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Escherichia coli thioredoxin is normally a cytoplasmic protein involved in the reduction of disulfide bonds. However, thioredoxin can be translocated to the periplasm when it is attached to a cotranslational signal sequence. When exported to the periplasm, it can partially replace the activity of DsbA in promoting the formation of disulfide bonds. In contrast, when thioredoxin is fused to a posttranslational signal sequence, very little of it appears in the periplasm. We propose that this absence of posttranslational export is due to the rapid folding of thioredoxin in the cytoplasm. We sought mutants of thioredoxin that retarded its folding in the cytoplasm, which we accomplished by fusing thioredoxin to a posttranslational signal sequence and selecting for mutants in which thioredoxin was exported to the periplasm, where it could replace DsbA. The collection of mutants obtained represents a limited number of amino acid changes in the protein. In vitro studies on purified mutant proteins show that all but one are defective in the kinetics and thermodynamics of protein folding. We propose that the slower folding of the thioredoxin mutant proteins in the cytoplasm allows their export by a posttranslational pathway. We discuss some implications of this class of mutants for aspects of the folding pathway of thioredoxin and for its mechanism of export. In particular, the finding that a folding mutant that allows protein translocation alters an amino acid at the C terminus of the protein suggests that the degree to which thioredoxin folds during its translation must be severely restricted. in vivo protein folding | protein folding | protein translocation | thioredoxin folding

Research has been conducted on the crystal structure of the tyrosyl-tRNA synthetase C-terminal domain from Bacillus stearothermophilus. The characterization of its conformational state is discussed.

The kinetics of refolding of oxidized Escherichia coli thioredoxin (Trx) in vitro were examined using an active site titration fluorescence spectroscopy and stopped-flow methods combined with circular dichroism and fluorescence spectroscopy. Results showed the existence of early folding intermediates with 'molten globule or pre-molten globule' properties. The formation of secondary structures was indicated by the ellipticity measurements at 222 nm where about 68% of the total change related to refolding occurred during the dead time of the stopped-flow instrument.

Protein recognition is important in most biological phenomena, and complementary protein fragments reassembled into native-like small structures, provide good models to study the interactions. The association/folding process between the complementary N-(1-73) and C-fragment (74-108) of oxidized Escherichia coli thioredoxin (Trx) has been studied, by comparison of the kinetics of formation, using stopped-flow and manual mixing experiments and fluorescence and CD spectroscopy.

Circular dichroism and fluorescence spectroscopy were used to examine the kinetic mechanism involved in the folding and assembly of the alpha and beta subunits of bacterial luciferase. The subunits were found to undergo multiple transitions during refolding. The concentration-dependent formation of their heterodimer complex was also confirmed to be a kinetically preferred process as demonstrated in the in vitro test wherein the two subunits were allowed to interact in a folding-refolding mixture.

Circular dichroism and nuclear magnetic resonance experiments are performed to evaluate the folding properties of the annexin I domain in pure aqueous solution. Results indicate that the folding process observed in the protein is due to helix propensity. This finding suggests that important information about the folding pathway are encoded in the protein sequence of annexin I domain.

Spectroscopic studies on isolated 101 residue long, C-terminal proteolytic domain (F2) of the Escherichia coli tryptophan synthase beta chain reveals that hydrogen bonds stabilize the residues of F2 associated with secondary structures. NMR data reveal the oscillation of F2 between various conformations in rapid equilibrium. Analysis of the kinetics of amide proton exchange by one-dimensional NMR reveals a higher level of proton protection.

High-resolution NMR and circular dichroism spectroscopic studies help examine the conformational properties of four peptides corresponding to alpha-helical regions of Rhodospirillium cytochrome c2 and bovine calcium binding protein. A new computation procedure helps predict the conformations that agree with experimental results. All these peptides that correspond to protein segments have a well-defined alpha-helical conformation at room temperature and at low TFE concentrations. Folding mechanisms of these proteins are discussed.

The folding mechanisms of guanidine-unfolded hen egg white lysozyme having intact disulfides at pH 7.8 and 20 degrees Celsius was probed by analyzing the kinetics of regain in the far- and near-ultraviolet (UV) spectra during renaturation. Results indicate that changes in the far-UV ellipticity during rapid and slow phases reflect alterations in the side chains rather than in the secondary structure of the native lysozyme. Renaturation of the lysozyme to the native configuration completes in less than four minutes.

The kinetics of early folding of isolated F2-V8, a 101-residue polypeptide corresponding to the C-terminal residues of Escherichia coli tryptophan synthase beta chains, was investigated. Stopped-flow kinetic studies on the appearance of the far-ultraviolet circular dichroism signal and on the binding of 1-anilino-8-naphthalenesulfonate during the refolding of guanidine-unfolded F2-V8 showed that the isolated F2-V8 folds into a condensed state with non-native secondary structures in less than four milliseconds. This conformation results from a hydrophobic collapse of the polypeptide chain.

Fibronectin (FN), an extracellular matrix (ECM) protein, is found soluble in corporal fluids or as an insoluble fibrillar component incorporated in the ECM. This phenomenon activates the protein to promote intermolecular interactions with other FN and implicates structural changes that expose FN binding sites.

Results show that the formation of disulfide bridges correctly in vivo is positively influenced by the maltose binding protein, which in addition also assists folding of the passenger proteins. Data demonstrate that the maltose binding protein facilitates the oxidative folding of the disulfide-rich C-terminal fragment of of Plasmodium falciparum merozoite surface protein 1.