Equilibrium unfolding of kinetically stable serine protease milin: the presence of various active and inactive dimeric intermediates

Kinetically stable homodimeric serine protease milin reveals high conformational stability against temperature, pH and chaotrope [urea, guanidine hydrochloride (GuHCl) and guanidine isothiocynate (GuSCN)] denaturation as probed by circular dichroism, fluorescence, differential scanning calorimetry and activity measurements. GuSCN induces complete unfolding in milin, whereas temperature, urea and GuHCl induce only partial unfolding even at low pH, through several intermediates with distinct characteristics. Some of these intermediates are partially active (viz. in urea and 2 M GuHCl at pH 7.0), and some exhibited strong ANS binding as well. All three tryptophans in the protein seem to be buried in a rigid, compact core as evident from intrinsic fluorescence measurements coupled to equilibrium unfolding experiments. The protein unfolds as a dimer, where the unfolding event precedes dimer dissociation as confirmed by hydrodynamic studies. The solution studies performed here along with previous biochemical characterization indicate that the protein has α-helix and β-sheet rich regions or structural domains that unfold independently, and the monomer association is isologous. The complex unfolding pathway of milin and the intermediates has been characterized. The physical, physiological and probable therapeutic importance of the results has been discussed.     

Dimeric procaspase-3 unfolds via a four-state equilibrium process.

We have examined the folding and assembly of a catalytically inactive mutant of procaspase-3, a homodimeric protein that belongs to the caspase family of proteases. The caspase family, and especially caspase-3, is integral to apoptosis. The equilibrium unfolding data demonstrate a plateau between 3 and 5 M urea, consistent with an apparent three-state unfolding process. However, the midpoint of the second transition as well as the amplitude of the plateau are dependent on the protein concentration. Overall, the data are well described by a four-state equilibrium model in which the native dimer undergoes an isomeration to a dimeric intermediate, and the dimeric intermediate dissociates to a monomeric intermediate, which then unfolds. By fitting the four-state model to the experimental data, we have determined the free energy change for the first step of unfolding to be 8.3 +/- 1.3 kcal/mol. The free energy change for the dissociation of the dimeric folding intermediate to two monomeric intermediates is 10.5 +/- 1 kcal/mol. The third step in the unfolding mechanism represents the complete unfolding of the monomeric intermediate, with a free energy change of 7.0 +/- 0.5 kcal/mol. These results show two important points. First, dimerization of procaspase-3 occurs as a result of the association of two monomeric folding intermediates, demonstrating that procaspase-3 dimerization is a folding event. Second, the stability of the dimer contributes significantly to the conformational free energy of the protein (18.8 of 25.8 kcal/mol).     

Equilibrium unfolding of dimeric human prostatic acid phosphatase involves an inactive monomeric intermediate

Guanidine hydrochloride (GdnHCl)-induced unfolding of human prostatic acid phosphatase (hPAP), a homodimer of 50 kDa subunit molecular weight, was investigated with activity measurements, size exclusion HPLC, tryptophan fluorescence, 1-anilinonaphtalene-8-sulfonate (ANS) binding and reactivity with 2-(4'-maleimidoanilino)naphthalene-6-sulfonate (MIANS). Equilibrium analysis was performed to shed light on the role of dimerization in the folding and stability of the catalytically active oligomeric protein. Unfolding was reversible, as verified by activity measurements and tryptophan fluorescence. The noncoincidence of the unfolding curves obtained by different techniques suggests the occurrence of a multiphasic process.The reaction of hPAP inactivation is accompanied by dissociation of the dimer into two monomers. The midpoint of this transition is at 0.65 M GdnHCl with 4.24+/-0.12 kcalmol(-1) free energy change. Binding of ANS to the inactive phosphatase monomer, especially remarkable in the region from 0.8 to 1.25M GdnHCl, suggests that the hydrophobic probe indicates exposition of the intersubunit hydrophobic surface and a loosening of the monomer's tertiary structure. Strong fluorescence of thiol group derivatives, the products of their reaction with MIANS, appears in a limited range of GdnHCl concentrations (1.2-1.6M). This shows that in the relaxed structure of the intermediate, the reagent is allowed to penetrate into the hydrophobic environment of the partially hidden thiol groups.The equilibrium unfolding reaction of hPAP, as monitored by tryptophan fluorescence, does not depend on the protein concentration and displays a single transition curve with a midpoint at 1.7 M GdnHCl and value of DeltaG(unf)(H(2)O)=3.38+/-0.08 kcalmol(-1) per monomer, a result implying that this transition is related to the conformational change of the earlier dissociated and already inactive subunit of the protein.     

