Proteolysis approach without chemical modification for a simple and rapid analysis of disulfide bonds using thermostable protease‐immobilized microreactors

Disulfide bonds in proteins are important not only for the conformational stability of the protein but also for the regulation of oxidation–reduction in signal transduction. The conventional method for the assignment of disulfide bond by chemical cleavage and/or proteolysis is a time‐consuming multi‐step procedure. In this study, we report a simple and rapid analysis of disulfide bond from protein digests that were prepared by the thermostable protease‐immobilized microreactors. The feasibility and performance of this approach were evaluated by digesting lysozyme and BSA at several temperatures. The proteins which stabilize their conformations by disulfide bonds were thermally denatured during proteolysis and were efficiently digested by the immobilized protease but not by free protease. The digests were directly analyzed by ESI‐TOF MS without any purification or concentration step. All four disulfide bonds on lysozyme and 10 of 17 on BSA were assigned from the digests by the trypsin‐immobilized microreactor at 50°C. The procedure for proteolysis and the assignment were achieved within 2 h without any reduction and alkylation procedure. From the present results, the proteolysis approach by the thermostable protease‐immobilized microreactor provides a strategy for the high‐throughput analysis of disulfide bond in proteomics.     

A novel engineered interchain disulfide bond in the constant region enhances the thermostability of adalimumab Fab

We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH₁ and CL domains was deleted by substitution of Cys with Ala (Fab_ΔSS). DSC measurements showed that the Tm values of Fab_ΔSS were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of Fab_ΔSS. The resulting Fab (mutSS Fab_ΔSS) had the mutations H:V177C and L:Q160C in Fab_ΔSS, confirming the formation of the disulfide bond between CH₁ and CL. The thermostability of mutSS Fab_ΔSS was approximately 5 °C higher than that of Fab_ΔSS. Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of Fab_ΔSS and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation.     

pH dependence of the stability of barstar to chemical and thermal denaturation.

Equilibrium unfolding of barstar with guanidine hydrochloride (GdnHCl) and urea as denaturants as well as thermal unfolding have been carried out as a function of pH using fluorescence, far-UV and near-UV CD, and absorbance as probes. Both GdnHCl-induced and urea-induced denaturation studies at pH 7 show that barstar unfolds through a two-state F<->U mechanism and yields identical values for delta GU, the free energy difference between the fully folded (F) and unfolded (U) forms, of 5.0 +/- 0.5 kcal.mol-1 at 25 degrees C. Thermal denaturation of barstar also follows a two-state F<->U unfolding transition at pH 7, and the value of delta GU at 25 degrees C is similar to that obtained from chemical denaturation. The pH dependence of denaturation by GdnHCl is complex. The Cm value (midpoint of the unfolding transition) has been used as an index for stability in the pH range 2-10, because barstar does not unfold through a two-state transition on denaturation by GdnHCl at all pH values studied. Stability is maximum at pH 2-3, where barstar exists in a molten globule-like form that forms a large soluble oligomer. The stability decreases with an increase in pH to 5, the isoelectric pH of the protein. Above pH 5, the stability increases as the pH is raised to 7. Above pH 8, it again decreases as the pH is raised to 10. The decrease in stability from pH 7 to 5 in wild-type (wt) barstar, which is shown to be characterized by an apparent pKa of 6.2 +/- 0.2, is not observed in H17Q, a His 17-->Gln 17 mutant form of barstar. This decrease in stability has therefore been correlated with the protonation of His 17 in barstar. The decrease in stability beyond pH 8 in wt barstar, which is characterized by an apparent pKa of 9.2 +/- 0.2, is not detected in BSCCAA, the Cys 40 Cys 82-->Ala 40 Ala 82 double mutant form of barstar. Thus, this decrease in stability has been correlated with the deprotonation of at least one of the two cysteines present in wt barstar. The increase in stability from pH 5 to 3 is characterized by an apparent pKa of 4.6 +/- 0.2 for wt barstar and BSCCAA, which is similar to the apparent pKa that characterizes the structural transition leading to the formation of the A form. The use of Cm as an index of stability has been supported by thermal denaturation studies.(ABSTRACT TRUNCATED AT 400 WORDS)     

