@article{milam_nicely_feeney_mattos_clark_2007, title={Rapid folding and unfolding of Apaf-1 CARD}, volume={369}, ISSN={["0022-2836"]}, DOI={10.1016/j.jmb.2007.02.105}, abstractNote={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. Interestingly, the results showed that the secondary structure is less stable than the tertiary structure, based on the transition mid-points for unfolding. Single mixing and sequential mixing stopped-flow studies showed that Apaf-1 CARD folds and unfolds rapidly and suggest a folding mechanism that contains parallel channels with two unfolded conformations folding to the native conformation. Kinetic simulations show that a slow folding phase is described by a third conformation in the unfolded ensemble that interconverts with one or both unfolded species. Overall, the native ensemble is formed rapidly upon refolding. This is in contrast to other CARDs in which folding appears to be dominated by formation of kinetic traps.}, number={1}, journal={JOURNAL OF MOLECULAR BIOLOGY}, author={Milam, Sara L. and Nicely, Nathan I. and Feeney, Brett and Mattos, Carla and Clark, A. Clay}, year={2007}, month={May}, pages={290–304} }
 @article{feeney_soderblom_goshe_clark_2006, title={Novel protein purification system utilizing an N-terminal fusion protein and a caspase-3 cleavable linker}, volume={47}, ISSN={1046-5928}, url={http://dx.doi.org/10.1016/j.pep.2005.10.005}, DOI={10.1016/j.pep.2005.10.005}, abstractNote={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. To address these issues, we created a fusion protein system that utilizes a highly efficient enzyme and leaves no residual amino acids on the target protein after removal of the affinity tag. We designed a glutathione S -transferase (GST)-fusion protein vector with a caspase-3 consensus cleavage sequence located between the N-terminal GST tag and a target protein. We show that the enzyme efficiently cleaves the fusion protein without leaving excess amino acids on the target protein. In addition, we used an engineered caspase-3 enzyme that is highly stable, has increased activity relative to the wild-type enzyme, and contains a poly-histidine tag that allows for efficient removal of the enzyme after cleavage of the fusion protein. Although we have developed this system using a GST tag, the system is amenable to any commercially available affinity tag.}, number={1}, journal={Protein Expression and Purification}, publisher={Elsevier BV}, author={Feeney, Brett and Soderblom, Erik J. and Goshe, Michael B. and Clark, A. Clay}, year={2006}, month={May}, pages={311–318} }
 @article{feeney_pop_swartz_mattos_clark_2006, title={Role of loop bundle hydrogen bonds in the maturation and activity of (pro) caspase-3}, volume={45}, ISSN={["0006-2960"]}, DOI={10.1021/bi0611964}, abstractNote={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. The new interactions are predicted to stabilize the active site. One unresolved issue is the extent to which the loop bundle residues also stabilize the procaspase active site. We examined the effects of replacing four loop bundle residues (E167, D169, E173, and Y203) on the biochemical and structural properties of the (pro)caspase. We show that replacing the residues affects the activity of the procaspase as well as the mature caspase, with D169A and E167A replacements having the largest effects. Replacement of D169 prevents caspase-3 autoactivation, and its cleavage at D175 no longer leads to an active enzyme. In addition, the E173A mutation, when coupled to a second mutation in the procaspase, D175A, may alter the substrate specificity of the procaspase. The mutations affected the active site environment as assessed by changes in fluorescence emission, accessibility to quencher, and cleavage by either trypsin or V8 proteases. High-resolution X-ray crystallographic structures of E167A, D173A, and Y203F caspases show that changes in the active site environment may be due to the increased flexibility of several residues in the N-terminus of the small subunit. Overall, the results show that these residues are important for stabilizing the procaspase active site as well as that of the mature caspase.}, number={44}, journal={BIOCHEMISTRY}, author={Feeney, Brett and Pop, Cristina and Swartz, Paul and Mattos, Carla and Clark, A. Clay}, year={2006}, month={Nov}, pages={13249–13263} }
