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.     

A kinetically stable plant subtilase with unique peptide mass fingerprints and dimerization properties

Milin, a potent molluscicide from the latex of Euphorbia milii, holds promise in medicinal biochemistry. Electrophoresis, size exclusion chromatography, mass spectrometry and other biochemical characteristics identify milin as a homodimeric, plant subtilisin-like serine protease, the first of its kind. The subunits of milin are differentially glycosylated affecting dimer association, solubility and proteolytic activity. The dimeric dissociation is SDS-insensitive and strongly temperature dependent but does not appear to be linked by disulfide bridges. N-terminal sequence of acid hydrolyzed peptide fragments shows no homology to known serine protease. Peptide mass fingerprinting and de novo sequencing of the tryptic fragments also did not identify putative domains in the protein. Milin seems to be a novel plant enzyme with subunit association partly similar to human herpes virus serine proteases and partly to penicillin binding proteins. Its behaviour on SDS-PAGE gels and other properties is like "kinetically stable" proteins. Such subunit association and properties might play a critical role in its physiological function and in controlling Schistosomiasis.     

Distinct secretases, a cysteine protease and a serine protease, generate the C termini of amyloid beta-proteins Abeta1-40 and Abeta1-42, respectively

The carboxy-terminal ends of the 40- and 42-amino acids amyloid beta-protein (Abeta) may be generated by the action of at least two different proteases termed gamma(40)- and gamma(42)-secretase, respectively. To examine the cleavage specificity of the two proteases, we treated amyloid precursor protein (APP)-transfected cell cultures with several dipeptidyl aldehydes including N-benzyloxycarbonyl-Leu-leucinal (Z-LL-CHO) and the newly synthesized N-benzyloxycarbonyl-Val-leucinal (Z-VL-CHO). All dipeptidyl aldehydes tested inhibited production of both Abeta1-40 and Abeta1-42. Changes in the P1 and P2 residues of these aldehydes, however, indicated that the amino acids occupying these positions are important for the efficient inhibition of gamma-secretases. Peptidyl aldehydes inhibit both cysteine and serine proteases, suggesting that the two gamma-secretases belong to one of these mechanistic classes. To differentiate between the two classes of proteases, we treated our cultures with the specific cysteine protease inhibitor E-64d. This agent inhibited production of secreted Abeta1-40, with a concomitant accumulation of its cellular precursor indicating that gamma(40)-secretase is a cysteine protease. In contrast, this treatment increased production of secreted Abeta1-42. No inhibition of Abeta production was observed with the potent calpain inhibitor I (acetyl-Leu-Leu-norleucinal), suggesting that calpain is not involved. Together, these results indicate that gamma(40)-secretase is a cysteine protease distinct from calpain, whereas gamma(42)-secretase may be a serine protease. In addition, the two secretases may compete for the same substrate. Dipeptidyl aldehyde treatment of cultures transfected with APP carrying the Swedish mutation resulted in the accumulation of the beta-secretase C-terminal APP fragment and a decrease of the alpha-secretase C-terminal APP fragment, indicating that this mutation shifts APP cleavage from the alpha-secretase site to the beta-secretase site.     

The effects of time,temperature, and pH on the stability of PDU bacterial microcompartments

Bacterial microcompartments (MCPs) are subcellular organelles that are composed of a protein shell and encapsulated metabolic enzymes. It has been suggested that MCPs can be engineered to encapsulate protein cargo for use as in vivo nanobioreactors or carriers for drug delivery. Understanding the stability of the MCP shell is critical for such applications. Here, we investigate the integrity of the propanediol utilization (Pdu) MCP shell of Salmonella enterica over time, in buffers with various pH, and at elevated temperatures. The results show that MCPs are remarkably stable. When stored at 4°C or at room temperature, Pdu MCPs retain their structure for several days, both in vivo and in vitro. Furthermore, Pdu MCPs can tolerate temperatures up to 60°C without apparent structural degradation. MCPs are, however, sensitive to pH and require conditions between pH 6 and pH 10. In nonoptimal conditions, MCPs form aggregates. However, within the aggregated protein mass, MCPs often retain their polyhedral outlines. These results show that MCPs are highly robust, making them suitable for a wide range of applications.     