蛋白质交联的研究进展

蛋白质共价交联不但存在于一些生理过程 ,还与一些神经性疾病的发病机理相关。本文综述国内外 3种观点 ,阐述蛋白质由功能体 /单体转变成交联的二聚体 /多聚体的分子机理     

N-glycosylation-negative catalase: a useful tool for exploring the role of hydrogen peroxide in the endoplasmic reticulum

Disulfide bond formation during protein folding of nascent proteins is associated with the generation of H₂O₂ in the endoplasmic reticulum (ER). Approaches to quantifying H₂O₂ directly within the ER failed because of the oxidative environment in the ER lumen, and ER-specific catalase expression to detoxify high H₂O₂ concentrations resulted in an inactive protein owing to N-glycosylation. Therefore, the N-glycosylation motifs at asparagine-244 and -439 of the human catalase protein were deleted by site-directed mutagenesis. The ER-targeted expression of these variants revealed that the deletion of the N-glycosylation motif only at asparagine-244 (N244) was associated with the maintenance of full enzymatic activity in the ER. Expression of catalase N244 in the ER (ER-Catalase N244) was ER-specific and protected the cells significantly against exogenously added H₂O₂. With the expression of ER-Catalase N244, a highly effective H₂O₂ inactivation within the ER was achieved for the first time. Catalase has a high H₂O₂-inactivation capacity without the need of reducing cofactors, which might interfere with the ER redox homeostasis, and is not involved in protein folding. With these characteristics ER-Catalase N244 is an ideal tool to explore the impact of ER-generated H₂O₂ on the generation of disulfide bonds or to study the induction of ER-stress pathways through protein folding overload and accumulation of H₂O₂.     

Modification of soybean sucrose synthase by S-thiolation with ENOD40 peptide A

The gene ENOD40 is expressed at an early stage of root nodule organogenesis and has been postulated to play a central regulatory role in the Rhizobium-legume interaction. In vitro translation of soybean ENOD40 mRNA showed that the gene encodes two peptides of 12 and 24aa residues (peptides A and B) that bind to sucrose synthase. Here we show that the small Cys-containing peptide A binds to sucrose synthase by disulfide bond formation, which may represent a novel form of posttranslational modification of this important metabolic enzyme. Assays using nanomolar concentrations of peptide A revealed that the monomeric reduced form of this peptide binds to purified sucrose synthase. Using a cysteinyl capture strategy combined with MALDI-TOF MS analysis we identified the Cys residue C264 of soybean sucrose synthase as the binding site of peptide A. Modification of sucrose synthase with ENOD40 peptide A activates sucrose cleavage activity whereas the synthesis activity of the enzyme is unaffected. The results are discussed in relation to the role of sucrose synthase in the control of sucrose utilization in nitrogen-fixing nodules.     

Protein disulfide isomerase homolog TrPDI2 contributing to cellobiohydrolase production in Trichoderma reesei

The majority of the cysteine residues in the secreted proteins form disulfide bonds via protein disulfide isomerase (PDI)-mediated catalysis, stabilizing the enzyme activity. The role of PDI in cellulase production is speculative, as well as the possibility of PDI as a target for improving enzyme production efficiency of Trichoderma reesei, a widely used producer of enzyme for the production of lignocellulose-based biofuels and biochemicals. Here, we report that a PDI homolog, TrPDI2 in T. reesei exhibited a 36.94% and an 11.81% similarity to Aspergillus niger TIGA and T. reesei PDI1, respectively. The capability of TrPDI2 to recover the activity of reduced and denatured RNase by promoting refolding verified its protein disulfide isomerase activity. The overexpression of Trpdi2 increased the secretion and the activity of CBH1 at the early stage of cellulase induction. In addition, both the expression level and redox state of TrPDI2 responded to cellulase induction in T. reesei, providing sustainable oxidative power to ensure cellobiohydrolase maturation and production. The results suggest that TrPDI2 may contribute to cellobiohydrolase secretion by enhancing the capability of disulfide bond formation, which is essential for protein folding and maturation.     