Preparation of monomeric acrylic ester intermediates using lipase catalysed transesterifications in organic solvents

Summary Acrylate monoesters were synthesized on a preparative scale by the regioselective enzymatic transesterification of a range of diols dissolved in ethyl acrylate using a commercial lipase fromChromobacterium viscosum.     

Synthesis of ppTppp via phosphotriester intermediates.

The bifunctional and crystalline phosphorylating agent morpholino-0,0-bis[1-benzotriazolyl]phosphate has been used for the preparation of a 3',5'-bis-phosphotriester intermediate of thymidine. The latter has been converted into ppTppp by the following consecutive steps; removal of the benzotriazolyl group followed by the addition of phosphoric acid and removal of the 2-(4-nitrophenyl)-ethyl group followed by the addition of pyrophosphoric acid.     

Design of a monomeric human glutathione transferase GSTP1, a structurally stable but catalytically inactive protein

By the introduction of 10 site-specific mutations in the dimer interface of human glutathione transferase P1-1 (GSTP1-1), a stable monomeric protein variant, GSTP1, was obtained. The monomer had lost the catalytic activity but retained the affinity for a number of electrophilic compounds normally serving as substrates for GSTP1-1. Fluorescence and circular dichroism spectra of the monomer and wild-type proteins were similar, indicating that there are no large structural differences between the subunits of the respective proteins. The GSTs have potential as targets for in vitro evolution and redesign with the aim of developing proteins with novel properties. To this end, a monomeric GST variant may have distinct advantages.     

Yoked complexes of human choriogonadotropin and the lutropin receptor: evidence that monomeric individual subunits are inactive

Human choriogonadotropin (hCG) contains an alpha-subunit, common to other members of the glycoprotein hormone family, and a unique beta-subunit that determines hormone specificity. It is generally thought that heterodimer formation is obligatory for full hormonal activity, although other studies have indicated that individual subunits and homodimeric hCGbeta were capable of low affinity binding to the LH receptor (LHR) and subsequent activation. Previously, we constructed two yoked hormone (hCG)-LHR complexes, where the two hormone subunits and the heptahelical receptor were engineered to form single polypeptide chains, i.e. N-beta-alpha-LHR-C and N-alpha-beta-LHR-C. Expression of both complexes led to constitutive stimulation of cAMP production. In the present study, we investigated whether the human alpha-subunit and hCGbeta can act as functional agonists when covalently attached to or coexpressed with the LH receptor. Our initial results showed that hCGbeta, but not alpha, was able to activate LHR with an increase in intracellular cAMP in human embryonic kidney 293 cells but not in Chinese hamster ovary or COS-7 cells. Further examination of this apparent cell-specific agonist activity of hCGbeta revealed that low levels of endogenous alpha-subunit were expressed in human embryonic kidney 293 cells, thus enabling sufficient amounts of active heterodimer to form with the transfected hCGbeta to activate LHR. The studies in Chinese hamster ovary and COS-7 cells clearly demonstrate that, even under experimental conditions where hormone-receptor interactions are maximized, individual subunits of hCG can not act as functional agonists, at least in their monomeric form.     

Synthesis of DNA via deoxynucleoside H-phosphonate intermediates.

Deoxynucleoside H-phosphonates are used in the chemical synthesis of deoxyoligonucleotides up to 107 bases in length. The biological activity of the synthetic DNA is assessed by cloning into M13 and sequencing. An improved synthesis of protected deoxynucleoside H-phosphonates is also described.     

Evolution of plant mitochondrial genomes via substoichiometric intermediates

Comparison of the modern fertile maize mitochondrial genome (N) with an ancestral maize mitochondrial genome (RU) reveals a 12 kb duplication (containing the atpA gene) in the modern genome that is absent from the ancestor. Cloning, mapping, and sequencing of the relevant portions of the ancestral genome shows that this duplication probably arose via a three-stage recombination process involving substoichiometric intermediates. Comparison with analogous observations on yeast mitochondrial genomes suggests that this three-stage model of genome reorganization can be generally applied to plant mitochondrial genomes to explain both deletions and the creation of novel repeats, common features of plant mitochondrial genome evolution.     

