Selective Removal of Individual Disulfide Bonds within a Potato Type II Serine Proteinase Inhibitor from Nicotiana alata Reveals Differential Stabilization of the Reactive-Site Loop

The 53-amino-acid trypsin inhibitor 1 from Nicotiana alata (T1) belongs to the potato type II family also known as the PinII family of proteinase inhibitors, one of the major families of canonical proteinase inhibitors. T1 contains four disulfide bonds, two of which (C4-C41 and C8-C37) stabilize the reactive-site loop. To investigate the influence of these two disulfide bonds on the structure and function of potato II inhibitors, we constructed two variants of T1, C4A/C41A-T1 and C8A/C37A-T1, in which these two disulfide bonds were individually removed and replaced by alanine residues. Trypsin inhibition assays show that wild-type T1 has a K_i of < 5 nM, C4A/C41A-T1 has a weaker K_i of ∼ 350 nM, and the potency of the C8A/C37A variant is further decreased to a K_i of ∼ 1.8 μM. To assess the influence of the disulfide bonds on the structure of T1, we determined the structure and dynamics of both disulfide variants by NMR spectroscopy. The structure of C4A/C41A-T1 and the amplitude of intrinsic flexibility in the reactive-site loop resemble that of the wild-type protein closely, despite the lack of the C4-C41 disulfide bond, whereas the timescale of motions is markedly decreased. The rescue of the structure despite loss of a disulfide bond is due to a previously unrecognized network of interactions, which stabilizes the structure of the reactive-site loop in the region of the missing disulfide bond, while allowing intrinsic motions on a fast (picosecond-nanosecond) timescale. In contrast, no comparable interactions are present around the C8-C37 disulfide bond. Consequently, the reactive-site loop becomes disordered and highly flexible in the structure of C8A/C37A-T1, making it unable to bind to trypsin. Thus, the reactive-site loop of T1 is stabilized differently by the C8-C37 and C4-C41 disulfide bonds. The C8-C37 disulfide bond is essential for the inhibitory activity of T1, whereas the C4-C41 disulfide bond is not as critical for maintaining the three-dimensional structure and function of the molecule but is responsible for maintaining flexibility of the reactive-site loop on a microsecond-nanosecond timescale.     

Role of disulfide bonds in conformational stability and folding of 5′-deoxy-5′-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus 5′-deoxy-5′-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138–C205 and C200–C262) and a CXC motif (C259–C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100 °C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102 °C and 99 °C respectively. Under reducing conditions these values decrease by 4 °C and 8 °C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90 °C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.     

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl]_1/2 at 3.4-5 M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1 mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys³⁶-Cys⁴⁹ and two disulfide bonds formed by two pair of consecutive cysteines, Cys²²-Cys²³ and Cys⁵⁶-Cys⁵⁷, a unique disulfide structure of polypeptide that has not been documented previously.     

A stable disulfide-free gene-3-protein of phage fd generated by in vitro evolution

Disulfide bonds provide major contributions to the conformational stability of proteins, and their cleavage often leads to unfolding. The gene-3-protein of the filamentous phage fd contains two disulfides in its N1 domain and one in its N2 domain, and these three disulfide bonds are essential for the stability of this protein. Here, we employed in vitro evolution to generate a disulfide-free variant of the N1-N2 protein with a high conformational stability. The gene-3-protein is essential for the phage infectivity, and we exploited this requirement for a proteolytic selection of stabilized protein variants from phage libraries. First, optimal replacements for individual disulfide bonds were identified in libraries, in which the corresponding cysteine codons were randomized. Then stabilizing amino acid replacements at non-cysteine positions were selected from libraries that were created by error-prone PCR. This stepwise procedure led to variants of N1-N2 that are devoid of all three disulfide bonds but stable and functional. The best variant without disulfide bonds showed a much higher conformational stability than the disulfide-containing wild-type form of the gene-3-protein. Despite the loss of all three disulfide bonds, the midpoints of the thermal transitions were increased from 48.5 degrees C to 67.0 degrees C for the N2 domain and from 60.0 degrees C to 78.7 degrees C for the N1 domain. The major loss in conformational stability caused by the removal of the disulfides was thus over-compensated by strongly improved non-covalent interactions. The stabilized variants were less infectious than the wild-type protein, probably because the domain mobility was reduced. Only a small fraction of the sequence space could be accessed by using libraries created by error-prone PCR, but still many strongly stabilized variants could be identified. This is encouraging and indicates that proteins can be stabilized by mutations in many different ways.     