Calpain proteolysis of free and bound forms of calponin, a troponin T-like protein in smooth muscle

Calponin, a novel homologue of troponin T, purified from chicken gizzard was found to be one of the most susceptible proteins among smooth muscle contraction-associated proteins to hydrolysis by calpain I purified from human red blood cells. The high susceptibility of calponin was comparable to that reported for troponin T. The rate of degradation of calponin, unlike caldesmon and myosin light chain kinase, was accelerated when bound to calmodulin. When calponin existed as a bound form in both reconstituted actin filament and native thin filament, the rate of proteolysis was markedly retarded, indicating close association of calponin with actin filament. These observations are compatible with the view that calponin is an integral part of the actin-linked contractile machinery in smooth muscle.     

Increase in the stability of serine acetyltransferase from Escherichia coli against cold inactivation and proteolysis by forming a bienzyme complex

Cysteine synthetase from Escherichia coli is a bienzyme complex composed of serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase-A (OASS). The effects of the complex formation on the stability of SAT against cold inactivation and proteolysis were investigated. SAT was reversibly inactivated on cooling to 0 degrees C. Ultracentrifugal analysis showed that SAT (a hexamer) was dissociated mostly into two trimers on cooling to 0 degrees C in the absence of OASS, while in the presence of OASS one trimer of the SAT subunits formed a complex with one dimer of OASS subunits. In the presence of OASS, not only the cold inactivation rate was reduced but also the reactivation rate was increased. Furthermore, SAT became stable against proteolytic attack by alpha-chymotrypsin and V8 protease by forming the complex with OASS. On the other hand, SAT was degraded by trypsin in the same manner both in the presence and in the absence of OASS. The different tendency in the stability against proteolysis with the different proteases was discussed with respect to the substrate specificity of the proteases and amino acid sequence of the C-terminal region of SAT that interacts with OASS.     

Purification and characterization of a putative virulence factor,serine protease,from Vibrio parahaemolyticus

Binding of immobilized collagen-I (Cn-I) and fibronectin (Fn) by Lactobacillus acidophilus CRL 639 depends on cell-surface proteins. Capsule formation during the stationary growth phase has a negative effect on adherence of Cn-I and Fn. However, cells from the exponential growth phase, which produce no capsule, exhibit maximal binding. Binding is sensitive to trypsin, proteinase K, pronase E, and heat. Gelatin and soluble Cn-I partially inhibit binding of Cn-I although various proteins, sugars and amino acids do not affect binding to Fn. These results indicate that protein-protein interactions mediate adhesion to extracellular matrix proteins. SDS-PAGE and Western blot analyses of surface proteins revealed that several proteins including the major 43-kDa protein of the S-layer are expressed. Monoclonal antibodies showed that Fn binds to a 15-kDa protein, while Cn-I binds to proteins of 45 and 58 kDa.     

Three-dimensional, sequence order-independent structural comparison of a serine protease against the crystallographic database reveals active site similarities: potential implications to evolution and to protein folding.

We have recently developed a fast approach to comparisons of 3-dimensional structures. Our method is unique, treating protein structures as collections of unconnected points (atoms) in space. It is completely independent of the amino acid sequence order. It is unconstrained by insertions, deletions, and chain directionality. It matches single, isolated amino acids between 2 different structures strictly by their spatial positioning regardless of their relative sequential position in the amino acid chain. It automatically detects a recurring 3D motif in protein molecules. No predefinition of the motif is required. The motif can be either in the interior of the proteins or on their surfaces. In this work, we describe an enhancement over our previously developed technique, which considerably reduces the complexity of the algorithm. This results in an extremely fast technique. A typical pairwise comparison of 2 protein molecules requires less than 3 s on a workstation. We have scanned the structural database with dozens of probes, successfully detecting structures that are similar to the probe. To illustrate the power of this method, we compare the structure of a trypsin-like serine protease against the structural database. Besides detecting homologous trypsin-like proteases, we automatically obtain 3D, sequence order-independent, active-site similarities with subtilisin-like and sulfhydryl proteases. These similarities equivalence isolated residues, not conserving the linear order of the amino acids in the chains. The active-site similarities are well known and have been detected by manually inspecting the structures in a time-consuming, laborious procedure. This is the first time such equivalences are obtained automatically from the comparison of full structures. The far-reaching advantages and the implications of our novel algorithm to studies of protein folding, to evolution, and to searches for pharmacophoric patterns are discussed.     

