Free energy simulation to investigate the effect of amino acid sequence environment on the severity of osteogenesis imperfecta by glycine mutations in collagen

Molecular dynamics simulations were carried out to calculate the free energy change difference of two collagen-like peptide models for Gly --> Ser mutations causing two different osteogenesis imperfecta phenotypes. These simulations were performed to investigate the impact of local amino acid sequence environment adjacent to a mutation site on the stability of the collagen. The average free energy differences for a Gly --> Ser mutant relative to a wild type are 3.4 kcal/mol and 8.2 kcal/mol for a nonlethal site and a lethal site, respectively. The free energy change differences of mutant containing two Ser residues relative to the wild type at the nonlethal and lethal mutation sites are 4.6 and 9.8 kcal/mol, respectively. Although electrostatic interactions stabilize mutants containing one or two Ser residues at both mutation sites, van der Waals interactions are of sufficient magnitude to cause a net destabilization. The presence of Gln and Arg near the mutation site, which contain large and polar side chains, provide more destabilization than amino acids containing small and nonpolar side chains.     

Effect of osteogenesis imperfecta mutations on free energy of collagen model peptides: a molecular dynamics simulation

We have carried out stochastic boundary molecular dynamics simulations to estimate free energy changes for substitutions of Gly with Val, Arg and Trp residues in a collagen-like peptide. The relative free energy change differences of mutants containing a Val, an Arg and a Trp relative to the wild type are 5.7, 8.1 and 9.5 kcal/mol, respectively. The corresponding free energy change differences of mutants containing two mutated residues are on average 7.6, 10.5 and 14.7 kcal/mol, respectively. We show that the free energy change differences are correlated with the severity of OI from statistical analysis and mechanical properties of the individual tropocollagen molecules. This simulation result indicates an atomistic-level mechanistic understanding of the effect of OI mutations in terms of stability of the mutants relative to the wild type, which could eventually provide a new strategy for diagnosis and treatment of the disease.     

Generation of the oxidized form protects human brain type creatine kinase against cystine-induced inactivation

Cystine accumulation in cystinotic patients has been reported to inhibit brain type creatine kinase (BBCK), an important thiol-containing enzyme in energy homeostasis. In this research, we found that the oxidized form of BBCK (O-BBCK) was induced by cystine, and the intramolecular disulfide bond of O-BBCK was formed between Cys74 and Cys254. The wild type BBCK was found to be more resistant to the inactivation induced by cystine when compared to the single point mutant C74S or C254S. Meanwhile, the existence of GSH could protect the wild type BBCK more efficiently than the mutants. These observations suggested that the ability to generate the oxidized form could protect BBCK against the intracellular oxidative stress.     

Free energy perturbation study on a Trp-binding mutant (Ser88-->Cys) of the trp-repressor.

The Ser88-->Cys mutant of the trp-repressor showed a lower affinity for the corepressor than the wild-type repressor [delta delta G = 1.7 +/- 0.3 kcal/mol, Chou and Matthews (1989) J. Biol. Chem., 264, 18314-18319]. A molecular dynamics/free energy cycle perturbation study was performed to understand the origin of the decreased affinity. A value (delta delta G = 1.58 +/- 0.28 kcal/mol) comparable with the experimental value was obtained by the simulation. Free energy component analysis revealed that destabilization of the van der Waals interaction between Ser88 and Trp109 (corepressor) mainly contributed to the decreased affinity of the mutant. The rotational transition of the hydroxyl (sulfhydryl) group of Ser88 (Cys88) during the simulations affected the contributions of Arg84 and water to the free energy change in the aporepressor and those of Arg84 and Trp109 to that in the holorepressor. However, the contributions from different residues compensated each other, and the total free energy changes were almost invariable in the various simulations.     

Structure of a stabilizing disulfide bridge mutant that closes the active-site cleft of T4 lysozyme.

