Differential effects induced by α- and β-endosulfan in lipid bilayer organization are reflected in proton permeability

The effects of two insecticides isomers, α- and β-endosulfan, on the passive proton permeability of large unilamellar vesicles (LUV) reconstituted with dipalmitoylphosphatidylcholine (DPPC) or mitochondrial lipids were reported. In DPPC (LUV) gel phase, at 30 °C, the global kinetic constant (K) of proton permeability (proportional to the proton permeability) initially increased slightly with the increase of α-endosulfan/lipid molar ratio up to 0.143. In the range from 0.143 to 0.286, a discontinuity in the increment occurred and, above this range, the proton permeability increased substantially. In DPPC fluid phase, at 48 °C, the proton permeability showed a behavior identical to that observed in gel DPPC, with a sharp increase for α-endosulfan/lipid molar ratios ranging from 0.143 to 0.286. At these and higher concentrations, α-endosulfan induced phase separation in the plane of DPPC membranes, as revealed by differential scanning calorimetry (DSC). Conversely to α-endosulfan, β-endosulfan induced only a slight increase in the proton permeability, either in the fluid or the gel phase of DPPC, for all β-endosulfan/lipid molar ratios tested. Additionally, the effects of the endosulfan isomers on the proton permeability of mitochondrial fluid lipid dispersions, at 37 °C, are similar to those described for DPPC. The β-isomer induced a very small effect, and α-endosulfan, at low concentrations, increased slightly the proton permeability, but for insecticide/lipid molar ratios above 0.143 the permeability increased substantially. Consequently, the membrane physical state of synthetic and native lipid dispersions, as affected by the structural features of α- and β-endosulfan, influenced the proton permeability. The effects here observed in vitro suggest that the formation of lateral membrane domains may underlay the biological activity of α-endosulfan in vivo, contributing to its higher degree of toxicity as compared with β-endosulfan.     

Involvement of Val 315 located in the C-terminal region of thermolysin in its expression in Escherichia coli and its thermal stability

Thermolysin is a thermophilic and halophilic zinc metalloproteinase that consists of β-rich N-terminal (residues 1–157) and α-rich C-terminal (residues 158–316) domains. Expression of thermolysin variants truncated from the C-terminus was examined in E. coli culture. The C-terminal Lys316 residue was not significant in the expression, but Val315 was critical. Variants in which Val315 was substituted with fourteen amino acids were prepared. The variants substituted with hydrophobic amino acids such as Leu and Ile were almost the same as wild-type thermolysin (WT) in the expression amount, α-helix content, and stability. Variants with charged (Asp, Glu, Lys, and Arg), bulky (Trp), or small (Gly) amino acids were lower in these characteristics than WT. All variants exhibited considerably high activities (50–100% of WT) in hydrolyzing protein and peptide substrates. The expression amount, helix content, and stability of variants showed good correlation with hydropathy indexes of the amino acids substituted for Val315. Crystallographic study of thermolysin has indicated that V315 is a member of the C-terminal hydrophobic cluster. The results obtained in the present study indicate that stabilization of the cluster increases thermolysin stability and that the variants with higher stability are expressed more in the culture. Although thermolysin activity was not severely affected by the variation at position 315, the stability and specificity were modified significantly, suggesting the long-range interaction between the C-terminal region and active site.     

Semisynthesis of carboxy-terminal fragments of thermolysin

Enzyme-catalyzed synthesis of two polypeptide fragments, one of which is obtained by chemical synthesis, in the presence of proteolytic enzymes and in aqueous organic solvents constitutes a convenient procedure for the synthesis of proteins and their analogs. This novel semisynthetic procedure was investigated for preparing COOH-terminal fragments of the metallo-protease thermolysin. Fragment 205-316, obtained by autolysis of the protein in the presence of EDTA, was first cleaved selectively with Staphylococcus aureus V8 protease at the level of the single Glu302 residue into fragments 205-302 and 303-316. Upon incubation for 2-5 days of fragment 205-302 with a 5-fold excess of peptide 303-316, prepared by solid phase synthesis, with V8-protease in 0.1 M ammonium acetate, pH 6.0, containing 50% glycerol as organic cosolvent, enzyme-catalyzed reformation of the peptide bond was achieved in yields up to approximately 90% (based on fragment 205-302). The same procedure was used to prepare also the thermolysin fragments 205-315 and 205-311 by enzymatic coupling of fragment 205-302 to peptide 303-315 or 303-311, these last prepared by proteolytic digestion of the synthetic peptide 303-316. This procedure of semisynthesis opens up an approach for the site-directed modification of the tetrahelical COOH-terminal fragment 205-316 of thermolysin at the level of its helical segment encompassing residues 301-312 in the native, intact protein. Such analogs will be useful for examining structure-folding-stability relationships in this folded fragment possessing domain-like characteristics.     

