Rapid folding and unfolding of Apaf-1 CARD.

Caspase recruitment domains (CARDs) are members of the death domain superfamily and contain six antiparallel helices in an alpha-helical Greek key topology. We have examined the equilibrium and kinetic folding of the CARD of Apaf-1 (apoptotic protease activating factor 1), which consists of 97 amino acid residues, at pH 6 and pH 8. The results showed that an apparent two state equilibrium mechanism is not adequate to describe the folding of Apaf-1 CARD at either pH, suggesting the presence of intermediates in equilibrium unfolding.

Role of loop bundle hydrogen bonds in the maturation and activity of (Pro)caspase-3.

During maturation, procaspase-3 is cleaved at D175, which resides in a linker that connects the large and small subunits. The intersubunit linker also connects two active site loops that rearrange following cleavage and, in part, form the so-called loop bundle. As a result of chain cleavage, new hydrogen bonds and van der Waals contacts form among three active site loops.

Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker.

Coupled with over-expression in host organisms, fusion protein systems afford economical methods to obtain large quantities of target proteins in a fast and efficient manner. Some proteases used for these purposes cleave C-terminal to their recognition sequences and do not leave extra amino acids on the target. However, they are often inefficient and are frequently promiscuous, resulting in non-specific cleavages of the target protein.

Reassembly of active caspase-3 is facilitated by the propeptide.

Changes in ionic homeostasis are early events leading up to the commitment to apoptosis. Although the direct effects of cations on caspase-3 activity have been examined, comparable studies on procaspase-3 are lacking. In addition, the effects of salts on caspase structure have not been examined.

Evaluation of the DNA binding tendencies of the transition state regulator AbrB.

Global transition state regulator proteins represent one of the most diverse classes of prokaryotic transcription factors. One such transition state regulator, AbrB from Bacillus subtilis, is known to bind more than 60 gene targets yet displays specificity within this target set by binding each promoter with a different affinity. Microelectrospray ionization mass spectrometry (microESI-MS), circular dichroism, fluorescence, UV spectroscopy, and molecular modeling were used to elucidate differences among AbrB, DNA, and AbrB-DNA complexes.

Ionic interactions near the loop L4 are important for maintaining the active-site environment and the dimer stability of (pro)caspase 3.

We have examined the role of a salt bridge between Lys242 and Glu246 in loop L4 of procaspase 3 and of mature caspase 3, and we show that the interactions are required for stabilizing the active site. Replacing either of the residues with an alanine residue results in a complete loss of procaspase 3 activity. Although both mutants are active in the context of the mature caspase 3, the mutations result in an increase in K(m) and a decrease in kcat when compared with the wild-type caspase 3.

Mutations in the procaspase-3 dimer interface affect the activity of the zymogen.

The interface of the procaspase-3 dimer plays a critical role in zymogen maturation. We show that replacement of valine 266, the residue at the center of the procaspase-3 dimer interface, with glutamate resulted in an increase in enzyme activity of approximately 60-fold, representing a pseudoactivation of the procaspase. In contrast, substitution of V266 with histidine abolished the activity of the procaspase-3 as well as that of the mature caspase.

An uncleavable procaspase-3 mutant has a lower catalytic efficiency but an active site similar to that of mature caspase-3.

We have examined the enzymatic activity of an uncleavable procaspase-3 mutant (D9A/D28A/D175A), which contains the wild-type catalytic residues in the active site. The results are compared to those for the mature caspase-3. Although at pH 7.