Effects of cavity-creating mutations on conformational stability and structure of the dimeric 4-α-helical protein ROP: Thermal unfolding studies

The structural and energetic perturbations caused by cavity-creating mutations (Leu-41 → Val and Leu-41 → Ala) in the dimeric 4-α-helical-bundle protein ROP have been characterized by CD spectroscopy and differential scanning calorimetry (DSC). Deconvolution of the CD spectra showed a decrease in α -helicity as a result of the amino acid exchanges that follows qualitatively the overall decrease in conformational stability. Transition enthalpies are sensitive probes of the energetic change associated with point mutations. ΔH⁰ values at the respective transition temperatures, T_1/2 (71.0, 65.3, and 52.9°C at 0.5 mg/ml) decrease from 580 ± 20 to 461 ± 20 kJ/(mol of dimmer) and 335 ± 20 kJ/(mol of dimmer) for wildtype ROP (Steif, C., Weber, P., Hinz, H.-J., Flossdorf, J., Cesareni, G., Kokkinidis, M. Biochemistry 32:3867-3876, 1993), L⁴¹V, and L⁴¹A, respectively. The conformational stabilities at 25°C expressed by the standard Gibbs energies of denaturation, ΔG, are 71.7, 61.1, and 46.1 kJ/(mol of dimmer). The corresponding transition enthalpies have been obtained from extrapolation using the c(T)and c(T) functions. Their values at 25°C are 176.3, 101.9, and 141.7 kJ/(mol of dimmer) for wild-type ROP, L⁴¹V, and L⁴¹A, respectively. When the stability perturbation resulting from the cavity creating mutations is referred to the exchange of 1 mol of CH₂ group, the average ΔΔG value is ?5.0 ± 1 kJ/(mol of CH₂ group). This decrease in conformation stability suggests that dimeric ROP exhibits the same susceptibility to Leu → Yal and Leu → Ala exchanges as small monomeric proteins. Careful determinations of the partial specific heat capacities of wild-type and mutated protein solutions suggest that the mutational effects are predominantly manifested in the native rather than the unfolded state. © 1995 Wiley-Liss, Inc.     

Structure, conformational stability, and enzymatic properties of acylphosphatase from the hyperthermophile Sulfolobus solfataricus

The structure of AcP from the hyperthermophilic archaeon Sulfolobus solfataricus has been determined by (1)H-NMR spectroscopy and X-ray crystallography. Solution and crystal structures (1.27 A resolution, R-factor 13.7%) were obtained on the full-length protein and on an N-truncated form lacking the first 12 residues, respectively. The overall Sso AcP fold, starting at residue 13, displays the same betaalphabetabetaalphabeta topology previously described for other members of the AcP family from mesophilic sources. The unstructured N-terminal tail may be crucial for the unusual aggregation mechanism of Sso AcP previously reported. Sso AcP catalytic activity is reduced at room temperature but rises at its working temperature to values comparable to those displayed by its mesophilic counterparts at 25-37 degrees C. Such a reduced activity can result from protein rigidity and from the active site stiffening due the presence of a salt bridge between the C-terminal carboxylate and the active site arginine. Sso AcP is characterized by a melting temperature, Tm, of 100.8 degrees C and an unfolding free energy, DeltaG(U-F)H2O, at 28 degrees C and 81 degrees C of 48.7 and 20.6 kJ mol(-1), respectively. The kinetic and structural data indicate that mesophilic and hyperthermophilic AcP's display similar enzymatic activities and conformational stabilities at their working conditions. Structural analysis of the factor responsible for Sso AcP thermostability with respect to mesophilic AcP's revealed the importance of a ion pair network stabilizing particularly the beta-sheet and the loop connecting the fourth and fifth strands, together with increased density packing, loop shortening and a higher alpha-helical propensity.     

