized, purified, and renatured/oxidized using original protocols. Their enzymatic, spectral, and hydrodynamic characteristics confirmed that their conformations were very similar to that of native wild-type (WT) lysozyme.