An allosteric redox switch in domain V of β2-glycoprotein I controls membrane binding and anti-domain I autoantibody recognition

β₂-glycoprotein I (β₂GPI) is an abundant multidomain plasma protein that plays various roles in the clotting and complement cascades. It is also the main target of antiphospholipid antibodies (aPL) in the acquired coagulopathy known as antiphospholipid syndrome (APS). Previous studies have shown that β₂GPI adopts two interconvertible biochemical conformations, oxidized and reduced, depending on the integrity of the disulfide bonds. However, the precise contribution of the disulfide bonds to β₂GPI structure and function is unknown. Here, we substituted cysteine residues with serine to investigate how the disulfide bonds C32-C60 in domain I (DI) and C288-C326 in domain V (DV) regulate β₂GPI''s structure and function. Results of our biophysical and biochemical studies support the hypothesis that the C32-C60 disulfide bond plays a structural role, whereas the disulfide bond C288-C326 is allosteric. We demonstrate that absence of the C288-C326 bond, unlike absence of the C32-C60 bond, diminishes membrane binding without affecting the thermodynamic stability and overall structure of the protein, which remains elongated in solution. We also document that, while absence of the C32-C60 bond directly impairs recognition of β₂GPI by pathogenic anti-DI antibodies, absence of the C288-C326 disulfide bond is sufficient to abolish complex formation in the presence of anionic phospholipids. We conclude that the disulfide bond C288-C326 operates as a molecular switch capable of regulating β₂GPI''s physiological functions in a redox-dependent manner. We propose that in APS patients with anti-DI antibodies, selective rupture of the C288-C326 disulfide bond may be a valid strategy to lower the pathogenic potential of aPL.     

Conformational landscape and pathway of disulfide bond reduction of human alpha defensin

Human alpha defensins are a class of antimicrobial peptides with additional antiviral activity. Such antimicrobial peptides constitute a major part of mammalian innate immunity. Alpha defensins contain six cysteines, which form three well defined disulfide bridges under oxidizing conditions. Residues C3-C31, C5-C20, and C10-C30 form disulfide pairs in the native structure of the peptide. The major tissue in which HD5 is expressed is the crypt of the small intestine, an anaerobic niche that should allow for substantial pools of both oxidized and (partly) reduced HD5. We used ion mobility coupled to mass spectrometry to track the structural changes in HD5 upon disulfide bond reduction. We found evidence of stepwise unfolding of HD5 with sequential reduction of the three disulfide bonds. Alkylation of free cysteines followed by tandem mass spectrometry of the corresponding partially reduced states revealed a dominant pathway of reductive unfolding. The majority of HD5 unfolds by initial reduction of C5-C20, followed by C10-C30 and C3-C31. We find additional evidence for a minor pathway that starts with reduction of C3-C31, followed by C5-C20 and C10-C30. Our results provide insight into the pathway and conformational landscape of disulfide bond reduction in HD5.     

An engineered disulfide bridge mimics the effect of calcium to protect neutral protease against local unfolding

