Fluorescence investigation of the sex steroid binding protein of rabbit serum: steroid binding and subunit dissociation

The relationship between steroid binding and protein subunit interactions of rabbit sex steroid binding protein (rSBP) has been studied by steady-state and time-resolved fluorescence spectroscopy. The high-affinity (Ka approximately 10(8) M-1 at 4 degrees C), fluorescent estrogen d-1,3,5(10),6,8-estrapentaene-3,17 beta-diol [dihydroequilenin (DHE)] was used as a fluorescent probe of the steroid-binding site. Perturbation of the binding site with guanidinium chloride (Gdm.Cl) was monitored by changes in the steady-state fluorescence anisotropy of DHE as well as by changes in fluorescence quenching of DHE with acrylamide. The results of acrylamide quenching at 11 degrees C show that, while between 0 and 1 M Gdm.Cl the steroid-binding site is completely shielded from bulk solvent, there is decreased DHE binding. To study the subunit-subunit interactions, rSBP was covalently labeled with dansyl chloride in the presence of saturating 5 alpha-dihydrotestosterone (DHT), which yielded a dansyl-conjugated protein that retained full steroid-binding activity. The protein subunit perturbation was monitored by changes in the steady-state fluorescence anisotropy of the dansyl group. At 11 degrees C, the dansyl anisotropy perturbation, reflecting changes in global and segmental motions of the dimer protein, occurs at concentrations of Gdm.Cl above 1 M. The Gdm.Cl titration in the presence of steroids with equilibrium association constants less than 10(8) M-1 shows a plateau near 3 M Gdm.Cl at 11 degrees C; at this Gdm.Cl concentration, no DHE is bound. No plateau is observed at 21 degrees C. At higher Gdm.Cl concentrations, the dansyl fluorescence anisotropy decreases further and shows no steroid dependence. Recovery of steroid-binding activity (assayed by saturation binding with [3H]DHT), under renaturation conditions, is dependent on both steroid concentration and affinity. Both unlabeled and dansyl-labeled protein recovery the same amount of activity, and according to fluorescence anisotropy, dansyl-labeled rSBP re-forms a dimer upon dilution below 1 M or removal of Gdm.Cl. From the steroid requirement for recovery of steroid-binding activity, it appears that a conformational template is required for the dimeric protein to re-form a steroid-binding site with native-like properties.     

Production and purification of refolded recombinant human IL-7 from inclusion bodies

A recombinant form of human rhIL-7 was overexpressed in Escherichia coli HMS174 (DE3) pLysS under the control of a T7 promoter. The resulting insoluble inclusion bodies were separated from cellular debris by cross-flow filtration and solubilized by homogenization with 6 M guanidine HCl. Attempts at refolding rhIL-7 from solubilized inclusion bodies without prior purification of monomeric, denatured rhIL-7 were not successful. Denatured, monomeric rhIL-7 was therefore initially purified by size-exclusion chromatography using Prep-Grade Pharmacia Superdex 200. Correctly folded rhIL-7 monomer was generated by statically refolding the denatured protein at a final protein concentration of 80-100 microg/ml in 100 mM Tris, 2mM EDTA, 500 mM L-arginine, pH 9.0, buffer with 0.55 g/l oxidized glutathione at 2-8 degrees C for at least 48 h. The refolded rhIL-7 was subsequently purified by low-pressure liquid chromatography, using a combination of hydrophobic interaction, cation-exchange, and size-exclusion chromatography. The purified final product was >95% pure by SDS-PAGE stained with Coomassie brilliant blue, high-pressure size-exclusion chromatography (SEC-HPLC), and reverse-phase HPLC. The endotoxin level was <0.05 EU/mg. The final purified product was biologically active in a validated IL-7 dependent pre-B-cell bioassay. In anticipation of human clinical trials, this material is currently being evaluated for safety and efficacy in non-human primate toxicology studies.     