 @article{feeney_clark_2005, title={Reassembly of active caspase-3 is facilitated by the propeptide}, volume={280}, ISSN={["1083-351X"]}, DOI={10.1074/jbc.M505834200}, abstractNote={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. We have studied the effects of cations on the activities and conformations of caspase-3 and an uncleavable mutant of procaspase-3 that is enzymatically active. The results show that caspase-3 is more sensitive to changes in pH and ion concentrations than is the zymogen. This is due to the loss of both an intact intersubunit linker and the prodomain. The results show that, although the caspase-3 subunits reassemble to the heterotetramer, the activity return is low after the protein is incubated at or below pH 4.5 and then returned to pH 7.5. The data further show that the irreversible step in assembly results from heterotetramer rather than heterodimer dissociation and demonstrate that the active site does not form properly following reassembly. However, active-site formation is fully reversible when reassembly occurs in the presence of the prodomain, and this effect is specific for the propeptide of caspase-3. The data show that the prodomain facilitates both dimerization and active-site formation in addition to stabilizing the native structure. Overall, the results show that the prodomain acts as an intramolecular chaperone during assembly of the (pro)caspase subunits and increases the efficiency of formation of the native conformation. 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. We have studied the effects of cations on the activities and conformations of caspase-3 and an uncleavable mutant of procaspase-3 that is enzymatically active. The results show that caspase-3 is more sensitive to changes in pH and ion concentrations than is the zymogen. This is due to the loss of both an intact intersubunit linker and the prodomain. The results show that, although the caspase-3 subunits reassemble to the heterotetramer, the activity return is low after the protein is incubated at or below pH 4.5 and then returned to pH 7.5. The data further show that the irreversible step in assembly results from heterotetramer rather than heterodimer dissociation and demonstrate that the active site does not form properly following reassembly. However, active-site formation is fully reversible when reassembly occurs in the presence of the prodomain, and this effect is specific for the propeptide of caspase-3. The data show that the prodomain facilitates both dimerization and active-site formation in addition to stabilizing the native structure. Overall, the results show that the prodomain acts as an intramolecular chaperone during assembly of the (pro)caspase subunits and increases the efficiency of formation of the native conformation. Apoptosis is a type of cell death that occurs in eumetazoans, is responsible for maintaining the balance between cell growth and death, and can occur via two major pathways (1Earnshaw W.C. Martins L.M. Kaufmann S.H. Annu. Rev. Biochem. 1999; 68: 383-424Crossref PubMed Scopus (2417) Google Scholar). The extrinsic pathway occurs in response to death receptor ligation (2Tewari M. Quan L.T. O'Rouke K. Desnoyers S. Zeng Z. Beidler D.R. Poirier G.G. Salvesen G.S. Dixit V.M. Cell. 1995; 81: 801-809Abstract Full Text PDF PubMed Scopus (2259) Google Scholar) and results in a cascade of limited proteolytic cleavages by upstream activator caspases that ultimately lead to the activation of the executioner caspase-3. The intrinsic pathway triggers the release of ATP and cytochrome c from the mitochondria in response to antineoplastic drugs (3Fearnhead H.O. McCurrach M.E. O'Neill J. Zhang K. Lowe S.W. Lazebnik Y.A. Genes Dev. 1997; 11: 1266-1276Crossref PubMed Scopus (61) Google Scholar), growth factor withdrawal (4Deckwerth T.L. Johnson Jr., E.M. Ann. N. Y. Acad. Sci. 1993; 679: 121-131Crossref PubMed Scopus (47) Google Scholar), or ionizing radiation (5Datta R. Banach D. Kojima H. Talanian R.V. Alnemri E.S. Wong W.W. Kufe D.W. Blood. 1996; 88: 1936-1943Crossref PubMed Google Scholar). The intrinsic pathway results in a proteolytic cascade that activates caspase-9 and, subsequently, caspase-3 (6Segal M.S. Beem E. Am. J. Physiol. 2001; 281: C1196-C1204Crossref PubMed Google Scholar). Both pathways give rise to the proteolysis of structural and protective components of the cell, which in turn leads to the coordinated disassembly of the cell (7Salvesen G.S. Dixit V.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10964-10967Crossref PubMed Scopus (762) Google Scholar). A relatively well characterized event leading up to the commitment to apoptosis is the change in the ionic homeostasis of the cell. Apoptotic cells are generally characterized as having low [K+]i, normal to slightly increased [Na+]i, high [H+]i, and normal to moderately increased [Ca2+]i. The efflux of potassium (leading to the depletion of intracellular potassium) is a key early step in apoptosis (8Yu S.P. Prog. Neurobiol. (N. Y.). 2003; 70: 363-386Crossref PubMed Scopus (294) Google Scholar). Under normal conditions, [K+]i is ∼140 mm, and levels decrease to <50 mm in apoptotic cells (9Yu S.P. Canzoniero L.M.T. Choi D.W. Curr. Opin. Cell Biol. 2001; 13: 405-411Crossref PubMed Scopus (328) Google Scholar, 10Hughes Jr., F.M. Cidlowski J.A. Adv. Enzyme Regul. 