The engineered disulfide bridge between residues 21 and 142 of phage T4 lysozyme spans the active-site cleft and can be used as a switch to control the activity of the enzyme (Matsumura, M. & Matthews, B.W., 1989, Science 243, 792-794). In the oxidized form the disulfide increases the melting temperature of the protein by 11 degrees C at pH 2. The crystal structure of this mutant lysozyme has been determined in both the reduced and oxidized forms. In the reduced form, the crystal structure of the mutant is shown to be extremely similar to that of wild type. In the oxidized form, however, the formation of the disulfide bridge causes the alpha-carbons of Cys 21 and Cys 142, on opposite sides of the active-site cleft, to move toward each other by 2.5 A. In association with this movement, the amino-terminal domain of the protein undergoes a rigid-body rotation of 5.1 degrees relative to the carboxy-terminal domain. This rotation occurs about an axis passing through the junction of the amino-terminal and carboxy-terminal domains and is also close to the axis that best fits the apparent thermal motion of the amino-terminal domain seen previously in crystals of wild-type lysozyme. Even though the engineered Cys 21-Cys 142 disulfide links together the amino-terminal and carboxy-terminal domains of T4 lysozyme, it does not reduce the apparent mobility of the one domain relative to the other. The pronounced "hinge-bending" mobility of the amino-terminal domain that is suggested by the crystallographic thermal parameters of wild-type lysozyme persists in the oxidized (and reduced) mutant structures. In the immediate vicinity of the introduced disulfide bridge the mutant structure is more mobile (or disordered) than wild type, so much so that the exact conformation of Cys 21 remains obscure. As with the previously described disulfide bridge between residues 9 and 164 of T4 lysozyme (Pjura, P.E., Matsumura, M., Wozniak, J.A., & Matthews, B.W., 1990, Biochemistry 29, 2592-2598), the engineered cross-link substantially enhances the stability of the protein without making the folded structure more rigid.     

Stabilization of Escherichia coli ribonuclease H by introduction of an artificial disulfide bond.

To examine the effect of the introduction of a disulfide bond on the stability of Escherichia coli ribonuclease H, a disulfide bond was engineered between Cys13, which is present in the wild-type enzyme, and Cys44, which is substituted for Asn44 by site-directed mutagenesis. The disulfide bond was only formed between these residues upon oxidation in vitro with redox buffer. The conformational and thermal stabilities were estimated from the guanidine hydrochloride and thermal denaturation curves, respectively. The oxidized (cross-linked) mutant enzyme showed a Tm of 62.3 degrees C, which was 11.8 degrees C higher than that observed for the wild-type enzyme. The free energy change of unfolding in the absence of denaturant, delta G[H2O], and the mid-point of the denaturation curve, [D]1/2, of the oxidized mutant enzyme were also increased by 2.1-2.8 kcal/mol and 0.36-0.48 M, respectively. Introduction of a disulfide bond thus greatly enhanced both the thermal and conformational stabilities of the enzyme. In addition, kinetic analyses for the enzymatic activities of mutant enzymes suggest that Thr43 and Asn44 are involved in the substrate-binding site of the enzyme.     

The role of transmembrane domain III in the lactose permease of Escherichia coli.

Deletion of putative transmembrane helix III from the lactose permease of Escherichia coli results in complete loss of transport activity. Similarly, replacement of this region en bloc with 23 contiguous Ala, Leu, or Phe residues abolishes active lactose transport. The observations suggest that helix III may contain functionally important residues; therefore, this region was subjected to Cys-scanning mutagenesis. Using a functional mutant devoid of Cys residues (C-less permease) each residue from Tyr 75 to Leu 99 was individually replaced with Cys. Twenty-one of the 25 mutants accumulate lactose to > 70% of the steady-state exhibited by C-less permease, and an additional 3 mutants transport to lower, but significant levels (40-60% of C-less). Cys replacement for Leu 76 results in low transport activity (18% of C-less). However, when placed in the wild-type background, mutant Leu 76-->Cys exhibits highly significant rates of transport (55% of wild type) and steady-state levels of lactose accumulation (65% of wild type). Immunoblots reveal that the mutants are inserted into the membrane at concentrations comparable to wild type. Studies with N-ethylmaleimide show that mutant Gly 96-->Cys is rapidly inactivated, whereas the other single-Cys mutants are not altered significantly by the alkylating agent. Moreover, the rate of inactivation of Gly 96-->Cys permease is enhanced at least 2-fold in the presence of beta-galactopyranosyl 1-thio-beta, D-galactopyranoside. The observations demonstrate that although no residue per se appears to be essential, structural properties of helix III are important for active lactose transport.     