Biophysical analysis of phaseolin denaturation induced by urea, guanidinium chloride, pH, and temperature.

The structural stability of phaseolin was determined by using absorbance, circular dichroism (CD), fluorescence emission, and fluorescence polarization anisotropy to monitor denaturation induced by urea, guanidinium chloride (GdmCl),pH changes, increasing temperature, or a combination thereof. Initial results indicated that phaseolin remained folded to a similar extent in the presence or absence of 6.0 M urea or GdmCl at room temperature. In 6.0 M GdmCl, phaseolin denatures at approximately 65°C when probed with absorbance, CD, and fluorescence polarization anisotropy. The transition occurs at lower temperatures by decreasingpH. Kinetic measurements of denaturation using CD indicated that the denaturation is slow below 55°C and is associated with an activation energy of 52 kcal/mol in 6.0 M GdmCl. In addition, kinetic measurement using fluorescence emission indicated that the single tryptophan residue was sensitive to at least two steps of the denaturation process. The fluorescence emission appeared to reflect some other structural perturbation than protein denaturation, as fluorescence inflection occurred approximately 5°C prior to the changes observed in absorbance, CD, and fluorescence polarization anisotropy.     

Stereoselectivity and conformational stability of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110: the effect of pH and temperature

The effect of pH and temperature on structure, stability, activity and enantioselectivity of haloalkane dehalogenase DbjA from Bradyrhizobium japonicum USDA110 was investigated in this study. Conformational changes have been assessed by circular dichroism spectroscopy, functional changes by kinetic analysis, while quaternary structure was studied by gel filtration chromatography. Our study shows that the DbjA enzyme is highly tolerant to pH changes. Its secondary and tertiary structure was not affected by pH in the ranges 5.3-10.3 and 6.2-10.1, respectively. Oligomerization of DbjA was strongly pH-dependent: monomer, dimer, tetramer and a high molecular weight cluster of the enzyme were distinguished in solution at different pH conditions. Moreover, different oligomeric states of DbjA possessed different thermal stabilities. The highest melting temperature (T(m) = 49.1 ± 0.2 °C) was observed at pH 6.5, at which the enzyme occurs in dimeric form. Maximal activity was detected at 50 °C and in the pH interval 7.7-10.4. While pH did not have any effect on enantiodiscriminination of DbjA, temperature significantly altered DbjA enantioselectivity. A decrease in temperature results in significantly enhanced enantioselectivity. The temperature dependence of DbjA enantioselectivity was analysed with 2-bromobutane, 2-bromopentane, methyl 2-bromopropionate and ethyl 2-bromobutyrate, and differential activation parameters Δ(R-S)ΔH and Δ(R-S)ΔS were determined. The thermodynamic analysis revealed that the resolution of β-bromoalkanes was driven by both enthalpic and entropic terms, while the resolution of α-bromoesters was driven mainly by an enthalpic term. Unique catalytic activity and structural stability of DbjA in a broad pH range, combined with high enantioselectivity with particular substrates, make this enzyme a very versatile biocatalyst. Enzyme EC3.8.1.5 haloalkane dehalogenase.     