1999; 39: 157-171Crossref PubMed Scopus (202) Google Scholar). The decrease in [K+]i and associated water movement are contributing factors to the change in cell volume observed during apoptosis. Normal potassium concentrations are inhibitory to apoptosis and may act by inhibiting an apoptotic nuclease, NUC18 (10Hughes Jr., F.M. Cidlowski J.A. Adv. Enzyme Regul. 1999; 39: 157-171Crossref PubMed Scopus (202) Google Scholar). In addition, normal potassium levels inhibit the cytochrome c-dependent activation of procaspase-3, but do not inhibit the activity of caspase-3 (6Segal M.S. Beem E. Am. J. Physiol. 2001; 281: C1196-C1204Crossref PubMed Google Scholar, 11Bilney A.J. Murray A.W. FEBS Lett. 1998; 424: 221-224Crossref PubMed Scopus (29) Google Scholar). Although free potassium is a major contributor to intracellular ionic strength, Hughes and Cidlowski (10Hughes Jr., F.M. Cidlowski J.A. Adv. Enzyme Regul. 1999; 39: 157-171Crossref PubMed Scopus (202) Google Scholar) showed that substitution of potassium with other monovalent ions results in similar effects. This indicates that the response is not specific to potassium cations, but is instead controlled by the ionic strength. Thus far, potassium is the only cation that has been described as having a role in the inhibition of apoptosis, although studies of the direct effects of cations on caspase-3 activation and apoptotic commitment are incomplete at best. Although the results are less clear, studies with other ions, such as magnesium, zinc, copper, and iron, show that these ions also may have a role in apoptosis in certain tissues (reviewed in Ref. 9Yu S.P. Canzoniero L.M.T. Choi D.W. Curr. Opin. Cell Biol. 2001; 13: 405-411Crossref PubMed Scopus (328) Google Scholar). In most cases, the effects of changes in ionic homeostasis occur at a stage prior to the maturation of caspase-3 and, in some cases, may result from the prevention of the formation of the apoptosome. For example, fluctuations in ion levels may inhibit the expression of caspase-9 as well as prevent the recruitment of caspase-9 to the apoptosome (6Segal M.S. Beem E. Am. J. Physiol. 2001; 281: C1196-C1204Crossref PubMed Google Scholar). Putney and co-workers (12Bian X. Hughes Jr., F.M. Huang Y. Cidlowski J.A. Putney Jr., J.W. Am. J. Physiol. 1997; 272: C1241-C1249Crossref PubMed Google Scholar) showed that Ca2+-ATPase inhibitors initiate apoptosis by decreasing [Ca2+]i, and Segal and Beem (6Segal M.S. Beem E. Am. J. Physiol. 2001; 281: C1196-C1204Crossref PubMed Google Scholar) demonstrated a 50% inhibition of caspase activity in cytosolic extracts with ∼60 mm CaCl2. However, Stennicke and Salvesen (13Stennicke H.R. Salvesen G.S. J. Biol. Chem. 1997; 272: 25719-25723Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar) found that calcium has no effect on caspase-3 activity at 100 mm and suggested that the effects of calcium on apoptosis are unlikely to be due to an effect on caspases. In contrast, the cleavage of poly(ADP-ribose) polymerase by caspase-3 has been shown to be inhibited by zinc at concentrations as low as 0.1 mm (14Perry D.K. Smyth M.J. Stennicke H.R. Salvesen G.S. Duriez P. Poirier G.G. Hannun Y.A. J. Biol. Chem. 1997; 272: 18530-18533Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). This is due to a direct inactivation of caspase-3 by zinc (14Perry D.K. Smyth M.J. Stennicke H.R. Salvesen G.S. Duriez P. Poirier G.G. Hannun Y.A. J. Biol. Chem. 1997; 272: 18530-18533Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). Although studies of the direct effects of cations on caspase-3 activity remain incomplete, comparable studies on the activity of procaspase-3 are lacking. Until recently, procaspase-3 was thought to be enzymatically inactive; thus, studies of the effects of ions on procaspase activity were not warranted. Recently, however, it was shown by Nicholson and co-workers (15Roy S. Bayly C.I. Gareau Y. Houtzager V.M. Kargman S. Keen S.L.C. Rowland K. Seiden I.M. Thornberry N.A. Nicholson D.W. Proc. Natl. Acad. Sci. U. S. A. 2001; 98 (S. L. C.): 6132-6137Crossref PubMed Scopus (165) Google Scholar) that an uncleavable procaspase-3 mutant contains catalytic activity. In this mutant, the three processing sites (Asp9, Asp28, and Asp175) have been replaced with glutamate. Likewise, we showed previously that a procaspase-3 mutant with the processing sites replaced with alanine is active (see Fig. 1) (16Bose K. Pop C. Feeney B. Clark A.C. Biochemistry. 2003; 42: 12298-12310Crossref PubMed Scopus (53) Google Scholar). In the maturation of procaspase-3, the cleavage at Asp175 separates the large and small subunits and results in a large increase in activity, whereas the cleavages at Asp9 and Asp28 remove the prodomain (17Denault J.