Structural analysis of three His32 mutants of DsbA: support for an electrostatic role of His32 in DsbA stability.

DsbA, a 21-kDa protein from Escherichia coli, is a potent oxidizing disulfide catalyst required for disulfide bond formation in secreted proteins. The active site of DsbA is similar to that of mammalian protein disulfide isomerases, and includes a reversible disulfide bond formed from cysteines separated by two residues (Cys30-Pro31-His32-Cys33). Unlike most protein disulfides, the active-site disulfide of DsbA is highly reactive and the oxidized form of DsbA is much less stable than the reduced form at physiological pH. His32, one of the two residues between the active-site cysteines, is critical to the oxidizing power of DsbA and to the relative instability of the protein in the oxidized form. Mutation of this single residue to tyrosine, serine, or leucine results in a significant increase in stability (of approximately 5-7 kcal/mol) of the oxidized His32 variants relative to the oxidized wild-type protein. Despite the dramatic changes in stability, the structures of all three oxidized DsbA His32 variants are very similar to the wild-type oxidized structure, including conservation of solvent atoms near the active-site residue, Cys30. These results show that the His32 residue does not exert a conformational effect on the structure of DsbA. The destabilizing effect of His32 on oxidized DsbA is therefore most likely electrostatic in nature.     

Thermal stability of wild type and disulfide bridge containing mutant of poplar plastocyanin

A comparative study of the thermal stability of wild type poplar plastocyanin and of a mutant form containing a disulfide bridge between residues 21 and 25 was performed using differential scanning calorimetry and optical spectroscopic techniques. For wild type plastocyanin the transition temperature, determined from the calorimetric profiles, is 62.7 degrees C at the scan rate of 60 degrees C/h, whereas for the mutant it is reduced to 58.0 degrees C. In both cases, the endothermic peak is followed by an exothermic one at higher temperatures. The unfolding process monitored by optical absorption at 596 nm also reveals a reduced thermal stability of the mutated plastocyanin compared to the wild type protein, with transition temperatures of 54.8 and 58.0 degrees C, respectively. For both proteins, the denaturation process was found to be irreversible and dependent on the scan rate preventing the thermodynamic analysis of the unfolding process. In parallel, small conformational changes between wild type and mutant plastocyanin emerge from fluorescence spectroscopy measurements. Here, a difference in the interaction of the two proteins between the microenvironment surrounding the fluorophores and the solvent was proposed. The destabilization observed in the disulfide containing mutant of plastocyanin suggests that the double mutation, Ile21Cys and Glu25Cys, introduces strain into the protein which offsets the stabilizing effect expected from the formation of a covalent crosslink.     

Essential role of the conformational flexibility of helices 1 and 5 on the lipid binding activity of apolipophorin-III.