A folding pattern that is stable to thermal cycling is achieved by long term storage of recombinant human beta-casein with four extra N-terminals amino-acid residues at -20 degrees C

Studies have followed the turbidity (OD400 nm) of beta-casein (CN) as temperature (T) increased from 4 to 37 degrees C. Native non-phosphorylated beta-CN showed a turbidity increase above 25 degrees C and precipitated at about 22 degrees C in 5mM Ca+2. These patterns were reproducible upon T-cycling while those of recombinant beta-CN proteins are not. Here, a wild-type recombinant that was thermally stable after being frozen in solution and stored at -20 degrees C for a prolonged period of time was denatured with guanidine HCl and refolded by dialysis against buffer. This protein was again not stable to T-cycling. A recombinant mutant with four extra N-terminal amino acids was very stable to T-cycling, both with and without 5mM Ca+2. However, it was still much different than the native protein. These results indicate that there are probably many energy minima for this protein and emphasize the possibility of "chaperon-like" conditions for proper folding of human beta-CN.     

Catalysis of oxidative protein folding by small-molecule diselenides

The production of recombinant, disulfide-containing proteins often requires oxidative folding in vitro. Here, we show that diselenides, such as selenoglutathione, catalyze oxidative protein folding by O 2. Substantially lower concentrations of a redox buffer composed of selenoglutathione and the thiol form of glutathione can consequently be used to achieve the same rate and yield of folding as a standard glutathione redox buffer. Further, the low p K a of selenols extends the pH range for folding by selenoglutathione to acidic conditions, where glutathione is inactive. Harnessing the catalytic power of diselenides may thus pave the way for more efficient oxidative protein folding.     

Role of cations in stability of acidic protein Desulfovibrio desulfuricans apoflavodoxin

Apoflavodoxin from the sulfate reducing bacteria Desulfovibrio desulfuricans is a small, acidic protein with a net charge of -19 at neutral pH. Here, we show that monovalent cations in biologically relevant amounts have dramatic effects on apoflavodoxin stability. The effect is largest for Gdm(+) and decreases as a function of increased cation charge density (Gdm(+)>NH(4)(+)K(+) approximately Cs(+) approximately Na(+)>Li(+)). A linear correlation of stabilizing effects with cation hydration properties suggests an important role of dehydration in efficient cation interaction with the protein. The effects on stability are due to preferential binding of one cation to native apoflavodoxin and results in an increase in thermal midpoint of 20 degrees C and the free energy of unfolding (at 20 degrees C) increases fivefold. Tuning of biophysical properties (such as folding and ligand/cofactor binding) of acidic proteins by cation binding may be important in vivo.     

Expression of the A subunit of protein phosphatase 2A and characterization of its interactions with the catalytic and regulatory subunits.

The alpha form of the A subunit of human protein phosphatase 2A was expressed in insect cells following infection with a recombinant baculovirus. A alpha was expressed as a soluble protein that comprised approximately 10% of total cellular protein. The expressed A alpha subunit was purified by chromatography on amino-hexyl-Sepharose and Mono Q with a yield of 2 mg/500-ml culture. The recombinant protein had the same apparent molecular mass as the bovine cardiac protein and was devoid of myosin light chain phosphatase activity. Biological activity of expressed A was assessed by assays of complex formation with the catalytic (C) and B subunits, purified from bovine cardiac tissue, and by inhibition of phosphatase activity. Purified A alpha had a high apparent affinity for C (IC50 = 0.10 nM) and bound with a stoichiometry of 1 mol of A/mol of C. Interaction of A alpha with the catalytic subunit caused a maximal inhibition of myosin light chain and phosphorylase phosphatase activities of 50 and 79%, respectively. The AC complex prepared by reconstitution of recombinant A alpha with C had the same electrophoretic mobility in nondenaturing polyacrylamide gels and the same elution volume when chromatographed on a size exclusion column as the native AC complex purified from cardiac muscle. Similar chromatographic profiles were also observed for the heterotrimer reconstituted from recombinant A alpha, purified B and C, and the native bovine cardiac heterotrimeric holoenzyme. Cross-linking of the native enzyme and the reconstituted heterotrimer generated the same pattern of high molecular weight species. Immunological analyses of these complexes demonstrated that distinct cross-linked forms composed of ABC, AC, AB, and BC were obtained. These results suggest that each of the three subunits of protein phosphatase 2A forms direct contacts with both of the others.     