-B. Salvesen G.S. Chem. Rev. 2002; 102: 4489-4499Crossref PubMed Scopus (268) Google Scholar). We further showed that the uncleavable procaspase is ∼200-fold less active than mature caspase-3 and that the lower activity is a result of a low catalytic efficiency rather than a difference in substrate binding (16Bose K. Pop C. Feeney B. Clark A.C. Biochemistry. 2003; 42: 12298-12310Crossref PubMed Scopus (53) Google Scholar). Here, we describe the effects of physiologically relevant salts on the activity of procaspase-3 and the accompanying changes in the active-site environment. In addition, it has been noted that the procaspase-3 homodimer is more stable than the caspase-3 heterotetramer between pH 4 and 7 (18Feeney B. Pop C. Tripathy A. Clark A.C. Biochem. J. 2004; 384: 515-525Crossref PubMed Scopus (16) Google Scholar, 19Bose K. Clark A.C. Protein Sci. 2005; 14: 24-36Crossref PubMed Scopus (21) Google Scholar), although it is not clear whether this is due to the presence of the propeptide or to an intact intersubunit linker in the procaspase. Indeed, Denault and Salvesen (20Denault J.-B. Salvesen G.S. J. Biol. Chem. 2003; 278: 34042-34050Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) showed that the propeptide of caspase-7 plays a role in stabilizing the zymogen and alters the properties of procaspase-7 as a substrate for caspase-7. By analogy, the lower stability of caspase-3 may be due to the loss of the propeptide upon maturation. We investigated whether the prodomain or the intact intersubunit linker affects dimer stability, and we further investigated the roles that salts and pH play in the stability of the (pro)caspase-3 dimer. Based on the results, we propose a model for the salt- and propeptide-dependent assembly of the caspase subunits. Materials—Caspase-3, -6, and -7 prodomains were synthesized by the Peptide Facility at the University of North Carolina (Chapel Hill, NC). Sodium chloride, ammonium chloride, Tris base, and β-mercaptoethanol were from Fisher. Zinc chloride, magnesium chloride, dithiothreitol (DTT), 2The abbreviations used are: DTTdithiothreitolCHAPS3-[(3-cholamidopropy-l)dimethylammonio]-1-propanesulfonic acidprocaspase-3(D3A)procaspase-3(D9A,D28A,D175A)PIPES1,4-piperazinediethanesulfonic acidIMCsintramolecular chaperones. sodium citrate, and monobasic and dibasic potassium phosphate were from Sigma. Potassium chloride and sucrose were from Mallinckrodt Chemical Works. Calcium chloride was from Acros Organics. CHAPS and Ac-DEVD-7-amino-4-trifluoromethylcoumarin were from Calbiochem, and EDTA was from EM Science. dithiothreitol 3-[(3-cholamidopropy-l)dimethylammonio]-1-propanesulfonic acid procaspase-3(D9A,D28A,D175A) 1,4-piperazinediethanesulfonic acid intramolecular chaperones. Mutagenesis—A mutation was introduced into the background of pro-less caspase-3 using the template pHC329 (21Pop C. Chen Y.-R. Smith B. Bose K. Bobay B. Tripathy A. Franzen S. Clark A.C. Biochemistry. 2001; 40: 14224-14235Crossref PubMed Scopus (61) Google Scholar) and the D175A forward and reverse primers (referred to as primers 1 and 2) as described previously (16Bose K. Pop C. Feeney B. Clark A.C. Biochemistry. 2003; 42: 12298-12310Crossref PubMed Scopus (53) Google Scholar). This generated plasmid pHC32902 and produces an uncleavable pro-less procaspase-3 with the D175A mutation. In this mutant, the N-terminal 28 amino acids were replaced previously with methionine (21Pop C. Chen Y.-R. Smith B. Bose K. Bobay B. Tripathy A. Franzen S. Clark A.C. Biochemistry. 2001; 40: 14224-14235Crossref PubMed Scopus (61) Google Scholar). Construction of plasmid pHC33209, which produces procaspase-3(D9A,D28A,D175A), was described previously (16Bose K. Pop C. Feeney B. Clark A.C. Biochemistry. 2003; 42: 12298-12310Crossref PubMed Scopus (53) Google Scholar). The D9A,D28A double mutant was produced using the template pHC33209 and primer 1 (5′-GTGGCATTGAGACAGACAGTGGTGTTGATGATG-3′) and primer 2 (5′-CATCATCAACACCACTGTCTGTCTCAATGCCAC-3′). The resulting plasmid is called pHC33260. In this plasmid, the NheI site that was incorporated previously for screening plasmid pHC33209 was removed, and its absence was used for screening. All plasmids were sequenced (both DNA strands) to confirm the mutations. A schematic diagram of the proteins produced from these plasmids and used in the studies presented here is shown in Fig. 1. Protein Purification—Caspase-3, uncleavable procaspase-3(D9A,D28A,D175A) (called procaspase-3(D3A)) (16Bose K. Pop C. Feeney B. Clark A.C. Biochemistry. 2003; 42: 12298-12310Crossref PubMed Scopus (53) Google Scholar), pro-less procaspase-3(D175A), and procaspase-3(D9A,D28A) were purified following overexpression in Escherichia coli as described previously (16Bose K. Pop C. Feeney B. Clark A.C. Biochemistry. 