It has been recently postulated that the conformational flexibility of helices 1 and 5 of Locusta migratoria apoLp-III could play an important role in early steps of binding of this apolipoprotein to a lipid surface (Soulages, J. L., and Arrese, E. L. (2000) J. Biol. Chem. 275, 17501-17509). To test this model, we have designed a double Cys mutant in which a disulfide bond linking helices 1 and 5 could be formed, resulting in an apolipoprotein with reduced conformational flexibility of its N- and C-terminal helices. Substitution of Thr(18) and Ala(147) by Cys residues provided a protein that under nonreducing conditions was fully oxidized. The far-UV CD spectra of this mutant in the reduced and oxidized states indicated that their secondary structures were identical to the structure of the wild type recombinant apoLp-III, which contains no Cys residues. Near-UV CD studies confirmed the formation of a disulfide bond and the absence of structural perturbations. The lipid binding activity of the reduced mutant, as determined by its ability to form discoidal lipoproteins, was nearly identical to that of the wild type protein. Contrarily, the disulfide form of the mutant was not able to form discoidal lipoproteins with liposomes of either dimirystoylphosphatidylcholine or dimyristoylphosphatidylglycerol. It is concluded that the separation of the helices 1 and 5 constitutes one of the key steps along the complex pathway for the formation of the final apolipoprotein lipid-bound state. It is inferred that the conformational flexibility of helices 1 and 5 is a key property of apoLp-III, allowing the exposure of hydrophobic protein regions and the interaction of the hydrophobic faces of the amphipathic alpha-helices with the lipoprotein lipid surface.     

Increasing the conformational stability by replacement of heme axial ligand in c-type cytochrome

To investigate the role of the heme axial ligand in the conformational stability of c-type cytochrome, we constructed M58C and M58H mutants of the red alga Porphyra yezoensis cytochrome c(6) in which the sixth heme iron ligand (Met58) was replaced with Cys and His residues, respectively. The Gibbs free energy change for unfolding of the M58H mutant in water (DeltaG degrees (unf)=1.48 kcal/mol) was lower than that of the wild-type (2.43 kcal/mol), possibly due to the steric effects of the mutation on the apoprotein structure. On the other hand, the M58C mutant exhibited a DeltaG degrees (unf) of 5.45 kcal/mol, a significant increase by 3.02 kcal/mol compared with that of wild-type. This increase was possibly responsible for the sixth heme axial bond of M58C mutant being more stable than that of wild-type according to the heme-bound denaturation curve. Based on these observations, we propose that the sixth heme axial ligand is an important key to determine the conformational stability of c-type cytochromes, and the sixth Cys heme ligand will give stabilizing effects.     

Dramatic stabilization of the native state of human carbonic anhydrase II by an engineered disulfide bond

To find a disulfide pair that could stabilize the enzyme human carbonic anhydrase II (HCA II), we grafted the disulfide bridge from the related and unusually stable carbonic anhydrase form from Neisseria gonorrhoeae (NGCA) into the human enzyme. Thus, the two Cys residues at positions 23 and 203 were engineered into a pseudo-wild-type form of HCA II (C206S), giving the mutant C206S/A23C/L203C. The disulfide bond was not formed spontaneously. The native state of the reduced form of the mutant was markedly destabilized (2.9 kcal/mol) compared to that of HCA II. Formation of a disulfide bridge was achieved by treatment by oxidized glutathione. This led to a significant stabilization of the native conformation. Compared to HCA II the unfolding midpoint for the variant was increased from 0.9 to 1.7 M guanidine HCl, corresponding to a stabilization of 3.7 kcal/mol. This makes the human enzyme almost as stable as the model protein NGCA, for which the unfolding of the native state has a midpoint at 2.1 M guanidine HCl. The stabilized protein underwent, contrary to all other investigated variants of HCA II, an apparent two-state unfolding transition, as judged from intrinsic Trp fluorescence measurements. A molten-globule intermediate is nevertheless formed but is suppressed because of the high denaturant pressure it faces upon rupture of the native state.     