The extreme thermal stabilization achieved by the introduction of a disulfide bond (G8C/N60C) into the cysteine-free wild-type-like mutant (pWT) of the neutral protease from Bacillus stearothermophilus[Mansfeld J, Vriend G, Dijkstra BW, Veltman OR, Van den Burg B, Venema G, Ulbrich-Hofmann R & Eijsink VG (1997) J Biol Chem272, 11152-11156] was attributed to the fixation of the loop region 56-69. In this study, the role of calcium ions in the guanidine hydrochloride (GdnHCl)-induced unfolding and autoproteolysis kinetics of pWT and G8C/N60C was analyzed by fluorescence spectroscopy, far-UV CD spectroscopy and SDS/PAGE. First-order rate constants (kobs) were evaluated by chevron plots (ln kobs vs. GdnHCl concentration). The kobs of unfolding showed a difference of nearly six orders of magnitude (DeltaDeltaG# = 33.5 kJ.mol(-1) at 25 degrees C) between calcium saturation (at 100 mM CaCl2) and complete removal of calcium ions (in the presence of 100 mM EDTA). Analysis of the protease variant W55F indicated that calcium binding-site III, situated in the critical region 56-69, determines the stability at calcium ion concentrations between 5 and 50 mM. In the chevron plots the disulfide bridge in G8C/N60C shows a similar effect compared with pWT as the addition of calcium ions, suggesting that the introduced disulfide bridge fixes the region (near calcium binding-site III) that is responsible for unfolding and subsequent autoproteolysis. Owing to the presence of the disulfide bridge, the DeltaDeltaG# is 13.2 kJ.mol(-1) at 25 degrees C and 5 mM CaCl2. Non-linear chevron plots reveal an intermediate in unfolding probably caused by local unfolding of the loop 56-69. The occurrence of this intermediate is prevented by calcium concentrations of > 5 mM, or the introduction of the disulfide bridge G8C/N60C.     

Changing the determinants of protein stability from covalent to non-covalent interactions by in vitro evolution: a structural and energetic analysis

The three disulfide bonds of the gene-3-protein of the phage fd are essential for the conformational stability of this protein, and it unfolds when they are removed by reduction or mutation. Previously, we used an iterative in vitro selection strategy to generate a stable and functional form of the gene-3-protein without these disulfides. It yielded optimal replacements for the disulfide bonds as well as several stabilizing second-site mutations. The best selected variant showed a higher thermal stability compared with the disulfide-bonded wild-type protein. Here, we investigated the molecular basis of this strong stabilization by solving the crystal structure of this variant and by analyzing the contributions to the conformational stability of the selected mutations individually. They could mostly be explained by improved side-chain packing. The R29W substitution alone increased the midpoint of the thermal unfolding transition by 14 deg and the conformational stability by about 25 kJ mol^− 1. This key mutation (i) removed a charged side chain that forms a buried salt bridge in the disulfide-containing wild-type protein, (ii) optimized the local packing with the residues that replace the C46-C53 disulfide and (iii) improved the domain interactions. Apparently, certain residues in proteins indeed play key roles for stability.     

Role of disulfide bonds in conformational stability and folding of 5'-deoxy-5'-methylthioadenosine phosphorylase II from the hyperthermophilic archaeon Sulfolobus solfataricus

Sulfolobus solfataricus 5'-deoxy-5'-melthylthioadenosine phosphorylase II (SsMTAPII), is a hyperthermophilic hexameric protein with two intrasubunit disulfide bonds (C138-C205 and C200-C262) and a CXC motif (C259-C261). To get information on the role played by these covalent links in stability and folding, the conformational stability of SsMTAPII and C262S and C259S/C261S mutants was studied by thermal and guanidinium chloride (GdmCl)-induced unfolding and analyzed by fluorescence spectroscopy, circular dichroism, and SDS-PAGE. No thermal unfolding transition of SsMTAPII can be obtained under nonreducing conditions, while in the presence of the reducing agent Tris-(2-carboxyethyl) phosphine (TCEP), a Tm of 100°C can be measured demonstrating the involvement of disulfide bridges in enzyme thermostability. Different from the wild-type, C262S and C259S/C261S show complete thermal denaturation curves with sigmoidal transitions centered at 102°C and 99°C respectively. Under reducing conditions these values decrease by 4°C and 8°C respectively, highlighting the important role exerted by the CXC disulfide on enzyme thermostability. The contribution of disulfide bonds to the conformational stability of SsMTAPII was further assessed by GdmCl-induced unfolding experiments carried out under reducing and nonreducing conditions. Thermal unfolding was found to be reversible if the protein was heated in the presence of TCEP up to 90°C but irreversible above this temperature because of aggregation. In analogy, only chemical unfolding carried out in the presence of reducing agents resulted in a reversible process suggesting that disulfide bonds play a role in enzyme denaturation. Thermal and chemical unfolding of SsMTAPII occur with dissociation of the native hexameric state into denatured monomers, as indicated by SDS-PAGE.     