2003; 42: 12298-12310Crossref PubMed Scopus (53) Google Scholar). Protein concentrations were determined using ϵ280 = 26,500 m–1 cm–1 (21Pop C. Chen Y.-R. Smith B. Bose K. Bobay B. Tripathy A. Franzen S. Clark A.C. Biochemistry. 2001; 40: 14224-14235Crossref PubMed Scopus (61) Google Scholar). Enzymatic Activity Versus Salt Concentration—Protein was incubated at 25 °C in buffer containing 50 mm Tris-HCl (pH 7.5) and 10 mm DTT for >1 h, and substrate (Ac-DEVD-7-amino-4-trifluoromethylcoumarin) was then added. Hydrolysis of the substrate was monitored as described previously (16Bose K. Pop C. Feeney B. Clark A.C. Biochemistry. 2003; 42: 12298-12310Crossref PubMed Scopus (53) Google Scholar, 21Pop C. Chen Y.-R. Smith B. Bose K. Bobay B. Tripathy A. Franzen S. Clark A.C. Biochemistry. 2001; 40: 14224-14235Crossref PubMed Scopus (61) Google Scholar). Salts were added to the buffer such that the final concentrations are those shown in the figures. Assays were performed in duplicate in the same buffer, and relative activity was determined versus salt concentration by comparing the initial velocity in the absence or presence of the salt. The concentration of substrate was 50 μm, and 10 nm caspase-3 or 50 nm procaspase-3 was used for each assay. Because DTT chelates Zn2+, the DTT was replaced with 20 mm β-mercaptoethanol in the assay buffer as described previously (13Stennicke H.R. Salvesen G.S. J. Biol. Chem. 1997; 272: 25719-25723Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). We showed previously that one can measure ∼50-fold less enzymatic activity than the maximal activity observed for procaspase-3(D3A) (or 10,000-fold less than that of mature caspase-3), establishing the sensitivity of the assay (16Bose K. Pop C. Feeney B. Clark A.C. Biochemistry. 2003; 42: 12298-12310Crossref PubMed Scopus (53) Google Scholar). Changes in Fluorescence Emission as a Function of pH and Salt—To determine the structural effects of different cations on caspase-3 and procaspase-3, the proteins (2 μm) were incubated in 50 mm citrate (pH 3.0–6.2) or 50 mm phosphate (pH 6.0–9.0) containing NaCl, KCl, NH4Cl, MgCl2, or CaCl2 at 50–1000 mm. Samples were excited at 280 nm, and fluorescence emission was acquired between 305 and 400 nm (PTI C-61 spectrofluorometer, Photon Technology International). The average emission wavelength was then calculated at each pH as described previously (16Bose K. Pop C. Feeney B. Clark A.C. Biochemistry. 2003; 42: 12298-12310Crossref PubMed Scopus (53) Google Scholar, 18Feeney B. Pop C. Tripathy A. Clark A.C. Biochem. J. 2004; 384: 515-525Crossref PubMed Scopus (16) Google Scholar, 22Royer C.A. Mann C.J. Matthews C.R. Protein Sci. 1993; 2: 1844-1852Crossref PubMed Scopus (173) Google Scholar) and compared with the spectra of the wild-type protein. Salt Titrations at Low pH—As described in Fig. 3, salts affected the average emission wavelength for caspase-3 but not for procaspase-3 at low pH. Because of this, we examined whether the effect was cooperative, reversible, or dependent on the protein concentration. To examine cooperativity, caspase-3 was incubated in 20 mm citrate (pH 3.0) and in the absence or presence of 2 m NaCl, KCl, NH4Cl, MgCl2, or CaCl2. Using an Olis titrator coupled to the PTI C-61 spectrofluorometer, a solution of caspase-3 in buffer containing 2 m salt was titrated into a solution of caspase-3 without salts. The solutions were mixed and incubated for 20 min between each measurement to allow equilibration. The salt-dependent conformational changes at pH 3.0 were determined by calculating the change in the fluorescence average emission wavelength (22Royer C.A. Mann C.J. Matthews C.R. Protein Sci. 1993; 2: 1844-1852Crossref PubMed Scopus (173) Google Scholar) at each concentration of salt. Reversibility was examined using 2 μm protein and a two-phase reverse titration. The first phase consisted of salt concentrations between 1000 and 250 mm, and the second phase consisted of salt concentrations between 250 and 50 mm. Two additional points were taken manually to obtain 25 and 12.5 mm salt concentrations. We observed that the data from forward or reverse titrations were superimposable, demonstrating that the transitions are reversible. The protein concentration dependence was examined by performing the titrations at several protein concentrations (2–12 μm). Reversibility of Activity as a Function of Salt and pH—To examine the reversibility of enzymatic activity following incubation at low pH as well as at high salt concentrations, both caspase-3 and procaspase-3(D3A) were dialyzed for 4 h at 25 °C in 50 mm citrate (pH 3.0) plus 1 m salt or in 50 mm Tris-HCl (pH 7.5) plus 1 m salt for monovalent cations or 0.5 m salt for divalent cations. All buffers contained 1 mm DTT. The proteins were then dialyzed for 9 h at 25 °C in 50 mm Tris-HCl (pH 7.5) and 1 mm DTT. In all cases, the volume of the dialysis buffer was >500-fold that of the protein sample. The enzymatic activity was measured in 50 mm Tris-HCl (pH 7.5), 0.1% CHAPS, 1% sucrose, and 10 mm DTT (assay buffer) (16Bose K. Pop C. Feeney B. Clark A.C. Biochemistry. 