In metalloproteins, metal centers serve as active sites for a range of functional purposes and as important structural elements to facilitate protein folding and assembly. It is challenging to observe the reversible unfolding and refolding of metalloproteins because of a loss or decomposition of the metal center. Here, the reversible unfolding-refolding of the iron-sulfur protein rubredoxin was...

MOTIVATION Single-molecule force spectroscopy has facilitated the experimental investigation of biomolecular force-coupled kinetics, from which the kinetics at zero force can be extrapolated via explicit theoretical models. The atomic force microscope (AFM) in particular is routinely used to study protein unfolding kinetics, but only rarely protein folding kinetics. The discrepancy arises becau...

Enteropeptidase is a serine protease used in different biotechnological applications. For many applications the smaller light chain can be used to avoid the expression of the rather large holoenzyme. Recombinant human enteropeptidase light chain (hEPL) shows high activity but low solubility and refolding yields, currently limiting its use in biotechnological applications. Here we describe sever...

Mutant human lysozymes (HLZ) lacking two disulfide bonds were constructed to study the importance of each disulfide bond on oxidative refolding. To avoid destabilization, a calcium-binding site was introduced. Five of the six species of two-disulfide mutants could be obtained with enzymatic activity. Based on the information obtained from refolding and unfolding experiments, the order of import...

Equilibrium and kinetic studies of the unfolding-refolding of goat spleen cathepsin B induced by urea are reported. Tryptophan fluorescence and enzyme activity were monitored. The activity of cathepsin B is lost reversibly at 1.2 M-urea. The enzyme unfolds in two main stages, having a stable intermediate (Y) between its native (N) and fully denatured (D) states. Enzyme activity and kinetic stud...

Expression of recombinant proteins as inclusion bodies in bacteria is one of the most efficient ways to produce cloned proteins, as long as the inclusion body protein can be successfully refolded. Aggregation is the leading cause of decreased refolding yields. Developments during the past year have advanced our understanding of the mechanism of aggregation in in vitro protein folding. New addit...

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