The folding process of acylphosphatase from Escherichia coli is remarkably accelerated by the presence of a disulfide bond

The acylphosphatase from Escherichia coli (EcoAcP) is the first AcP so far studied with a disulfide bond. A mutational variant of the enzyme lacking the disulfide bond has been produced by substituting the two cysteine residues with alanine (EcoAcP mutational variant C5A/C49A, mutEcoAcP). The native states of the two protein variants are similar, as shown by far-UV and near-UV circular dichroism and dynamic light-scattering measurements. From unfolding experiments at equilibrium using intrinsic fluorescence and far-UV circular dichroism as probes, EcoAcP shows an increased conformational stability as compared with mutEcoAcP. The wild-type protein folds according to a two-state model with a very fast rate constant (k_F^H2O = 72,600 s^− 1), while mutEcoAcP folds ca 1500-fold slower, via the accumulation of a partially folded species. The correlation between the hydrophobicity of the polypeptide chain and the folding rate, found previously in the AcP-like structural family, is maintained only when considering the mutant but not the wild-type protein, which folds much faster than expected from this correlation. Similarly, the correlation between the relative contact order and the folding rate holds only for mutEcoAcP. The correlation also holds for EcoAcP, provided the relative contact order value is recalculated by considering the disulfide bridge as an alternate path for the backbone to determine the shortest sequence separation between contacting residues. These results indicate that the presence of a disulfide bond in a protein is an important determinant of the folding rate and allows its contribution to be determined in quantitative terms.     

Conformational stability of α-amylase from malted sorghum (Sorghum bicolor): Reversible unfolding by denaturants

α-Amylase from Sorghum bicolor, is reversibly unfolded by chemical denaturants at pH 7.0 in 50 mM Hepes containing 13.6 mM calcium and 15 mM DTT. The isothermal equilibrium unfolding at 27 °C is characterized by two state transition with ΔG (H₂O) of 16.5 kJ mol⁻¹ and 22 kJ mol⁻¹, respectively, at pH 4.8 and pH 7.0 for GuHCl and ΔG (H₂O) of 25.2 kJ mol⁻¹ at pH 4.8 for urea. The conformational stability indicators such as the change in excess heat capacity (ΔC_p), the unfolding enthalpy (H_g) and the temperature at ΔG = 0 (T_g) are 17.9 ± 0.7 kJ mol⁻¹ K⁻¹, 501.2 ± 18.2 kJ mol⁻¹ and 337.3 ± 6.9 K at pH 4.8 and 14.3 ± 0.5 kJ mol⁻¹ K⁻¹, 509.3 ± 21.7 kJ mol⁻¹ and 345.4 ± 4.8 K at pH 7.0, respectively. The reactivity of the conserved cysteine residues, during unfolding, indicates that unfolding starts from the ‘B’ domain of the enzyme. The oxidation of cysteine residues, during unfolding, can be prevented by the addition of DTT. The conserved cysteine residues are essential for enzyme activity but not for the secondary and tertiary fold acquired during refolding of the denatured enzyme. The pH dependent stability described by ΔG (H₂O) and the effect of salt on urea induced unfolding confirm the role of electrostatic interactions in enzyme stability.     

Redox changes accompanying storage protein mobilization in moist chilled and warm incubated walnut kernels prior to germination