2003; 42: 12298-12310Crossref PubMed Scopus (53) Google Scholar, 21Pop C. Chen Y.-R. Smith B. Bose K. Bobay B. Tripathy A. Franzen S. Clark A.C. Biochemistry. 2001; 40: 14224-14235Crossref PubMed Scopus (61) Google Scholar) using the substrate and protein concentrations described above. The initial velocity was compared with that of a control protein that had been stored at –20 °C (i.e. not dialyzed for 13 h). Activity as a Function of pH and Protein Concentration—The pH at which caspase-3 loses activity irreversibly was determined by dialyzing the protein (10 μm) for 4 h at 25 °C in 50 mm citrate (pH 3.0–6.0) or in 50 mm Tris-HCl (pH 6.5–7.5) containing 1 mm DTT every 0.5 pH units. Samples were then dialyzed for 9 h at 25 °C in 50 mm Tris-HCl (pH 7.5) and 1 mm DTT. The enzymatic activity was measured in assay buffer and compared with that of a control sample of caspase-3 that had been incubated for 13 h at 25 °C in 50 mm Tris-HCl (pH 7.5) and 1 mm DTT. In all cases, the volume of the dialysis buffer was >500-fold that of the protein sample. To investigate the effect of protein concentration on solubility and return of activity in the pH-dependent folding of caspase-3, protein concentrations ranging from 2 to ∼50 μm were used. Samples of caspase-3 were incubated at 25 °C in 50 mm citrate (pH 4) and 1 mm DTT for 24 h to dissociate the heterotetramer (18Feeney B. Pop C. Tripathy A. Clark A.C. Biochem. J. 2004; 384: 515-525Crossref PubMed Scopus (16) Google Scholar, 19Bose K. Clark A.C. Protein Sci. 2005; 14: 24-36Crossref PubMed Scopus (21) Google Scholar), and the protein was then refolded by dialyzing the samples against 50 mm Tris-HCl (pH 7.5) and 1 mm DTT at 25 °C for 24 h. The samples were centrifuged at 14,000 rpm at 25 °C for 5 min, and the supernatant was transferred to a new tube. The absorbance at 280 nm was compared with that at time 0. The enzymatic activity was measured in assay buffer. The final protein concentrations in the enzymatic assays were as follows: caspase-3, 10 nm; pro-less procaspase-3(D175A), 50 nm; procaspase-3(D9A,D28A), 10 nm; and procaspase-3(D3A), 50 nm. To measure the loss of enzymatic activity over time, protein (1 μm) was incubated in 50 mm Tris-HCl (pH 7.5) at 25 °C for the times indicated in Fig. 7. In separate experiments, DTT (1 or 10 mm), KCl (1 m), MgCl2 (0.5 m), or caspase-3 propeptide (15 μm) was added to the buffer. Aliquots were removed at various times, and the enzymatic activity was measured in assay buffer. The final protein concentrations were 10 nm in the assays. To examine the effect of the prodomain concentration on the return of activity, the caspase-3, -6, or -7 prodomain was added to caspase-3 or pro-less procaspase-3(D175A) at 2–200-fold excess concentrations. The (pro)caspase concentrations were 10 μm. In these experiments, the proteins were dialyzed initially in 50 mm citrate (pH 4.0) for ∼24 h at 25 °C. Following the initial dialysis, the samples were then dialyzed against 50 mm Tris-HCl (pH 7.5) for ∼24 h at 25 °C, and the enzymatic activity was measured as described above. All buffers contained 1 mm DTT. In these experiments, a dialysis membrane with an Mr 1000 cutoff was used. The final protein concentrations in the enzymatic assays were as follows: caspase-3, 10 nm; and pro-less procaspase-3(D175A), 50 nm. Activity Versus Salt Concentration—We examined the activities of caspase-3 and procaspase-3(D3A) in the presence of the monovalent cations Na+, K+, and NH+4 at concentrations up to 500 mm, and the results are shown in Fig. 2. As was observed previously (13Stennicke H.R. Salvesen G.S. J. Biol. Chem. 1997; 272: 25719-25723Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar), the activity of caspase-3 increased slightly (∼20%) in the presence of low concentrations of Na+ (∼25 mm) (Fig. 2A). This was true also for NH+4 (∼50 mm). However, K+ had little to no effect on the activity of caspase-3. In contrast, there was no effect of Na+ or NH+4 on the activity of procaspase-3(D3A) (Fig. 2B). Unlike the activity of caspase-3, that of procaspase-3 was sensitive to the presence of K+ and decreased to ∼50% in the presence of >25 mm K+. In the presence of Mg2+, the activities of caspase-3 and procaspase-3 decreased to ∼40%, and the midpoints of the transitions were similar, ∼100 mm (Fig. 2, C and D). In addition, the activities of both proteins decreased in the presence of Ca2+ (Fig. 2, C and D). For caspase-3, the activity decreased to ∼50% between 10 and 100 mm Ca2+ and then decreased further at higher Ca2+ concentrations. For procaspase-3, the activity decreased to ∼25% in