NMR assignment of the periplasmic oxidoreductase DsbH from Chlamydia

Chlamydia use a complex of outer envelope proteins, which are highly cross-linked by disulfide bonds, to protect their infectious developmental form from lysis. Reported herein are the NMR chemical shift assignments of DsbH, a novel disulfide oxidoreductase from Chlamydia.     

Thermal stability of beta-lactoglobulin in the presence of aqueous solution of alcohols and polyols

A systematic study concerning the effect of aqueous solution of alcohols and polyols with four carbon atoms on β-lactoglobulin stability is presented. The protein was chosen due to its functional properties and applications in food and pharmaceutical industries and because its structure and properties in aqueous solution have been widely described. The alcohols having a four carbon chain were selected to examine the effect of the gradual increase in the number of OH groups on protein stability.

Protein thermal stability in water, buffers and dilute aqueous solutions of 1-butanol, 1,2-butanediol, 1,2,4-butanetriol and 1,2,3,4-butanetetrol was evaluated by fluorescence spectroscopy. The results were used to determine the temperature range in which the unfolding process is reversible and the protein denaturation temperature in acetate buffer pH 5.5 and in the aqueous mixed solvents. Thermodynamic results show that alcohol denaturating effect diminishes gradually as the number of OH groups increase.     

Conformational characterization of disulfide bonds: A tool for protein classification

Background

Throughout evolution, mutations in particular regions of some protein structures have resulted in extra covalent bonds that increase the overall robustness of the fold: disulfide bonds. The two strategically placed cysteines can also have a more direct role in protein function, either by assisting thiol or disulfide exchange, or through allosteric effects. In this work, we verified how the structural similarities between disulfides can reflect functional and evolutionary relationships between different proteins. We analyzed the conformational patterns of the disulfide bonds in a set of disulfide-rich proteins that included twelve SCOP superfamilies: thioredoxin-like and eleven superfamilies containing small disulfide-rich proteins (SDP).

Results

The twenty conformations considered in the present study were characterized by both structural and energetic parameters. The corresponding frequencies present diverse patterns for the different superfamilies. The least-strained conformations are more abundant for the SDP superfamilies, while the “catalytic” +/−RHook is dominant for the thioredoxin-like superfamily. The “allosteric” -RHSaple is moderately abundant for BBI, Crisp and Thioredoxin-like superfamilies and less frequent for the remaining superfamilies. Using a hierarchical clustering analysis we found that the twelve superfamilies were grouped in biologically significant clusters.

Conclusions

In this work, we carried out an extensive statistical analysis of the conformational motifs for the disulfide bonds present in a set of disulfide-rich proteins. We show that the conformational patterns observed in disulfide bonds are sufficient to group proteins that share both functional and structural patterns and can therefore be used as a criterion for protein classification.     

Thermodynamics and stability of the PAAD/DAPIN/PYRIN domain of IFI-16

The PAAD domain is a conserved domain recently identified in more than 35 human proteins that are involved in apoptosis and inflammatory signaling pathways. Structural studies have confirmed that this domain belongs to the death domain superfamily which includes PAAD/CARD/DED/DD families. Recently, the 3D structures determined by NMR of NALP1 and ASC PAAD domain, members of the PAAD family, have shown that it is composed of a 6 helix bundle as with other death domain family members. However, helix-3 in the solved structures is unordered in solution. In this study we compare the thermodynamic, folding and stability properties of different members of the PAAD and CARD families and investigate structural conformational changes induced by the helix inducers trifluoroethanol and SDS on the PAAD domain of IFI16 and on the CARD domain of RAIDD. We show that inside the PAAD and CARD families, members have similar thermodynamic properties, however, the DeltaG of folding for PAAD and CARD members are, respectively, -1.4 and -5.5 kcal mol(-1). This difference is attributed to less alpha helical content for PAAD due to the unfolding of helix-3 that lowers bonded energy and increases disorder when compared to CARD members. Despite identical fold between PAAD and CARD families but limited sequence identity, there are striking differences in the thermodynamics of both families.     