Modeling the deamidation of asparagine residues via succinimide intermediates

Density functional theory (B3LYP/6-31G*) has been used to study the cyclization, deamidation and hydrolysis reactions of a model peptide. Single point energy calculations with the polarized continuum model drastically lower the activation energy for cyclization in a basic medium. Confirmation of the experimental results that cyclization is slower than deamidation in acidic media and the opposite is true in basic media has enabled us to propose mechanisms for both processes.     

Site-specific integration of Agrobacterium tumefaciens T-DNA via double-stranded intermediates

Agrobacterium tumefaciens-mediated genetic transformation involves transfer of a single-stranded T-DNA molecule (T strand) into the host cell, followed by its integration into the plant genome. The molecular mechanism of T-DNA integration, the culmination point of the entire transformation process, remains largely obscure. Here, we studied the roles of double-stranded breaks (DSBs) and double-stranded T-DNA intermediates in the integration process. We produced transgenic tobacco (Nicotiana tabacum) plants carrying an I-SceI endonuclease recognition site that, upon cleavage with I-SceI, generates DSB. Then, we retransformed these plants with two A. tumefaciens strains: one that allows transient expression of I-SceI to induce DSB and the other that carries a T-DNA with the I-SceI site and an integration selection marker. Integration of this latter T-DNA as full-length and I-SceI-digested molecules into the DSB site was analyzed in the resulting plants. Of 620 transgenic plants, 16 plants integrated T-DNA into DSB at their I-SceI sites; because DSB induces DNA repair, these results suggest that the invading T-DNA molecules target to the DNA repair sites for integration. Furthermore, of these 16 plants, seven plants incorporated T-DNA digested with I-SceI, which cleaves only double-stranded DNA. Thus, T-strand molecules can be converted into double-stranded intermediates before their integration into the DSB sites within the host cell genome.     

Synthesis of phospholipids and nucleoside-phospholipid conjugates via hydrogenphosphonate intermediates

Chemical synthesis of nucleoside-phospholipid conjugates based on hydrogenphosphonate chemistry has been achieved via coupling of 1,2-dipalmitoylglycero-3-H-phosphonate with suitable protected nucleosides or via coupling of nucleoside H-phosphonates with 1,2-dipalmitoylglycerol. It was also found that 1,2-dipalmitoylglycero-3-H-phosphonate, which is a stable compound, can serve as a convenient intermediate in the synthesis of various phospholipids.     

The delay phenomenon: the story unfolds

Our previous studies have shown that when a flap is delayed, there is dilation of existing vessels within the flap not ingrowth of new vessels. The maximal anatomic effect on the arterial tree occurs at the level of the reduced-caliber "choke" anastomotic vessels that link adjacent vascular territories. To further investigate the sequence of anatomic changes that occurs during the delay phenomenon, a large series of 200 rabbits and 17 dogs underwent a flap delay procedure in either skin or muscle and the tissues were examined at postoperative periods between 1 hour and 1 year by using well-established fluorescein, angiographic, light microscopic, immunohistochemical, and electron microscopic techniques. These data in the rabbit skin consistently demonstrated an initial period of vasoconstriction that resolved within 3 hours postoperatively and was followed by an active and progressive dilation of choke vessels that was most dramatic between 48 and 72 hours. In vivo intravenous fluorescein dye testing revealed an interesting parallel in that there was a temporary barrier to the flow of fluorescein that occurred at the level of the choke vessels immediately after the flap was raised and that this temporary barrier-continued to impede the flow toward the flap tip in rabbits where flaps had been delayed for periods up to 72 hours. Thereafter, there was no obstruction to the flow of fluorescein along the flap. During this early delay period of 3 days, light microscopy revealed a decrease in vessel wall thickness associated with an increase in lumen diameter. Over the next 4 days, the luminal diameter continued to dilate to a lesser extent and the vessel wall thickened. Immunohistochemical analysis showed increased cell division, maximal between 24 and 72 hours, in all layers of the choke vessel wall. During this same postoperative interval, transmission electron microscopy revealed phenotypic changes in smooth muscle cells from contractile to synthetic cells. Hypertrophy of the smooth muscle cells was also observed. The vascular endothelium, which initially showed evidence of denudation, was restored to a healthy intact appearance within the first week after delay. When followed for longer periods, long-term studies of the delayed flap of up to 1 year demonstrated dramatically a permanent dilation of the choke vessel lumen and a thickening of the choke vessel wall. In canine studies, one rectus abdominis muscle was delayed by ligating the deep inferior epigastric artery. The time sequence of choke vessel dilation, studied by sequential angiograms in vivo, was comparable to that of the rabbit skin model. To ascertain the permanence and irreversibility of this dilation, the normal circulation of the delayed rectus abdominis muscle was restored by reanastomosing the deep inferior epigastric artery. Even after a recovery period of up to 3 months, the choke vessels remained dilated and tortuous instead of reverting to their original narrow diameters. From this work, it is suggested that the choke vessel dilation seen in the delay period is a permanent and irreversible event. It is an active process associated with both an increase (hyperplasia) and an enlargement (hypertrophy) of the cells in all layers of the choke artery wall and a resultant increase in caliber of these vessels. The time sequence for delay appears to be similar in different species and in different tissues, suggesting the possibility of a universal process for delay.     