an apparent single transition, with a midpoint of ∼10 mm Ca2+. This is in contrast to results reported by Stennicke and Salvesen (13Stennicke H.R. Salvesen G.S. J. Biol. Chem. 1997; 272: 25719-25723Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar), who observed no effect on the activity of caspase-3 at concentrations up to 100 mm. It is not clear why there is a discrepancy in the results, although the experimental conditions are similar but not identical. For these studies, our assays were carried out at 25 °C rather than at 37 °C, and our buffer contained 50 mm Tris-HCl (pH 7.5) rather than 20 mm PIPES (pH 7.2) and did not contain sucrose. In addition, the control with which the activities were compared was taken from a fresh stock of protein that had been stored at –20 °C until just prior to use. At present, it is not clear which of these parameters would result in the discrepancy. Regardless, it is interesting to note that [Ca2+]i is ∼100 nm and either increases slightly or does not change significantly, depending on the cell type, during apoptosis (9Yu S.P. Canzoniero L.M.T. Choi D.W. Curr. Opin. Cell Biol. 2001; 13: 405-411Crossref PubMed Scopus (328) Google Scholar). Thus, in the effects described here, the Ca2+ concentrations are at least 4 orders of magnitude higher than those found in apoptotic cells. Our conclusion from these studies is that, although Ca2+ does affect the activities of both caspase-3 and procaspase-3 in vitro and at high concentrations, it is unlikely that Ca2+ fluctuations in apoptotic cells directly affect the activity of either protein. However, this is not true of K+ because the concentrations used in our experiments are comparable with those found in vivo. Therefore, although the activity of caspase-3 is unaffected by changes in the levels of K+, the activity of the procaspase would be expected to increase if the efflux of potassium during apoptosis resulted in concentrations below 25 mm. Caspase-3 is known to be inactivated by micromolar concentrations of Zn2+, and it has been suggested that Zn2+ coordinates one or both catalytic residues His121 and Cys163 (14Perry D.K. Smyth M.J. Stennicke H.R. Salvesen G.S. Duriez P. Poirier G.G. Hannun Y.A. J. Biol. Chem. 1997; 272: 18530-18533Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). As shown in Fig. 2E, caspase-3 was completely inactivated by ∼10 mm Zn2+, in agreement with previous studies (13Stennicke H.R. Salvesen G.S. J. Biol. Chem. 1997; 272: 25719-25723Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar, 14Perry D.K. Smyth M.J. Stennicke H.R. Salvesen G.S. Duriez P. Poirier G.G. Hannun Y.A. J. Biol. Chem. 1997; 272: 18530-18533Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). Surprisingly, procaspase-3(D3A) is much less sensitive to Zn2+. We observed that the procaspase was inactivated by Zn2+ concentrations between 300 and 400 μm, >3 orders of magnitude that required to inactivate caspase-3 (compare the midpoints of the curves in Fig. 2E). This is in agreement with our assertion that the catalytic residue Cys163 is less solvent-accessible in the procaspase than in the mature caspase (16Bose K. Pop C. Feeney B. Clark A.C. Biochemistry. 2003; 42: 12298-12310Crossref PubMed Scopus (53) Google Scholar). It has been shown that ionic effects on caspase activity are due to cations rather than anions (6Segal M.S. Beem E. Am. J. Physiol. 2001; 281: C1196-C1204Crossref PubMed Google Scholar). We confirmed this by examining chloride, acetate, sulfate, and phosphate anions using the sodium or magnesium salts of each. We observed no effect on the enzymatic activity of either caspase-3 or procaspase-3(D3A) beyond those described above for the cations (data not shown). Structural Changes as a Function of pH and Salt—The active site of procaspase-3(D3A) was shown to be distinctly different from that of mature caspase-3, as evidenced in part by the differences in pH-dependent activity profiles (16Bose K. Pop C. Feeney B. Clark A.C. Biochemistry. 2003; 42: 12298-12310Crossref PubMed Scopus (53) Google Scholar). Procaspase-3(D3A) exhibits maximal activity between pH 8.0 and 8.5, in contrast to caspase-3, which has a maximal activity between pH 7.2 and 7.8 (13Stennicke H.R. Salvesen G.S. J. Biol. Chem. 1997; 272: 25719-25723Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar, 16Bose K. Pop C. Feeney B. Clark A.C. Biochemistry. 2003; 42: 12298-12310Crossref PubMed Scopus (53) Google Scholar). In addition, we examined changes in the fluorescence average emission wavelength (〈λ 〉) (Fig. 3), which report on conformational changes in the protein that result from changes in pH (16Bose K. Pop C. Feeney B. Clark A.C. Biochemistry. 2003; 42: 12298-12310Crossref PubMed Scopus (53) Google Scholar, 18Feeney B. Pop C. Tripathy A. Clark A.C. Biochem. J. 2004; 384: 515-525Crossref PubMed Scopus (16) Google Scholar). (Pro)caspase-}, number={48}, journal={JOURNAL OF BIOLOGICAL CHEMISTRY}, author={Feeney, B and Clark, AC}, year={2005}, month={Dec}, pages={39772–39785} }
