The kinetics of refolding of Escherichia coli dihydrofolate reductase (EC 1.5.1.3) have been examined upon dilution of unfolded enzyme in 4.5 M urea to 1.29 M urea in 0.02 M phosphate buffer (pH 7.2) at 10 degrees C. Changes in the intrinsic protein fluorescence on refolding are characterized by four phases. Based on changes in the amplitudes of these phases, as a consequence of quenching of th...

This paper uses capillary electrophoresis to follow a globular metalloprotein--bovine carbonic anhydrase II (BCA, EC 4.2.1.1)--on unfolding upon treatment with sodium dodecyl sulfate (SDS) and refolding upon removal of SDS, both in the presence and the absence of its Zn(II) cofactor. This research demonstrates that the Zn(II) cofactor is not required for refolding into a nativelike conformation...

Acidic fibroblast growth factor (aFGF) is unstable at physiological temperatures in the absence of polyanions such as heparin. Therefore, the effect of temperature on the kinetics of refolding of aFGF has been examined in the presence and absence of several polyanions. The protein folds into its native state at temperatures up to 30 degrees C without polyanions with an activation energy of appr...

The PEB4 protein is an antigenic virulence factor implicated in host cell adhesion, invasion, and colonization in the food-borne pathogen Campylobacter jejuni. peb4 mutants have defects in outer membrane protein assembly and PEB4 is thought to act as a periplasmic chaperone. The crystallographic structure of PEB4 at 2.2-Å resolution reveals a dimer with distinct SurA-like chaperone and peptidyl...

The mitochondrial isozyme of aspartate aminotransferase (mAspAT), a dimeric pyridoxal phosphate (PLP)-dependent enzyme, is encoded by the nuclear genome and synthesized in the cytoplasm as a precursor protein (pmAspAT) containing a 29-residue amino-terminal signal peptide which is essential for its targeting and import into mitochondria. In the cytosolic-like environment of rabbit reticulocyte ...

The alpha-crystallins are members of the small heat shock protein family of molecular chaperones that have evolved to minimize intracellular protein aggregation; however, they are also implicated in a number of protein deposition diseases. In this study, we employed novel mass spectrometry techniques to investigate the changes in quaternary structure associated with this switch from chaperone t...

Because efficient folding in vivo and reconstitution in vitro of phage P22 tailspike protein is temperature-sensitive, and because a chaperone function of the GroE proteins for tailspike folding in vivo has been suggested by genetic observations, the interactions of purified Escherichia coli GroE proteins with phage P22 tailspikes during refolding in vitro were investigated. At elevated tempera...

The photoreceptor photoactive yellow protein (PYP) was used as a model system to study receptor activation and protein folding. Refolding was studied by stopped-flow absorbance spectroscopy for PYP with either a trans or a cis chromophore. Chromophore trans to cis isomerization, the mechanism of light detection by PYP, greatly affects the protein folding process. When the cis chromophore is pre...

Tryptophan was substituted for Tyr92 to create a sensitive and unique optical probe in order to study the unfolding and refolding kinetics of disulfide-intact bovine pancreatic ribonuclease A by fluorescence-detected stopped-flow techniques. The stability of the Trp mutant was found to be similar to that of wild-type RNase A when denatured by heat or GdnHCl, and the mutant was found to have 85%...

The role of specific disulfides in the refolding of bovine trypsinogen was examined with samples of the protein lacking one and two disulfide bonds. Disulfide 179 to 203 was reduced with 0.1 M sodium borohydride and the same bond and disulfide 122 to 189 was reduced with 0.002 M dithioerythritol. The newly formed sulfhydry1 groups were converted to the “‘C-labeled diand tetracarboxymethyl deriv...