Evidence that Highly Conserved Residues of <Emphasis Type="Italic">Delonix regia</Emphasis> Trypsin Inhibitor Are Important for Activity

Delonix regia trypsin inhibitor (DrTI) consists of a single-polypeptide chain with a molecular mass of 22 kDa and containing two disulfide bonds (Cys44–Cys89 and Cys139–Cys149). Sequence comparison with other plant trypsin inhibitors of the Kunitz family reveals that DrTI contains a negatively charged residue (Glu68) at the reactive site rather than the conserved Arg or Lys found in other Kunitz-type trypsin inhibitors. Site-directed mutagenesis yielded five mutants containing substitutions at the reactive site and at one of the disulfide bonds. Assay of the recombinant proteins showed mutant Glu68Leu and Glu68Lys to have only 4–5% of the wild-type activity. These provide evidence that the Glu68 residue is the reactive site for DrTI and various other Kunitz-type trypsin inhibitors. The Cys139Gly mutant lost its inhibitory activity, whereas the Cys44Gly mutant did not, indicating that the second disulfide bond (Cys139–Cys149) is critical to DrTI inhibitory activity, while the first disulfide bond (Cys44–Cys89) is not required.     

Molecular Dynamics/Free Energy Perturbation Studies of the Thermostable V74I Mutant of Ribonuclease HI

Abstract

Recent site-directed mutagenesis and thermodynamic studies have shown that the V74I mutant of Escherichia coli ribonuclease HI (RNase HI) is more stable than the wild type protein [Ishikawa et al., Biochemistry 32, 6171 (1993)]. In order to clarify the stabilization mechanism of this mutant, we calculated the free energy change due to the mutation Val 74→Ile in both the native and denatured states by free energy perturbations based on molecular dynamics (MD) simulations. We carried out inclusive MD simulations for the protein in water; i.e., fully solvated, no artificial constraints applied, and all long-range Coulomb interactions included. We found that the free energy of the mutant increased slightly relative to the wild type, in the native state by 1.60 kcal/mol, and in the denatured state by 2.25 kcal/mol. The unfolding free energy increment of the mutant (0.66 ± 0.19 kcal/mol) was in good agreement with the experimental value (0.6 kcal/mol). The hysteresis error in the free energy calculations, i.e., forward and reverse perturbations, was only ±0.19 kcal/mol. These results show that the V74I mutant is stabilized relative to the wild type by the increased free energy of the denatured state and not by a decrease in the free energy of the native state as had been proposed earlier based on the mutant X-ray structure. It was found that the stabilization was caused by a loss of solvation energy in the mutant denatured state and not by improved packing interactions inside the native protein.     

Folding and oxidation of recombinant human granulocyte colony stimulating factor produced in Escherichia coli. Characterization of the disulfide-reduced intermediates and cysteine----serine analogs.

The folding and oxidation of recombinant human granulocyte colony-stimulating factor solubilized from Escherichia coli inclusion bodies was investigated. During the folding process, two intermediates, I1 and I2, were detected by kinetic studies using high performance liquid chromatography. I1 exists transiently and disappears quickly with the concomitant formation of I2. In contrast, I2 requires a longer time to fold into the final oxidized form, N. CuSO4 catalysis increases the folding rate of I2 from I1, while CuSO4 and elevated temperature (37 degrees C) have a dramatic effect on the folding rate of N from I2. These observations suggest the following sequential oxidative folding pathway. [sequence: see text] Peptide map analysis of the iodoacetate-labeled intermediates revealed that I1 represents the fully reduced granulocyte colony-stimulating factor containing 5 free cysteines; I2 is the partially oxidized species containing a single Cys36-Cys42 disulfide bond; and N, the final folded form, has two disulfide bonds. The physicochemical properties and biological activities of I1, I2, N, and several Cys----Ser analogs made by site-directed mutagenesis were further investigated. In guanidine hydrochloride-induced denaturation studies, the disulfide-reduced intermediates and the analogs missing either of the disulfide bonds are conformationally less stable than those of the wild type molecule or the analog with the free Cys at position 17 changed to Ser. Recombinant human granulocyte colony stimulating factor lacking either disulfide bond or both has overall secondary and tertiary structures different from those of the wild type molecule and exhibits lower biological activity. These studies show that disulfide bond formation is crucial for maintaining the molecule in a properly folded and biologically active form.     