Stability of recombinant human alpha-1-antitrypsin produced in rice in infant formula

Human milk contains several biologically active proteins that benefit the breast-fed infant. In order to survive in the gastrointestinal tract, these proteins need to be protected against proteolysis. Since human milk contains relatively high concentrations of alpha-1-antitrypsin (AAT), we have expressed recombinant AAT in rice to explore the possibility of supplementing infant formula with this protein. The stability of recombinant AAT was examined by biochemical and functional assays, such as SDS-PAGE, Western blotting, ELISA, elastase and trypsin inhibition, following exposure to heat, low pH, and in vitro digestion, conducted in both phosphate buffered saline and infant formula. Native AAT is resistant to acidic environments down to pH 2 for 1 h and can withstand in vitro digestion modeled after conditions in the infant gut. Recombinant AAT is nearly as resistant as the native form in buffer, and is equally resilient in formula. Heat treatments (60 degrees C for 15 min, 72 degrees C for 20 sec, 85 degrees C for 3 min, and 137 degrees C for 20 sec) revealed that recombinant AAT is not as stable as native AAT in buffer, particularly at higher temperatures. While significantly less recombinant AAT is detected by ELISA after heating in formula, addition of bile extract can restore epitopes resulting in higher concentrations, suggesting protein aggregation that may not affect AAT activity. This study shows that recombinant AAT may survive the conditions of the infant stomach and duodenum and affect protein digestion in the infant small intestine.     

Identification and characterization of cellular receptors for the growth regulator, oncostatin M

Oncostatin M is a polypeptide growth regulator produced by activated T cells and phorbol ester-treated U937 cells. To identify specific cellular receptors for this factor, we have characterized the binding of 125I-labeled oncostatin M to a variety of normal and malignant mammalian cells. Recombinant oncostatin M was labeled with 125I with full retention of growth inhibitory activity on A375 melanoma cells. 125I-Oncostatin M bound to sensitive cells in a time- and temperature-dependent fashion. Binding was specifically inhibited by unlabeled native or recombinant oncostatin M, but not by other polypeptide growth factors tested. Binding to human leukemic and normal blood cells was generally less than to nonhematopoietic cells. With four different cell lines, maximal growth inhibition by oncostatin M was achieved at less than maximal binding site occupancy. Scatchard graphs of direct binding data were curvilinear and indicated that 125I-oncostatin M bound with higher apparent affinity at lower 125I-oncostatin M concentrations. Using a two binding site model, affinity constants of Kd1 = 11 +/- 11 pM and Kd2 = 1000 +/- 380 pM were extrapolated from binding data with A375 cells, and values of Kd1 = 3 +/- 2 pM and Kd2 = 400 +/- 44 pM from A549 cells. The major 125I-oncostatin M binding species in a number of mammalian cell lines was identified by chemical cross-linking as a specific protein(s) of Mr = 150,000-160,000. 125I-Oncostatin M was internalized (t1/2 = 30 min) and degraded subsequent to binding to a responsive cell line.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fully oxidized scrambled isomers are essential and predominant folding intermediates of cardiotoxin-III

Scrambled isomers (X-isomers) are fully oxidized, non-native isomers of disulfide proteins. They have been shown to represent important intermediates along the pathway of oxidative folding of numerous disulfide proteins. A simple method to assess whether X-isomers present as folding intermediate is to conduct oxidative folding of fully reduced protein in the alkaline buffer alone without any supplementing thiol catalyst or redox agent. Cardiotoxin-III (CTX-III) contains 60 amino acids and four disulfide bonds. The mechanism of oxidative folding of CTX-III has been systematically characterized here by analysis of the acid trapped folding intermediates. Folding of CTX-III was shown to proceed sequentially through 1-disulfide, 2-disulfide, 3-disulfide and 4-disulfide (scrambled) isomers as folding intermediates to reach the native structure. When folding of CTX-III was performed in the buffer alone, more than 97% of the protein was trapped as 4-disulfide X-isomers, unable to convert to the native structure due to the absence of thiol catalyst. In the presence of thiol catalyst (GSH) or redox agents (GSH/GSSG), the recovery of native CTX-III was 80-85%. These results demonstrate that X-isomers play an essential and predominant role in the oxidative folding of CTX-III.     