The glucagon receptor antagonist BI-32169 constitutes a new class of lasso peptides

The glucagon receptor antagonist BI-32169, recently isolated from Streptomyces sp., was described as a bicyclic peptide, although its primary structure comprises conserved elements of class I and class II lasso peptides. Tandem mass spectrometric and nuclear magnetic resonance spectroscopic studies revealed that BI-32169 is a lasso-structured peptide constituting the new class III of lasso peptides. The determined lasso fold opens new avenues to improve the promising biological activity of BI-32169.     

TMX, a human transmembrane oxidoreductase of the thioredoxin family: the possible role in disulfide-linked protein folding in the endoplasmic reticulum

Various proteins sharing thioredoxin (Trx)-like active site sequences (Cys-Xxx-Xxx-Cys) have been found and classified in the Trx superfamily. Among them, transmembrane Trx-related protein (TMX) was recently identified as a novel protein possessing an atypical active site sequence, Cys-Pro-Ala-Cys. In the present study, we describe the properties of this membranous Trx-related molecule. Endogenous TMX was detected as a protein of approximately 30 kDa with a cleavable signal peptide. TMX was enriched in membrane fractions and exhibited a similar subcellular distribution with calnexin localized in the endoplasmic reticulum (ER). The examination of membrane topology of TMX suggested that the N-terminal region containing the Trx-like domain was present in the ER lumen, where protein disulfide isomerase (PDI) was found to assist protein folding. Recombinant TMX showed PDI-like activity to refold scrambled RNase. These results indicate the possibility that TMX can modify certain molecules with its oxidoreductase activity and be involved in the redox regulation in the ER.     

If space is provided,bulky modification on the rim of azurin's beta-barrel results in folded protein

Pseudomonas aeruginosa azurin is a blue-copper protein with a beta-barrel fold. Here we report that, at conditions where thermal unfolding of apo-azurin is reversible, the reaction occurs in a single step with a transition midpoint (T(m)) of 69 degrees C (pH 7). The active-site mutation His117Gly creates a cavity in the beta-barrel near the surface but does not perturb the overall fold (T(m) of 64 degrees C, pH 7). Oxidation of the active-site cysteine (Cysteine-112) in wild-type azurin, which occurs readily at higher temperatures, results in a modified protein that cannot adopt a native-like structure. In sharp contrast, Cysteine-112 oxidation in His117Gly azurin yields a modified apo-azurin that appears folded and displays cooperative, reversible unfolding (T(m) approximately 55 degrees C, pH 7). We conclude that azurin's beta-barrel is a rigid structural element that constrains the structure of its surface; a bulky modification can only be accommodated if complementary space is provided.     

The intrachain disulfide bridge is responsible of the unusual stability properties of novel acylphosphatase from Escherichia coli

Acylphosphatase (AcP) activity in prokaryotes was classically attributed to some aspecific acid phosphatases. We identified an open reading frame for a putative AcP in the b0968 Escherichia coli gene and purified the recombinant enzyme after checking by RT-PCR that it was indeed expressed. EcoAcP has a predicted typical fold of the AcP family but displays a very low specific activity and a high structural stability differently from its mesophilic and similarly to its hyperthermophilic counterparts. Site directed mutagenesis suggests that, together with other structural features, the intrachain S–S bridge in EcoAcP is involved in a remarkable thermal and chemical stabilization of the protein without affecting its catalytic activity.     