Molecular dynamics simulations as a tool for improving protein stability

Haloalkane dehalogenase (DhlA) was used as a model protein to explore the possibility to use molecular dynamics (MD) simulations as a tool to identify flexible regions in proteins that can serve as a target for stability enhancement by introduction of a disulfide bond. DhlA consists of two domains: an alpha/beta-hydrolase fold main domain and a cap domain composed of five alpha-helices. MD simulations of DhlA showed high mobility in a helix-loop-helix region in the cap domain, involving residues 184-211. A disulfide cross-link was engineered between residue 201 of this flexible region and residue 16 of the main domain. The mutant enzyme showed substantial changes in both thermal and urea denaturation. The oxidized form of the mutant enzyme showed an increase of the apparent transition temperature from 47.5 to 52.5 degrees C, whereas the T(m,app) of the reduced mutant decreased by more than 8 degrees C compared to the wild-type enzyme. Urea denaturation results showed a similar trend. Measurement of the kinetic stability showed that the introduction of the disulfide bond caused a decrease in activation free energy of unfolding of 0.43 kcal mol(-1) compared to the wild-type enzyme and also indicated that the helix-loop-helix region was involved early in the unfolding process. The results show that MD simulations are capable of identifying mobile protein domains that can successfully be used as a target for stability enhancement by the introduction of a disulfide cross-link.     

The disulfide bonding pattern in ficolin multimers

Ficolin is a plasma lectin, consisting of a short N-terminal multimerization domain, a middle collagen domain, and a C-terminal fibrinogen-like domain. The collagen domains assemble the subunits into trimers, and the N-terminal domain assembles four trimers into 12-mers. Two cysteine residues in the N-terminal domain are thought to mediate multimerization by disulfide bonding. We have generated three mutants of ficolin alpha in which the N-terminal cysteines were substituted by serines (Cys4, Cys24, and Cys4/Cys24). The N-terminal cysteine mutants were produced in a mammalian cell expression system, purified by affinity chromatography, and analyzed under nondenaturing conditions to resolve the multimer structure of the native protein and under denaturing conditions to resolve the disulfide-linked structure. Glycerol gradient sedimentation and electron microscopy in nondenaturing conditions showed that plasma and recombinant wild-type protein formed 12-mers. The Cys4 mutant also formed 12-mers, but Cys24 and Cys4/Cys24 mutants formed only trimers. This means that protein interfaces containing Cys4 are stable as noncovalent protein-protein interactions and do not require disulfides, whereas those containing Cys24-Cys24 require the disulfides for stability. Proteins were also analyzed by nonreducing SDS-PAGE to show the covalent structure under denaturing conditions. Wild-type ficolin was covalently linked into 12-mers, whereas elimination of either Cys4 or Cys24 gave dimers and monomers. We present a model in which symmetric Cys24-Cys24 disulfide bonds between trimers are the basis for multimerization. The model may also be relevant to collectin multimers.     

Selectivity of tryptophan residues in mediating photolysis of disulfide bridges in goat alpha-lactalbumin

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues and four disulfide bonds. Illumination with near-UV light results in the cleavage of disulfide bridges and in the formation of free thiols. To obtain information about the reaction products, the illuminated protein was carbamidomethylated and digested with trypsin and the peptides were analyzed by mass spectrometry. Peptides containing Cys120Cam, Cys61Cam, or Cys91Cam were detected, as well as two peptides containing a new Cys-Lys cross-link. In one, Cys6 was cross-linked to Lys122, while the cross-link in the second was either a Cys91-Lys79 or Cys73-Lys93 cross-link; however, the exact linkage could not be defined. The results demonstrate photolytic cleavage of the Cys6-Cys120, Cys61-Cys77, and Cys73-Cys91 disulfide bonds. While photolysis of Cys6-Cys120 and Cys73-Cys91 disulfide bonds in GLA has been reported, cleavage of the Cys61-Cys77 disulfide bonds has not been previously detected. To examine the contribution of the individual Trp residues, we constructed the GLA mutants, W26F, W60F, W104F, and W118F, by replacing single Trp residues with phenylalanine (Phe). The substitution of each Trp residue led to less thiol production compared to that for wild-type GLA, showing that each Trp residue in GLA contributed to the photolytic cleavage of disulfide bridges. The specificity was expressed by the nature of the reaction products. No cleavage of the Cys6-Cys120 disulfide bridge was detected when the W26F mutant was illuminated, and no cleavage of the Cys73-Cys91 disulfide bridge was seen following illumination of W26F or W104F. In contrast, Cys61Cam, resulting from the cleavage of the Cys61-Cys77 disulfide bridge, was found following illumination of any of the mutants.     