Involvement of alpha-2-macroglobulin receptor in clearance of interleukin 8-alpha-2-macroglobulin complexes by human alveolar macrophages

The purpose of this study was to determine if interleukin 8 (IL-8) in complex with alpha2-macroglobulin (alpha-2-M) can be taken up by human alveolar macrophages. First, we demonstrated that human alveolar macrophages have receptors for alpha-2-M but not IL-8. The binding of(125)I-labeled alpha-2-M to the cells was specific and saturable, whereas(125)I-labeled recombinant human IL-8 (rhIL-8) did not bind to macrophages. However,(125)I-rhIL-8-alpha-2-M complexes bound to macrophages, and unlabeled alpha-2-M competed for the binding. We then cultured the cells in the presence of(125)I-rhIL-8-alpha-2-M complexes,(125)I-rhIL-8 alone or buffer for 24 h. Macrophages were lysed, and the released radioactivity measured. IL-8 concentrations in supernatants and cells were also measured using an IL-8 ELISA. When the macrophages were incubated with(125)I-rhIL-8-alpha-2-M complexes there was a significant amount of IL-8 associated with the cells. However, this was not the case when the cells were incubated with(125)I- rhIL-8 alone suggesting that only these complexes were taken-up by human alveolar macrophages. Furthermore, the clearance of complexes was specifically inhibited by a monoclonal antibody against the 515-kDa subunit of the alpha-2-M receptor (alpha-2-MR) but not by an isotopic mouse IgG1. The study shows an important clearance mechanism for IL-8 in the lung.     

The N-terminal stem region of bovine and human beta1,4-galactosyltransferase I increases the in vitro folding efficiency of their catalytic domain from inclusion bodies

Many recombinant proteins overexpressed in Escherichia coli are generally misfolded, which then aggregate and accumulate as inclusion bodies. The catalytic domain (CD) of bovine and human beta1,4-galactosyltransferase (beta4Gal-T), expressed in E. coli, it also accumulates as inclusion bodies. We studied the effect of the fusion of the stem region (SR), as an N-terminal extension of the catalytic domain, on the in vitro folding efficiencies of the inclusion bodies. The stem region fused to the catalytic domain (SRCD) increases the folding efficiency of recombinant protein with native fold compared to the protein that contains only the CD. During in vitro folding, also promotes considerably the solubility of the misfolded proteins, which do not bind to UDP-agarose columns and exhibit no galactosyltransferase activity. In contrast, the misfolded proteins that consist of only the CD are insoluble and precipitate out of solution. It is concluded that a protein domain that is produced in a soluble form does not guarantee the presence of the protein molecules in a properly folded and active form. The stem domain has a positive effect on the in vitro folding efficiency of the catalytic domain of both human and bovine beta4Gal-T1, suggesting that the stem region acts as a chaperone during protein folding. Furthermore, investigation of the folding conditions of the sulphonated inclusion bodies resulted in identifying a condition in which the presence of PEG-4000 and L-arginine, compared to their absence, increased the yields of native CD and SRCD 7- and 3-fold, respectively.     

Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue

The carboxyl methyltransferase, which is claimed to exclusively methylate the carboxyl group of the C-terminal leucine residue of the catalytic subunit of protein phosphatase 2A (Leu(309)), was purified from porcine brain. On the basis of tryptic peptides, the cDNA encoding the human homologue was cloned. The cDNA of this gene encodes for a protein of 334 amino acids with a calculated M(r) of 38 305 and a predicted pI of 5.72. Database screening reveals the presence of this protein in diverse phyla. Sequence analysis shows that the novel methyltransferase is distinct from other known protein methyltransferases, sharing only sequence motifs supposedly involved in the binding of adenosylmethionine. The recombinant protein expressed in bacteria is soluble and the biophysical, catalytic, and immunological properties are indistinguishable from the native enzyme. The methylation of PP2A by the recombinant protein is restricted to Leu(309) of PP2A(C). No direct effects on phosphatase activity changes were observed upon methylation of the dimeric or trimeric forms of PP2A.     

Folding of the multidomain human immunodeficiency virus type-I integrase.