Alterations in the redox state of storage proteins and the associated proteolytic processes were investigated in moist-chilled and warm-incubated walnut (Juglans regia L.) kernels prior to germination. The kernel total protein labeling with a thiol-specific fluorochrome i.e. monobromobimane (mBBr) revealed more reduction of 29–32 kDa putative glutelins, while in the soluble proteins, both putative glutelins and 41, 55 and 58 kDa globulins contained reduced disulfide bonds during mobilization. Thus, the in vivo more reduced disulfide bonds of storage proteins corresponds to greater solubility. After the in vitro reduction of walnut kernel proteins pre-treated by N-ethyl maleimide (NEM) with dithioerythrethiol (DTT) and bacterial thioredoxin, the 58 kDa putative globulin and a 6 kDa putative albumin were identified as disulfide proteins. Thioredoxin stimulated the reduction of the H₂O₂-oxidized 6 kDa polypeptide, but not the 58 kDa polypeptide by DTT. The solubility of 6 kDa putative albumin, 58 and 19–24 kDa putative globulins and glutelins, respectively, were increased by DTT. The in vitro specific mobilization of the 58 kDa polypeptide that occurred at pH 5.0 by the kernel endogenous protease was sensitive to the serine-protease inhibitor phenylmethylsulfonyl fluoride (PMSF) and stimulated by DTT. The specific degradation of the 58 kDa polypeptide might be achieved through thioredoxin-mediated activation of a serine protease and/or reductive unfolding of its 58 kDa polypeptide substrate. As redox changes in storage proteins occurred equally in both moist chilled and warm incubated walnut kernels, the regulatory functions of thioredoxins in promoting seed germination may be due to other germination related processes.     

The effect of additional disulfide bonds on the stability and folding of ribonuclease A

The significant contribution of disulfide bonds to the conformational stability of proteins is generally considered to result from an entropic destabilization of the unfolded state causing a faster escape of the molecules to the native state. However, the introduction of extra disulfide bonds into proteins as a general approach to protein stabilization yields rather inconsistent results. By modeling studies, we selected positions to introduce additional disulfide bonds into ribonuclease A at regions that had proven to be crucial for the initiation of the folding or unfolding process, respectively. However, only two out of the six variants proved to be more stable than unmodified ribonuclease A. The comparison of the thermodynamic and kinetic data disclosed a more pronounced effect on the unfolding reaction for all variants regardless of the position of the extra disulfide bond. Native-state proteolysis indicated a perturbation of the native state of the destabilized variants that obviously counterbalances the stability gain by the extra disulfide bond.     

Rectification ratio based determination of disulfide bonds of β2 extracellular loop of BK channel

Large-conductance Ca²⁺-activated K⁺ (BK) channels are composed of a pore-forming α and a variable number of auxiliary β subunits and play important roles in regulating excitability, action potential waveforms and firing patterns, particularly in neurons and endocrine and cardiovascular cells. The β2 subunits increase the diversity of gating and pharmacological properties. Its extracellular loop contains eight cysteine residues, which can pair to form a high-order structure, underlying the stability of the extracellular loop of β2 subunits and the functional effects on BK channels. However, how these cysteines form disulfide bonds still remains unclear. To address this, based on the fact that the rectification and association of BK α to β2 subunits are highly sensitive to disruption of the disulfide bonds in the extracellular loop of β2, we developed a rectification ratio based assay by combining the site-directed mutagenesis, electrophysiology and enzymatic cleavage. Three disulfide bonds: C1(C84)-C5(C113), C3(C101)-C7(C148) and C6(C142)-C8C(174) are successfully deduced in β2 subunit in complex with a BK α subunit, which are helpful to predict structural model of β2 subunits through computational simulation and to understand the interface between the extracellular domain of the β subunits and the pore-forming α subunit.     

Redox, mutagenic and structural studies of the glutaredoxin/arsenate reductase couple from the cyanobacterium Synechocystis sp. PCC 6803

The arsenate reductase from the cyanobacterium Synechocystis sp. PCC 6803 has been characterized in terms of the redox properties of its cysteine residues and their role in the reaction catalyzed by the enzyme. Of the five cysteines present in the enzyme, two (Cys13 and Cys35) have been shown not to be required for catalysis, while Cys8, Cys80 and Cys82 have been shown to be essential. The as-isolated enzyme contains a single disulfide, formed between Cys80 and Cys82, with an oxidation-reduction midpoint potential (E_m) value of − 165 mV at pH 7.0. It has been shown that Cys15 is the only one of the four cysteines present in Synechocystis sp. PCC 6803 glutaredoxin A required for its ability to serve as an electron donor to arsenate reductase, while the other three cysteines (Cys18, Cys36 and Cys70) play no role. Glutaredoxin A has been shown to contain a single redox-active disulfide/dithiol couple, with a two-electron, E_m value of − 220 mV at pH 7.0. One cysteine in this disulfide/dithiol couple has been shown to undergo glutathionylation. An X-ray crystal structure, at 1.8 Å resolution, has been obtained for glutaredoxin A. The probable orientations of arsenate reductase disulfide bonds present in the resting enzyme and in a likely reaction intermediate of the enzyme have been examined by in silico modeling, as has the surface environment of arsenate reductase in the vicinity of Cys8, the likely site for the initial reaction between arsenate and the enzyme.     