Novel insight into the mechanism of the vitamin K oxidoreductase (VKOR): electron relay through Cys43 and Cys51 reduces VKOR to allow vitamin K reduction and facilitation of vitamin K-dependent protein carboxylation

The vitamin K oxidoreductase (VKOR) reduces vitamin K to support the carboxylation and consequent activation of vitamin K-dependent proteins, but the mechanism of reduction is poorly understood. VKOR is an integral membrane protein that reduces vitamin K using membrane-embedded thiols (Cys-132 and Cys-135), which become oxidized with concomitant VKOR inactivation. VKOR is subsequently reactivated by an unknown redox protein that is currently thought to act directly on the Cys132-Cys135 residues. However, VKOR contains evolutionarily conserved Cys residues (Cys-43 and Cys-51) that reside in a loop outside of the membrane, raising the question of whether they mediate electron transfer from a redox protein to Cys-132/Cys-135. To assess a possible role, the activities of mutants with Ala substituted for Cys (C43A and C51A) were analyzed in intact membranes using reductants that were either membrane-permeable or -impermeable. Both reductants resulted in wild type VKOR reduction of vitamin K epoxide; however, the C43A and C51A mutants only showed activity with the membrane-permeant reductant. We obtained similar results when testing the ability of wild type and mutant VKORs to support carboxylation, using intact membranes from cells coexpressing VKOR and carboxylase. These results indicate a role for Cys-43 and Cys-51 in catalysis, suggesting a relay mechanism in which a redox protein transfers electrons to these loop residues, which in turn reduce the membrane-embedded Cys132-Cys135 disulfide bond to activate VKOR. The results have implications for the mechanism of warfarin resistance, the topology of VKOR in the membrane, and the interaction of VKOR with the carboxylase.     

Engineering subtilisin E for enhanced stability and activity in polar organic solvents

We examined the effect of a novel disulfide bond engineered in subtilisin E from Bacillus subtilis based on the structure of a thermophilic subtilisin-type serine protease aqualysin I. Four sites (Ser163/Ser194, Lys170/Ser194, Lys170/Glu195, and Pro172/Glu195) in subtilisin E were chosen as candidates for Cys substitutions by site-directed mutagenesis. The Cys170/Cys195 mutant subtilisin formed a disulfide bond in B. subtilis, and showed a 5-10-fold increase in specific activity for an authentic peptide substrate for subtilisin, N-succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide, compared with the single-Cys mutants. However, the disulfide mutant had a 50% decrease in catalytic efficiency due to a smaller k(cat) and was thermolabile relative to the wild-type enzyme, whereas it was greatly stabilized relative to its reduced form. These results suggest that an electrostatic interaction between Lys170 and Glu195 is important for catalysis and stability in subtilisin E. Interestingly, the disulfide mutant was found to be more stable in polar organic solvents, such as dimethylformamide and ethanol, than the wild-type enzyme, even under reducing conditions; this is probably due to the substitution of uncharged Cys by charged surface residues (Lys170 and Glu195). Further, the amino-terminal engineered disulfide bond (Gly61Cys/Ser98Cys) and the mutation Ile31Leu were introduced to enhance the stability and catalytic activity. A prominent 3-4-fold increase in the catalytic efficiency occurred in the quintet mutant enzyme over the range of dimethylformamide concentration (up to 40%).