Protein folding conditions were established for human immunodeficiency virus integrase (IN) obtained from purified bacterial inclusion bodies. IN was denatured by 6 M guanidine.HCl-5 mM dithiothreitol, purified by gel filtration, and precipitated by ammonium sulfate. The reversible solvation of precipitated IN by 6 M guanidine.HCl allowed for wide variation of protein concentration in the folding reaction. A 6-fold dilution of denatured IN by 1 M NaCl buffer followed by dialysis produced enzymatically active IN capable of 3' OH end processing, strand transfer, and disintegration using various human immunodeficiency virus-1 (HIV-1) long terminal repeat DNA substrates. The specific activities of folded IN preparations for these enzymatic reactions were comparable to those of soluble IN purified directly from bacteria. The subunit composition and enzymatic activities of IN were affected by the folding conditions. Standard folding conditions were defined in which monomers and protein aggregates sedimenting as dimers and tetramers wree produced. These protein aggregates were enzymatically active, whereas monomers had reduced strand transfer activity. Temperature modifications of the folding conditions permitted formation of mainly monomers. Upon assaying, these monomers were efficient for strand transfer and disintegration, but the oligomeric state of IN under the conditions of the assay is determinate. Our results suggest that monomers of the multidomain HIV-1 IN are folded correctly for various catalytic activities, but the conditions for specific oligomerization in the absence of catalytic activity are undefined.     

IL-3 promotes basophilic differentiation of KU812 cells through high affinity binding sites

The myeloid precursor cell line KU812 exhibits a constitutive potential to differentiate into basophilic cells. In the present study, the influence of recombinant human (rh)IL-2, rhIL-3, and recombinant human granulocyte-macrophage-CSF on basophilic differentiation of KU812-F cells was studied. Of all cytokines tested, rhIL-3 induced a significant increase in formation of metachromatically granulated cells (from 10% in control cultures up to 30% in cultures supplemented with 100 U/ml of rhIL-3) as well as dose-dependent (1.5- to 3 fold) increase in cellular histamine in KU812-F cell cultures. In addition, KU812-F cells exposed to rhIL-3 bound more IgE antibody than cells cultured in control medium with up to 3.3-fold increases in the mean fluorescence intensity on days 2 and/or 5 compared with control (p less than 0.001). RhIL-3 failed to induce significant changes in expression of the Tac-reactive subunit of the IL-2R (CD25), surface aminopeptidase N (CD13), ICAM-1 Ag (CD54), or CD40 Ag on KU812-F cells. To investigate the mechanism of IL-3 action on KU812-F cells, receptor analyses were performed by using 125I-radiolabeled rhIL-3. Quantitative binding studies and Scatchard plot analyses revealed the presence of a single class of 1910 to 2460 high affinity IL-3-binding sites per KU812-F cell with an apparent dissociation constant of 1.22 to 2.35 x 10(-9) M. Together, these results show that rhIL-3 promotes basophilic differentiation of KU812-F cells through a specific receptor.     

Oxidative protein folding in vitro: a study of the cooperation between quiescin-sulfhydryl oxidase and protein disulfide isomerase

The flavin-dependent quiescin-sulfhydryl oxidase (QSOX) inserts disulfide bridges into unfolded reduced proteins with the reduction of molecular oxygen to form hydrogen peroxide. This work investigates how QSOX and protein disulfide isomerase (PDI) cooperate in vitro to generate native pairings in two unfolded reduced proteins: ribonuclease A (RNase, four disulfide bonds and 105 disulfide isomers of the fully oxidized protein) and avian riboflavin binding protein (RfBP, nine disulfide bonds and more than 34 million corresponding disulfide pairings). Experiments combining avian or human QSOX with up to 200 muM avian or human reduced PDI show that the isomerase is not a significant substrate of QSOX. Both reduced RNase and RfBP can be efficiently refolded in an aerobic solution containing micromolar concentrations of reduced PDI and nanomolar levels of QSOX without any added oxidized PDI or glutathione redox buffer. Refolding of RfBP is followed continuously using the complete quenching of the fluorescence of free riboflavin that occurs on binding to apo-RfBP. The rate of refolding is half-maximal at 30 muM reduced PDI when the reduced client protein (1 muM) is used in the presence of 30 nM QSOX. The use of high concentrations of PDI, in considerable excess over the folding protein client, reflects the concentration prevailing in the lumen of the endoplasmic reticulum and allows the redox poise of these in vitro experiments to be set with oxidized and reduced PDI. In the absence of either QSOX or redox buffer, the fastest refolding of RfBP is accomplished with excess reduced PDI and just enough oxidized PDI to generate nine disulfides in the protein client. These in vitro experiments are discussed in terms of current models for oxidative folding in the endoplasmic reticulum.     

Formation of an active dimer during storage of interleukin-1 receptor antagonist in aqueous solution.