Bacterial production and purification of SGPI-1 and SGPI-2, two peptidic serine protease inhibitors from the desert locust, Schistocerca gregaria

The last decade, a new serine protease inhibitor family has been described in arthropods. Eight members were purified from the locusts Locusta migratoria (LMPI-1-2 and HI) and Schistocerca gregaria (SGPI-1-5) and 11 additional locust peptides were identified by cDNA cloning. Furthermore, the light chain of the 155-kDa heterodimeric protease inhibitor pacifastin, from the freshwater crayfish Pacifastacus leniusculus, was found to be composed of nine consecutive inhibitory domains (PLDs). These domains share a pattern of 6 conserved cysteine residues (Cys-Xaa(9-12)-Cys-Asn-Xaa-Cys-Xaa-Cys-Xaa(2-3)-Gly-Xaa(3-4)-Cys-Thr-Xaa3-Cys) with the locust inhibitors. So far, for most of the PLD-related peptides the biological functions remain obscure. To obtain sufficient amounts of material to perform physiological experiments, we have optimised the production of SGPI-1-2 via a bacterial (Escherichia coli) expression system. The cDNA sequences encoding these peptides were inserted in the pMAL-2pX vector, downstream of the gene encoding the maltose-binding protein (including a signal peptide). As a consequence, both peptides were expressed as fusion proteins (2-3 mg/l) and targeted to the periplasmic space. Following a one-step affinity purification, both fusion proteins were successfully cleaved by Factor Xa and after a methanol extraction, it took only one additional RP-HPLC run to purify both peptides to homogeneity. Finally, the formation of the disulphide bridges and the biological activity of the recombinant peptides were verified by mass spectrometry and a spectrophotometric protease inhibitor assay, respectively.     

The PiggyBac transposon enhances the frequency of CHO stable cell line generation and yields recombinant lines with superior productivity and stability

Generating stable, high-producing mammalian cell lines is a major bottleneck in the manufacture of recombinant therapeutic proteins. Conventional gene transfer methods for cell line generation rely on random plasmid integration, resulting in unpredictable and highly variable levels of transgene expression. As a consequence, a large number of stably transfected cells must be analyzed to recover a few high-producing clones. Here we present an alternative gene transfer method for cell line generation based on transgene integration mediated by the piggyBac (PB) transposon. Recombinant Chinese hamster ovary (CHO) cell lines expressing a tumor necrosis factor receptor:Fc fusion protein were generated either by PB transposition or by conventional transfection. Polyclonal populations and isolated clonal cell lines were characterized for the level and stability of transgene expression for up to 3 months in serum-free suspension culture. Pools of transposed cells produced up to fourfold more recombinant protein than did the pools generated by standard transfection. For clonal cell lines, the frequency of high-producers was greater following transposition as compared to standard transfection, and these clones had a higher volumetric productivity and a greater number of integrated transgenes than did those generated by standard transfection. In general, the volumetric productivity of the cell pools and individual cell lines generated by transposition was stable for up to 3 months in the absence of selection. Our results indicate that the PB transposon supports the generation of cell lines with high and stable transgene expression at an elevated frequency relative to conventional transfection. Thus, PB-mediated gene delivery is expected to reduce the extent of recombinant cell line screening.     

A 3-disulfide mutant of mouse prion protein expression, oxidative folding, reductive unfolding, conformational stability, aggregation and isomerization

The structure of wild-type mouse prion protein mPrP(23-231) consists of two distinctive segments with approximately equal size, a disordered and flexible N-terminal domain encompassing residues 23-124 and a largely structured C-terminal domain containing about 40% of helical structure and stabilized by one disulfide bond (Cys(178)-Cys(213)). We have expressed a mPrP mutant with 4 Ala/Ser-->Cys replacements, two each at the N-(Cys(36), Cys(112)) and C-(Cys(134), Cys(169)) domains. Our specific aims are to study the interaction between N- and C-domains of mPrP during the oxidative folding and to produce stabilized isomers of mPrP for further analysis. Oxidative folding of fully reduced mutant, mPrP(6C), generates one predominant 3-disulfide isomer, designated as N-mPrP(3SS), which comprises the native disulfide (Cys(178)-Cys(213)) and two non-native disulfide bonds (Cys(36)-Cys(134) and Cys(112)-Cys(169)) that covalently connect the N- and C-domains. In comparison to wild-type mPrP(23-231), N-mPrP(3SS) exhibits an indistinguishable CD spectra, a similar conformational stability in the absence of thiol and a reduced ability to aggregate. In the presence of thiol catalyst and denaturant, N-mPrP(3SS) unfolds and generates diverse isomers that are amenable to further isolation, structural and functional analysis.     