Function of the conserved triad residues in the class C beta-lactamase from Citrobacter freundii GN346

The conserved KTG triad in the class C beta-lactamase from Citrobacter freundii GN346 was examined as to its function by means of site-directed mutagenesis. The following conversions were performed; Lys-315 to arginine, alanine or glutamic acid, Thr-316 to valine, and Gly-317 to alanine, proline or isoleucine. The resultant mutant enzymes revealed that a basic amino acid at position 315 and a small uncharged residue at position 317 are essential for the enzyme activity, but a hydroxyl group at residue 316 is not required for the enzymatic catalysis. The kinetic properties of the purified Arg-315 and Val-316 enzymes provided information on the function of these residues.     

Structure-based design and application of an engineered glutathione transferase for the development of an optical biosensor for pesticides determination

In the present work, a structure-based design approach was used for the generation of a novel variant of synthetic glutathione transferase (PvGmGSTU) with higher sensitivity towards pesticides. Molecular modelling studies revealed Phe117 as a key residue that contributes to the formation of the hydrophobic binding site (H-site) and modulates the affinity of the enzyme towards xenobiotic compounds. Site-saturation mutagenesis of position Phe117 created a library of PvGmGSTU variants with altered kinetic and binding properties. Screening of the library against twenty-five different pesticides, showed that the mutant enzyme Phe117Ile displays 3-fold higher catalytic efficiency and exhibits increased affinity towards α-endosulfan, compared to the wild-type enzyme. Based on these catalytic features the mutant enzyme Phe117Ile was explored for the development of an optical biosensor for α-endosulfan. The enzyme was entrapped in alkosixylane sol-gel system in the presence of two pH indicators (bromocresol purple and phenol red). The sensing signal was based on the inhibition of the sol-gel entrapped GST, with subsequent decrease of released [H⁺] by the catalytic reaction, measured by sol–gel entrapped indicators. The assay response at 562?nm was linear in the range pH?=?4–7. Linear calibration curves were obtained for α-endosulfan in the range of 0–30?μΜ. The reproducibility of the assay response, expressed by relative standard deviation, was in the order of 4.1% (N?=?28). The method was successfully applied to the determination of α-endosulfan in real water samples without sample preparation steps.     

Genetically engineered V79 Chinese hamster cell expression of purified cytochrome P-450IIB1 monooxygenase activity

Chinese hamster V79 fibroblasts, frequently used as target cells in short-term tests for mutagenicity, do not possess measurable monooxygenase activity; in particular, enzymatic oxidation of testosterone (T) cannot be demonstrated. If these V79 cells, however, had been transfected with the cDNA-encoding rat liver cytochrome P-450IIB1 under control of the SV40 early promoter, they stably expressed monooxygenase activity. These so-called SD1 cells then oxidatively metabolized T at a rate of 27 pmol/mg protein/min, converting it to 16 alpha- and 16 beta-hydroxy-T as well as 4-androsten-3,17-dione as sole metabolites in a ratio of 1.1:1.0:1.6. The regio- and stereoselective conversion of T by SD1 cells, as well as the quantitative distribution of the metabolites, corresponds well with the results reported for pure cytochrome P-450IIB1 in a reconstituted system.     

Nuclear Localization Signals in Prototype Foamy Viral Integrase for Successive Infection and Replication in Dividing Cells