The degradation products of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) formed during storage at 30 degrees C in aqueous solution were characterized. Cationic exchange chromatography of the stored sample showed two major, new peaks eluting before (P1) and after (L2) the native protein, which were interconvertible. Size-exclusion chromatography and electrophoresis documented that both the P1 and L2 fractions were irreversible dimers, formed by noncovalent interactions. A competition assay with interleukin-1 indicated that on a per monomer basis the P1 and L2 dimers retained about two-thirds of the activity of the native monomer. Infrared and far-UV circular dichroism spectroscopies showed that only minor alterations in secondary structure arose upon the formation of the P1 dimer. However, alteration in the near-UV circular dichroism spectrum suggested the presence of disulfide bonds in the P1 dimer, which are absent in the native protein. Mass spectroscopy and tryptic mapping, before and after carboxymethylation, demonstrated that the P1 dimer contained an intramolecular disulfide bond between Cys-66 and Cys-69. Although conversion of native protein to the P1 dimer was irreversible in buffer alone, the native monomer could be regained by denaturing the P1 dimer with guanidine hydrochloride and renaturing it by dialysis, suggesting that the intramolecular disulfide bond does not interfere with refolding. Analysis of the time course of P1 formation during storage at 30 degrees C indicated that the process followed first-order, and not second-order, kinetics, suggesting that the rate-limiting step was not dimerization. It is proposed that a conformational change in the monomer is the rate-limiting step in the formation of the P1 dimer degradation product. Sucrose stabilized the native monomer against this process. This result can be explained by the general stabilization mechanism for this additive, which is due to its preferential exclusion from the protein surface.     

Modification of low density lipoproteins by polymorphonuclear cell elastase leads to enhanced uptake by human monocyte-derived macrophages via the low density lipoprotein receptor pathway

In previous studies we reported that polymorphonuclear cell (PMN) elastase cleaves apoB-100 of human plasma low density lipoprotein (LDL) into seven or eight large Mr fragments (1, Polacek, D., R.E. Byrne, G.M. Fless, and A.M. Scanu. 1986. J. Biol. Chem. 261: 2057-2063). In the present studies we examined the interaction of native and elastase-digested LDL (ED-LDL) with primary cultures of human monocyte-derived macrophages (HMD-M). For this purpose LDL was digested with purified PMN elastase, re-isolated by ultracentrifugation at d 1.063 g/ml to remove the enzyme, and radiolabeled with 125I. At all LDL concentrations in the medium, the degradation of 125I-labeled ED-LDL was 1.5- to 2.5-fold greater than that of 125I-labeled native LDL, and for both lipoproteins species it was further enhanced by prior incubation of the cells in autologous lipoprotein-deficient serum (ALPDS). ED-LDL incubated with HMD-M in a medium containing [14C]oleate stimulated cholesteryl [14C]oleate formation 2- to 3-fold more than native LDL. In competitive degradation experiments, unlabeled ED-LDL did not inhibit the degradation of 125I-labeled acetylated LDL, whereas it caused a 90% inhibition of the degradation of 125I-labeled native LDL. At 4 degrees C, the binding of both 125I-labeled native and 125I-labeled ED-LDL was specific and of a high affinity. At saturation (Bmax), the binding of 125I-labeled ED-LDL was 2-fold higher (68 ng/mg cell protein) than that of 125I-labeled native LDL (31 ng/mg), with Kd values of 6.5 x 10(-8) M and 2.1 x 10(-8) M, respectively. A possible explanation of the binding data was provided by electrophoretic analyses suggesting that ED-LDL was twice the size of native LDL and thus potentially capable of delivering proportionately more cholesterol to the cells. Taken together, the results indicate that 1) digestion of LDL by purified PMN elastase results in a greater mass of ED-LDL (relative to native LDL) being degraded per unit time by HMD-M; 2) uptake of ED-LDL occurs via the LDL receptor; and 3) LDL digested by PMN elastase undergoes a physical change that may be responsible for its unique interactions with HMD-M. We speculate that if this process were to occur in vivo during an inflammatory process, macrophages could acquire excess cholesterol and be transformed into foam cells which are considered to be precursors of the atherosclerotic process.     