Contribution of disulfide bonds to the conformational stability and catalytic activity of ribonuclease A.

Disulfide bonds between the side chains of cysteine residues are the only common crosslinks in proteins. Bovine pancreatic ribonuclease A (RNase A) is a 124-residue enzyme that contains four interweaving disulfide bonds (Cys26-Cys84, Cys40-Cys95, Cys58-Cys110, and Cys65-Cys72) and catalyzes the cleavage of RNA. The contribution of each disulfide bond to the conformational stability and catalytic activity of RNase A has been determined by using variants in which each cystine is replaced independently with a pair of alanine residues. Thermal unfolding experiments monitored by ultraviolet spectroscopy and differential scanning calorimetry reveal that wild-type RNase A and each disulfide variant unfold in a two-state process and that each disulfide bond contributes substantially to conformational stability. The two terminal disulfide bonds in the amino-acid sequence (Cys26-Cys84 and Cys58-Cys110) enhance stability more than do the two embedded ones (Cys40-Cys95 and Cys65-Cys72). Removing either one of the terminal disulfide bonds liberates a similar number of residues and has a similar effect on conformational stability, decreasing the midpoint of the thermal transition by almost 40 degrees C. The disulfide variants catalyze the cleavage of poly(cytidylic acid) with values of kcat/Km that are 2- to 40-fold less than that of wild-type RNase A. The two embedded disulfide bonds, which are least important to conformational stability, are most important to catalytic activity. These embedded disulfide bonds likely contribute to the proper alignment of residues (such as Lys41 and Lys66) that are necessary for efficient catalysis of RNA cleavage.     

Single amino acid substitutions in recombinant plant-derived human α1-proteinase inhibitor confer enhanced stability and functional efficacy

Background

Human α₁-proteinase inhibitor (α₁-PI) is the most abundant serine protease inhibitor in the blood and the heterologous expression of recombinant α₁-PI has great potential for possible therapeutic applications. However, stability and functional efficacy of the recombinant protein expressed in alternate hosts are of major concern.

Methods

Five variants of plant-expressed recombinant α₁-PI protein were developed by incorporating single amino acid substitutions at specific sites, namely F51C, F51L, A70G, M358V and M374I. Purified recombinant α₁-PI variants were analyzed for their expression, biological activity, oxidation-resistance, conformational and thermal stability by DAC-ELISA, porcine pancreatic elastase (PPE) inhibition assays, transverse urea gradient (TUG) gel electrophoresis, fluorescence spectroscopy and far-UV CD spectroscopy.

Results

Urea-induced unfolding of recombinant α₁-PI variants revealed that the F51C mutation shifted the mid-point of transition from 1.4 M to 4.3 M, thus increasing the conformational stability close to the human plasma form, followed by F51L, A70G and M374I variants. The variants also exhibited enhanced stability for heat denaturation, and the size-reducing substitution at Phe51 slowed down the deactivation rate ~ 5-fold at 54 °C. The M358V mutation at the active site of the protein did not significantly affect the conformational or thermal stability of the recombinant α₁-PI but provided enhanced resistance to oxidative inactivation.

Conclusions

Our results suggest that single amino acid substitutions resulted in improved stability and oxidation-resistance of the plant-derived recombinant α₁-PI protein, without inflicting the inhibitory activity of the protein.

General significance

Our results demonstrate the significance of engineered modifications in plant-derived recombinant α₁-PI protein molecule for further therapeutic development.     