We identified four basic amino acid residues as nuclear localization signals (NLS) in the C-terminal domain of the prototype foamy viral (PFV) integrase (IN) protein that were essential for viral replication. We constructed seven point mutants in the C-terminal domain by changing the lysine and arginine at residues 305, 308, 313, 315, 318, 324, and 329 to threonine or proline, respectively, to identify residues conferring NLS activity. Our results showed that mutation of these residues had no effect on expression assembly, release of viral particles, or in vitro recombinant IN enzymatic activity. However, mutations at residues 305 (R → T), 313(R → T), 315(R → P), and 329(R → T) lead to the production of defective viral particles with loss of infectivity, whereas non-defective mutations at residues 308(R → T), 318(K → T), and 324(K → T) did not show any adverse effects on subsequent production or release of viral particles. Sub-cellular fractionation and immunostaining for viral protein PFV-IN and PFV-Gag localization revealed predominant cytoplasmic localization of PFV-IN in defective mutants, whereas cytoplasmic and nuclear localization of PFV-IN was observed in wild type and non-defective mutants. However sub-cellular localization of PFV-Gag resulted in predominant nuclear localization and less presence in the cytoplasm of the wild type and non-defective mutants. But defective mutants showed only nuclear localization of Gag. Therefore, we postulate that four basic arginine residues at 305, 313, 315 and 329 confer the karyoplilic properties of PFV-IN and are essential for successful viral integration and replication.     

The alkene monooxygenase from Xanthobacter Py2 is a binuclear non-haem iron protein closely related to toluene 4-monooxygenase

The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter Py2 have been sequenced. The predicted amino acid sequence of the first ORF shows homology with the iron binding subunits of binuclear non-haem iron containing monooxygenases including benzene monooxygenase, toluene 4-monooxygenase (>60% sequence similarity) and methane monooxygenase (>40% sequence similarity) and that the necessary sequence motifs associated with iron co-ordination are also present. Secondary structure prediction based on the amino acid sequence showed that the predominantly α-helical structure that surrounds the binuclear iron binding site was conserved allowing the sequence to be modelled on the co-ordinates of the methane monooxygenase α-subunit. Significant differences in the residues forming the hydrophobic cavity which forms the substrate binding site are discussed with reference to the differences in reaction specificity and stereospecificity of binuclear non-haem iron monooxygenases.     

The study of the bacteriophage T5 deoxynucleoside monophosphate kinase active site by site-directed mutagenesis

The amino acid residues essential for the enzymatic activity of bacteriophage T5 deoxyribonucleoside monophosphate kinase were determined using a computer model of the enzyme active site. By site-directed mutagenesis, cloning, and gene expression in E. coli, a series of proteins were obtained with single substitutions of the conserved active site amino acid residues—S13A, D16N, T17N, T17S, R130K, K131E, Q134A, G137A, T138A, W150F, W150A, D170N, R172I, and E176Q. After purification by ion exchange and affine chromatography electrophoretically homogeneous preparations were obtained. The study of the enzymatic activity with natural acceptors of the phosphoryl group (dAMP, dCMP, dGMP, and dTMP) demonstrated that the substitutions of charged amino acid residues of the NMP binding domain (R130, R172, D170, and E176) caused nearly complete loss of enzymatic properties. It was found that the presence of the OH-group at position 17 was also important for the catalytic activity. On the basis of the analysis of specific activity variations we assumed that arginine residues at positions 130 and 172 were involved in the binding to the donor γ-phosphoryl and acceptor α-phosphoryl groups, as well as the aspartic acid residue at position 16 of the ATP-binding site (P-loop), in the binding to some acceptors, first of all dTMP. Disproportional changes in enzymatic activities of partially active mutants, G137A, T138A, T17N, Q134A, S13A, and D16N, toward different substrates may indicate that different amino acid residues participate in the binding to various substrates.     

The Nuclear Magnetic Resonance Solution Structure of the Mixed Disulfide between Escherichia coli Glutaredoxin(C14S) and Glutathione

The determination of the nuclear magnetic resonance (NMR) solution structure of the mixed disulfide between the mutant Escherichia coli glutaredoxin Grx(C14S) and glutathione (GSH), Grx(C14S)-SG, is described, the binding site for GSH on Grx(C14S) is located, and the non-bonding interactions between -SG and the protein are characterized. Based on nearly complete sequence-specific NMR assignments, 1010 nuclear Overhauser enhancement upper distance constraints and 116 dihedral angle constraints were obtained as the input for the structure calculations, for which the distance geometry program DIANA was used followed by energy minimization in a waterbath with the AMBER force field in the program OPAL. The -SG moiety was found to be localized on the surface of the protein in a cleft bounded by the amino acid residues Y13, T58, V59, Y72, T73 and D74. Hydrogen bonds have been identified between -SG and the residues V59 and T73 of Grx(C14S), and the formation of an additional hydrogen bond with Y72 and electrostatic interactions with the side-chains of D74 and K45 are also compatible with the NMR, conformational constraints. Comparison of the reduced and oxidized forms of Grx with Grx(C14S)-SG shows that the mixed disulfide more closely resembles the oxidized form of the protein. Functional implications of this observation are discussed. Comparisons are also made with the related proteins bacteriophage T4 glutaredoxin and glutathione S-transferase.     