Equilibrium and kinetic folding of an alpha-helical Greek key protein domain: caspase recruitment domain (CARD) of RICK

We have characterized the equilibrium and kinetic folding of a unique protein domain, caspase recruitment domain (CARD), of the RIP-like interacting CLARP kinase (RICK) (RICK-CARD), which adopts a alpha-helical Greek key fold. At equilibrium, the folding of RICK-CARD is well described by a two-state mechanism representing the native and unfolded ensembles. The protein is marginally stable, with a DeltaG(H)()2(O) of 3.0 +/- 0.15 kcal/mol and an m-value of 1.27 +/- 0.06 kcal mol(-1) M(-1) (30 mM Tris-HCl, pH 8, 1 mM DTT, 25 degrees C). While the m-value is constant, the protein stability decreases in the presence of moderate salt concentrations (below 200 mM) and then increases at higher salt concentrations. The results suggest that electrostatic interactions are stabilizing in the native protein, and the favorable Coulombic interactions are reduced at low ionic strength. Above 200 mM salt, the results are consistent with Hofmeister effects. The unfolding pathway of RICK-CARD is complex and contains at least three non-native conformations. The refolding pathway of RICK-CARD also is complex, and the data suggest that the unfolded protein folds via two intermediate conformations prior to reaching the native state. Overall, the data suggest the presence of kinetically trapped, or misfolded, species that are on-pathway both in refolding and in unfolding.     

Characterization of Lys-698-to-Met substitution in human plasminogen catalytic domain

Streptokinase (SK) is a human plasminogen (Pg) activator secreted by streptococci. The activation mechanism of SK differs from that of physiological Pg activators in that SK is not a protease and cannot proteolytically activate Pg. Instead, it forms a tight complex with Pg that proteolytically activates other Pg molecules. The residue Lys-698 of human Pg was hypothesized to participate in triggering activation in the SK-Pg complex. Here, we report a study of the Lys-698 to Met substitution in the catalytic domain of Pg (microPg) containing the proteolytic activation-resistant background (R561A). While it remains competent in forming a complex with SK, maintaining a comparable equilibration dissociation constant (K(D)), the recombinant protein shows a nearly 60-fold reduction in amidolytic activity relative to its R561A background when mixed with native SK. A 2.3 A crystal structure of this mutant microPg confirmed the correct folding of this recombinant protein. Combined with other biochemical data, these results support the premise that Lys-698 of human Pg plays a functional role in the so-called N-terminal insertion activation mechanism by SK.     

Independent intramolecular mediators of folding, activity, and inhibition for the Plasmodium falciparum cysteine protease falcipain-2

The Plasmodium falciparum cysteine protease falcipain-2 is a trophozoite hemoglobinase and potential antimalarial drug target. Unlike other studied papain family proteases, falcipain-2 does not require its prodomain for folding to active enzyme. Rather, folding is mediated by an amino-terminal extension of the mature protease. As in related enzymes, the prodomain is a potent inhibitor of falcipain-2. We now report further functional evaluation of the domains of falcipain-2 and related plasmodial proteases. The minimum requirement for folding of falcipain-2 and four related plasmodial cysteine proteases was inclusion of a 14-15-residue amino-terminal folding domain, beginning with a conserved Tyr. Chimeras of the falcipain-2 catalytic domain with extensions from six other plasmodial proteases folded normally and had kinetic parameters (k(cat)/K(m) 124,000-195,000 M(-1) s(-1)) similar to those of recombinant falcipain-2 (k(cat)/K(m) 120,000 M(-1) s(-1)), indicating that the folding domain is functionally conserved across the falcipain-2 subfamily. Correct folding also occurred when the catalytic domain was refolded with a separate prodomain-folding domain construct but not with an isolated folding domain peptide. Thus, the prodomain mediated interaction between the other two domains when they were not covalently bound. The prodomain-catalytic domain interaction was independent of the active site, because it was blocked by free inactive catalytic domain but not by the active site-binding peptide leupeptin. The folded catalytic domain retained activity after purification from the prodomain-folding domain construct (k(cat)/K(m) 168,000 M(-1) s(-1)), indicating that the folding domain is not required for activity once folding has been achieved. Activity was lost after nonreducing gelatin SDS-PAGE but not native gelatin PAGE, indicating that correct disulfide bonds are insufficient to direct appropriate folding. Our results identify unique features of the falcipain-2 subfamily with independent mediation of activity, folding, and inhibition.