 @article{bobay_benson_naylor_feeney_clark_goshe_strauch_thompson_cavanagh_2004, title={Evaluation of the DNA Binding Tendencies of the Transition State Regulator AbrB†}, volume={43}, ISSN={0006-2960 1520-4995}, url={http://dx.doi.org/10.1021/bi048399h}, DOI={10.1021/bi048399h}, abstractNote={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. MicroESI-MS analysis of AbrB confirmed its stable macromolecular state as being tetrameric and verified the same stoichiometric state in complex with DNA targets. MicroESI-MS, circular dichroism, and fluorescence provided relative binding affinities for AbrB-DNA interactions in a qualitative manner. UV spectroscopy was used in a quantitative manner to determine solution phase dissociation constants for AbrB-DNA complexes. General DNA structural parameters for all known natural AbrB binding sequences were also studied and significant similarities in topological constraints (stretch, opening, and propeller twist) were observed. It is likely that these parameters contribute to the differential binding proclivities of AbrB. In addition to providing an improved understanding of transition state regulator-DNA binding properties and structural tendencies of target promoters, this comprehensive and corroborative spectroscopic study endorses the use of microESI-MS for rapidly ascertaining qualitative binding trends in noncovalent systems in a high-throughput manner.}, number={51}, journal={Biochemistry}, publisher={American Chemical Society (ACS)}, author={Bobay, Benjamin G. and Benson, Linda and Naylor, Stephen and Feeney, Brett and Clark, A. Clay and Goshe, Michael B. and Strauch, Mark A. and Thompson, Richele and Cavanagh, John}, year={2004}, month={Dec}, pages={16106–16118} }
 @article{feeney_pop_tripathy_clark_2004, title={Ionic interactions near the loop L4 are important for maintaining the active-site environment and the dimer stability of (pro)caspase 3}, volume={384}, number={Dec 15 2004}, journal={Biochemical Journal (London, England : 1984)}, author={Feeney, B. and Pop, C. and Tripathy, A. and Clark, A. C.}, year={2004}, pages={515–525} }
 @article{bose_pop_feeney_clark_2003, title={An uncleavable procaspase-3 mutant has a lower catalytic efficiency but an active site similar to that of mature caspase-3}, volume={42}, ISSN={["0006-2960"]}, DOI={10.1021/bi034998x}, abstractNote={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 7.5 and 25 degrees C the K(m) values are similar, the catalytic efficiency (k(cat)) is approximately 130-fold lower in the zymogen. The mature caspase-3 demonstrates a maximum activity at pH 7.4, whereas the maximum activity of procaspase-3 occurs at pH 8.3. The pK(a) values of both catalytic groups, H121 and C163, are shifted to higher pH for procaspase-3. We developed limited proteolysis assays using trypsin and V8 proteases, and we show that these assays allow the examination of amino acids in three of five active site loops. In addition, we examined the fluorescence emission of the two tryptophanyl residues in the active site over the pH range of 2.5-9 as well as the response to several quenching agents. Overall, the data suggest that the major conformational change that occurs upon maturation results in formation of the loop bundle among loops L4, L2, and L2'. The pK(a) values of both catalytic groups decrease as a result of the loop movements. However, loop L3, which comprises the bulk of the substrate binding pocket, does not appear to be unraveled and solvent-exposed, even at lower pH.}, number={42}, journal={BIOCHEMISTRY}, author={Bose, K and Pop, C and Feeney, B and Clark, AC}, year={2003}, month={Oct}, pages={12298–12310} }
 @article{pop_feeney_tripathy_clark_2003, title={Mutations in the procaspase-3 dimer interface affect the activity of the zymogen}, volume={42}, ISSN={["0006-2960"]}, DOI={10.1021/bi034999p}, abstractNote={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. While the mutations do not affect the dimeric properties of the procaspase, we show that the V266E mutation may affect the formation of a loop bundle that is important for stabilizing the active site. In contrast, the V266H mutation affects the positioning of loop L3, the loop that forms the bulk of the substrate binding pocket. In some cases, the amino acids affected by the mutations are >20 A from the interface. Overall, the results demonstrate that the integrity of the dimer interface is important for maintaining the proper active site conformation.}, number={42}, journal={BIOCHEMISTRY}, author={Pop, C and Feeney, B and Tripathy, A and Clark, AC}, year={2003}, month={Oct}, pages={12311–12320} }