Structural and evolutionary consequences of unpaired cysteines in trypsinogen

Vertebrate trypsins usually contain six disulfide bonds but human trypsin 1 (PRSS1) contains only five and human trypsin 2 (PRSS2) contains only four. To elucidate possible evolutionary pathways leading to the loss of disulfide bonds, we have constructed mutants lacking one or two cysteines of four disulfide bonds (C22-C157, C127-C232, C136-C201, and C191-C220) in rat anionic trypsinogen and followed their expression in the periplasm of Escherichia coli. When both cysteines of any of the above-mentioned disulfide bonds were replaced by alanines we found, as expected, proteolytically active enzymes. In the case of C127-C232 (missing from both human trypsins) and C191-C220 both single mutants gave active enzymes although their yield was significantly reduced. In contrast, only one of the single mutants of disulfide bonds C22-C157 and C136-C201 (missing from human trypsin 2) was expressed in E. coli. In the case of these disulfide bonds, we obtained no expression when the solvent accessible molecular surface of the free cysteine residue was the smaller one, indicating that a buried unpaired cysteine was more deleterious than one on the surface of the molecule.     

Structure and stability of the neurotoxin PV2 from the eggs of the apple snail Pomacea canaliculata

There is little information on the egg proteins of gastropod mollusks. Here we focus on PV2, a novel neurotoxin from snail eggs, studying its size, shape, structure, and stability, using small angle X-ray scattering (SAXS), absorption and fluorescence spectroscopy, circular dichroism, electron microscopy and partial proteolysis. Results indicate that PV2 is a compact and well folded oligomer of 130 × 44 Å. It is an octamer of four 98 kDa heterodimers composed of 67 and 31 kDa subunits. Subunits are held together by disulfide bonds. Dimers are assembled into native PV2 by non-covalent forces. The larger subunit is more susceptible to proteolysis, indicating it is less compactly folded and/or more exposed. Quenching of tryptophan fluorescence showed a single class of tryptophyl side chains occluded in hydrophobic regions. Native structure shows loss of secondary structure (α+β) at 6 M urea or 60–70 °C; the effects on the quaternary structure suggest an unfolding without disassembling of the protein. The 3D model of PV2 presented here is the first for an egg proteinaceous neurotoxin in animals.     

Structure and conformation of the disulfide bond in dimeric lung surfactant peptides SP-B1-25 and SP-B8-25

Raman spectroscopy was used to determine the conformation of the disulfide linkage between cysteine residues in the homodimeric construct of the N-terminal alpha helical domain of surfactant protein B (dSP-B_1-25). The conformation of the disulfide bond between cysteine residues in position 8 of the homodimer of dSP-B_1-25 was compared with that of a truncated homodimer (dSP-B_8-25) of the peptide having a disulfide linkage at the same position in the alpha helix. Temperature-dependent Raman spectra of the S-S stretching region centered at ∼ 500 cm^− 1 indicated a stable, although highly strained disulfide conformation with a χ(CS-SC) dihedral angle of ± 10° for the dSP-B_1-25 dimer. In contrast, the truncated dimer dSP-B_8-25 exhibited a series of disulfide conformations with the χ(CS-SC) dihedral angle taking on values of either ± 30° or 85± 20°. For conformations with χ(CS-SC) close to the ± 90° value, the Raman spectra of the 8-25 truncated dimers exhibited χ(SS-CC) dihedral angles of 90/180° and 20-30°. In the presence of a lipid mixture, both constructs showed a ν(S-S) band at ∼ 488 cm^− 1, corresponding to a χ(CS-SC) dihedral angle of ± 10°. Polarized infrared spectroscopy was also used to determine the orientation of the helix and β-sheet portion of both synthetic peptides. These calculations indicated that the helix was oriented primarily in the plane of the surface, at an angle of ∼ 60-70° to the surface normal, while the β structure had ∼ 40° tilt. This orientation direction did not change in the presence of a lipid mixture or with temperature. These observations suggest that: (i) the conformational flexibility of the disulfide linkage is dependent on the amino acid residues that flank the cysteine disulfide bond, and (ii) in both constructs, the presence of a lipid matrix locks the disulfide bond into a preferred conformation.