Isolation and structural characterization of monomeric and dimeric forms of replicative intermediates of Kilham rat virus DNA.

Two virus-specific species of newly synthesized DNA were isolated from rat fibroblast cell cultures infected with the Kilham rat virus (RV). These two DNA species were purified; their behavior on hydroxyapatite chromatography and their sedimentation coefficients in sucrose gradients were determined. One of the two species corresponds to the linear double-stranded form of the RV DNA, and the other corresponds to the dimeric duplex form. After denaturation, a fraction of both species showed an intramolecular renaturation; these molecules are composed of viral strand covalently linked to complementary strand. Models for the structure of both species are posposed. Both species may be considered as double-strand replicative intermediates of the single-stranded RV DNA.     

Reversible equilibrium unfolding of triosephosphate isomerase from Trypanosoma cruzi in guanidinium hydrochloride involves stable dimeric and monomeric intermediates

The reversible guanidinium hydrochloride-induced unfolding of Trypanosoma cruzi triosephosphate isomerase (TcTIM) was characterized under equilibrium conditions. The catalytic activity was followed as a native homodimeric functional probe. Circular dichroism, intrinsic fluorescence, and size-exclusion chromatography were used as secondary, tertiary, and quaternary structural probes, respectively. The change in ANS fluorescence intensity with increasing denaturant concentrations was also determined. The results show that two stable intermediates exist in the transition from the homodimeric native enzyme to the unfolded monomers: one (N(2*)) is a slightly more expanded, non-native, and active dimer, and the other is a partially expanded monomer (M) that binds ANS. Spectroscopic and activity data were used to reach a thermodynamic characterization. The results indicate that the Gibbs free energies for the partial reactions are 4.5 (N(2) <==> N(2*)), 65.8 (N(2*) <==> 2M), and 17.8 kJ/mol (M <==> U). It appears that TcTIM monomers are more stable than those found for other TIM species (except yeast TIM), where monomer stability is only marginal. These results are compared with those for the guanidinium hydrochloride-induced denaturation of TIM from different species, where despite the functional and three-dimensional similarities, a remarkable heterogeneity exists in the unfolding pathways.     

Synthesis of leukotriene A4 methyl ester via acetylene intermediates

Rac-leukotriene A4 methyl ester has been synthesized from propargylic alcohol and 1-heptyne. The synthetic strategy involves the assembly of carbon chain by acetylenide anion condensations and the introduction of (Z)-double bonds by the triple bond hydrogenation.     

Glycosylation of myelin glycoproteins in peripheral nerve via lipid intermediates

Membrane preparations from chick peripheral nervous system (PNS) catalyzed the transfer of [³H]glucose from UDP-[³H]glucose into glucosylphosphoryl dolichol. The initial rate of glucosylphosphoryl dolichol formation in a non-myelin membrane fraction from actively myelinating chick PNS was 11 fold higher than that from adult. Exogenous dolichyl monophosphate stimulated glucosylphosphoryl dolichol synthesis in both fractions. The higher level of glucosylphosphoryl dolichol synthesis corresponded to the onset of myelination in chick PNS. Exogenous dolichyl monophosphate also stimulated the labeling of glucosylated oligosaccharide lipids and glycoproteins in the fraction. On SDS polyacrylamide gel electrophoresis, the relative mobility of the major and minor radioactive glycoprotein corresponded with that of the P0 and PASII glycoprotein in PNS myelin, respectively. The results suggest that myelin glycoproteins in PNS are glycosylated via lipid intermediates.