半胱氨酸氧化还原状态的协同性

以2002年4月份的Culled Protein Data Bank数据库中的639条蛋白质多肽链为研究对象,统计分析了其含有的584条二硫键的形成特征,发现半胱氨酸氧化还原状态表现出明显的协同性现象：含有二硫键的蛋白质中几乎所有的半胱氨酸都以氧化态形式存在。这一协同性可以通过蛋白质全局水平上的20种氨基酸组分的百分含量很好地加以说明,由此来预测半胱氨酸的氧化还原状态准确率最高可达84．5％。结果表明半胱氨酸是否形成二硫键主要取决于蛋白质全局的而非局部的结构信息。     

Disulfide bond formation in prokaryotes: History,diversity and design

The formation of structural disulfide bonds is essential for the function and stability of a great number of proteins, particularly those that are secreted. There exists a variety of dedicated cellular catalysts and pathways from archaea to humans that ensure the formation of native disulfide bonds. In this review we describe the initial discoveries of these pathways and report progress in recent years in our understanding of the diversity of these pathways in prokaryotes, including those newly discovered in some archaea. We will also discuss the various successful efforts to achieve laboratory-based evolution and design of synthetic disulfide bond formation machineries in the bacterium Escherichia coli. These latter studies have also led to new more general insights into the redox environment of the cytoplasm and bacterial cell envelope. This article is part of a Special Issue entitled: Thiol-Based Redox Processes.     

Characterization and analysis of thermal denaturation of antibodies by size exclusion high-performance liquid chromatography with quadruple detection

Size exclusion chromatography (SEC) coupled with online light scattering, viscometry, refractometry, and UV-visible spectroscopy provides a very powerful tool for studying protein size, shape, and aggregation. This technique can be used to determine the molecular weight of the component peaks independent of the retention times in the SEC column and simultaneously measure the hydrodynamic radius and polydispersity of the protein. We applied this technology by coupling an Agilent Chemstation high-performance liquid chromatography system with a diode array UV-visible detector and a Viscotek 300 EZ Pro triple detector (combination of a light scattering detector, refractometer, and differential pressure viscometer) to characterize and compare the molecular properties of a number of monoclonal antibodies. Our studies reveal that different monoclonal immunoglobulin Gs (IgGs) and chimeric IgGs show slightly different retention times and therefore different molecular weights in gel filtration analysis. However, when they are analyzed by light scattering, refractometry, and viscometry, different IgGs have comparable molecular weight, molecular homogeneity (polydispersity), and size. Gel filtration coupled with UV or refractive index detection suggests that antibodies purified and formulated for preclinical and clinical development are more than 95% monomer with little or no detectable soluble aggregates. Light scattering measurements showed the presence of trace amounts of soluble aggregate in all the IgG preparations. The different IgG molecules showed different susceptibility to heat and pH. One of the murine antibodies was considerably less stable than the others at 55 degrees C. The application of this powerful technology for the characterization of monoclonal antibodies of therapeutic potential is discussed.     

Reversible denaturation of the soybean Kunitz trypsin inhibitor

The soybean Kunitz trypsin inhibitor (SKTI) is a beta-sheet protein with unusual stability to chemical and thermal denaturation. Different spectroscopic criteria were used to follow the thermal denaturation and renaturation of SKTI. Upon heating to 70 degrees C, changes in UV difference spectra showed increased absorbance at 292 and 297 nm, attributable to perturbation of aromatic residues. Cooling the protein resulted in restoration of the native spectrum unless reduced with dithiothreitol. Far- and near-UV CD spectra also indicate thermal unfolding involving the core tryptophan and tyrosine residues. Both CD and UV-absorbance data suggest a two-state transition with the midpoint at approximately 65 degrees C. CD data along with the increased fluorescence intensity of the reporter fluorophore, 1-anilino-8-naphthalenesulfonate with SKTI, between 60 and 70 degrees C, are consistent with a transition of the native inhibitor to an alternate conformation with a more molten state. Even after heating to 90 degrees C, subsequent cooling of SKTI resulted in >90% of native trypsin inhibition potential. These results indicate that thermal denaturation of SKTI is readily reversible to the native form upon cooling and may provide a useful system for future protein folding studies in the class of disordered beta-sheet proteins.