Comparative study of mature and zymogen mite cysteine protease stability and pH unfolding

Background

Papain-like proteases (CA1) are synthesized as inactive precursors carrying an N-terminal propeptide, which is further removed under acidic conditions to generate active enzymes.

Methods

To have a better insight into the mechanism of activation of this protease family, we compared the pH unfolding of the zymogen and the mature form of the mite cysteine protease Der p 1.

Results

We showed that the presence of the propeptide does not significantly influence the pH-induced unfolding of the catalytic domain but does affect its fluorescence properties by modifying the exposure of the tryptophan 192 to the solvent. In addition, we demonstrated that the propeptide displays weaker pH stability than the protease domain confirming that the unfolding of the propeptide is the key event in the activation process of the zymogen.

General significance

Finally, we show, using thermal denaturation and enzymatic activity measurements, that whatever the pH value, the propeptide does not stabilize the structure of the catalytic domain but very interestingly, prevents its autolysis.     

Posttranslational modifications of serine protease TMPRSS13 regulate zymogen activation,proteolytic activity,and cell surface localization

TMPRSS13, a member of the type II transmembrane serine protease (TTSP) family, harbors four N-linked glycosylation sites in its extracellular domain. Two of the glycosylated residues are located in the scavenger receptor cysteine-rich (SRCR) protein domain, while the remaining two sites are in the catalytic serine protease (SP) domain. In this study, we examined the role of N-linked glycosylation in the proteolytic activity, autoactivation, and cellular localization of TMPRSS13. Individual and combinatory site-directed mutagenesis of the glycosylated asparagine residues indicated that glycosylation of the SP domain is critical for TMPRSS13 autoactivation and catalytic activity toward one of its protein substrates, the prostasin zymogen. Additionally, SP domain glycosylation-deficient TMPRSS13 displayed impaired trafficking of TMPRSS13 to the cell surface, which correlated with increased retention in the endoplasmic reticulum. Importantly, we showed that N-linked glycosylation was a critical determinant for subsequent phosphorylation of endogenous TMPRSS13. Taken together, we conclude that glycosylation plays an important role in regulating TMPRSS13 activation and activity, phosphorylation, and cell surface localization.     

Lyophilization and enhanced stability of fluorescent protein nanoparticles

Protein nanoparticles (PNPs) that are nanostructured biomaterials with intrinsic biological function have been widely employed as three-dimensional nanobiomaterials for sensitive bioassays, MRI contrast, semiconductor devices, template for hybrid materials, etc., and stable and long-term maintenance of PNPs seems to be of crucial importance. We evaluated the stability of PNPs and the efficacy of lyophilization for the long-term stability of PNPs, especially using green fluorescent protein nanoparticles (gFPNPs) as a model PNP. Fluorescence intensities and TEM images of gFPNPs were analyzed to monitor their functional and structural stabilities. Unlike the green fluorescent protein monomers (eGFP) that were gradually inactivated in aqueous solution, gFPNP in the same aqueous solution retained the initial fluorescence activity and spherical nanoparticle structure even for 2 weeks at 4 °C. To ensure stable and long-term maintenance of gFPNPs, gFPNPs in aqueous solution were converted to the dried solid forms through lyophilization. It is notable that fluorescence activity and nanoparticle structure of gFPNPs that were lyophilized with both Tween 80 and sucrose were very stably maintained even for 10 weeks at various storage temperatures (−20 °C, 4 °C, 25 °C, and 37 °C). During the period of 10 weeks, the fluorescence of gFPNP was always more than 80% level of initial fluorescence at a wide range of temperature. Although this stability study was focused on gFPNPs, the developed optimal lyophilization conditions for gFPNPs can be applied in general to stable and long-term maintenance of many other PNP-derived biomaterials.