Differential rate constants of racemization of aspartyl and asparaginyl residues in human alpha A-crystallin mutants

Asp58 and Asp151 in alpha A-crystallin of human eye lenses become highly inverted and isomerized to d-beta-Asp residues with age. Racemization was previously shown to proceed rapidly when the residue on the carboxyl side of the Asp residue is small. Asn was also demonstrated to be more susceptible to racemization than Asp in protein. In this study, the changes of rate constants for racemization at Asp58 and Asp151 and at Asn58 and Asn151 were investigated using D58N, S59T, D151N and A152V mutants obtained through site-directed mutagenesis. The rate constant of racemization at Asn151 in D151N was found to be 1.5 times more rapid than Asp151 in the wild-type. For A152V, the rate constant at Asp151 was 1/4 that of the wild-type. There were no significant differences in the rate constants of racemization for both Asp58 and Asn58 residues. The aggregate size of D58N, S59T and D151N mutants increased or increased in polydispersity and their chaperone activities decreased. The size and chaperone activity of A152V was unchanged. These results suggest that structures close to Asp58 and Asp151 residues in the protein affect the rate constant of Asp racemization and the size and chaperone function of alpha A-crystallin.     

Protection against experimental encephalomyelitis. Idiotypic autoregulation induced by a nonencephalitogenic T cell clone expressing a cross-reactive T cell receptor V gene.

The recovery process in experimental autoimmune encephalomyelitis (EAE) in Lewis rats is characterized by an increasing diversity of T cell clones directed at secondary epitopes of myelin basic protein. Of particular interest, residues 55 to 69 of guinea pig basic protein could induce protection against EAE. A nonencephalitogenic T cell clone, C455-69, that was specific for this epitope transferred protection against both active and passive EAE. Clone C4 was found to express V beta 8.6 in its Ag receptor, and residues 39 to 59 of the TCR V beta 8.6 sequence were found to be highly crossreactive with the corresponding residues 39 to 59 of TCR V beta 8.2, which is known to induce protective anti-idiotypic T cells and antibodies. Like the TCR V beta 8.2 peptide, the V beta 8.6 sequence induced autoregulation and provided effective treatment of established EAE. Thus, the EAE-protective effect of the guinea pig basic protein 55-69 sequence was most likely mediated by T cell clones such as C4 that could efficiently induce anti-TCR immunity directed at a cross-reactive regulatory idiotope.     

The role of loop 7 in mediating calcineurin regulation

Calcineurin (CN) is a heterodimer protein consisting of a 61 kDa catalytic subunit A and a 19 kDa regulatory subunit B. It plays a critical role in T-cell activation and is involved in many cellular processes. Regulation of CN is rather complex, including a number of factors such as divalent metal ions (primarily Ca(2+) and Mn(2+)), calmodulin (CaM) and autoinhibition (AI) segment. Previously, we reported that a loop 7 deletion mutant (V314) in subunit A exhibited high phosphatase activity, although the mechanism for the surprising activity enhancement and whether the activity change applies to other loop 7 residues were not known. In order to probe the role of loop 7, we have carried out extensive mutagenesis experiments, followed by systematic activity assays under a number of regulatory conditions. All mutants, including single deletion mutants Y315, N316 and double deletion mutant V314Y315, showed increased phosphatase activity. Significantly, activities of the mutants containing the V314 deletion, namely V314 and V314Y315, were no longer regulated by regulatory subunit B. These results, along with the structure analysis, suggest that loop 7 as a whole plays an important role in mediating CN's regulation through bridging the regulatory subunit and catalytic core and interaction with the AI segment of CN.     

A Structural Hinge in Eukaryotic MutY Homologues Mediates Catalytic Activity and Rad9-Rad1-Hus1 Checkpoint Complex Interactions

The DNA glycosylase MutY homologue (MYH or MUTYH) removes adenines misincorporated opposite 8-oxoguanine as part of the base excision repair pathway. Importantly, defects in human MYH (hMYH) activity cause the inherited colorectal cancer syndrome MYH-associated polyposis. A key feature of MYH activity is its coordination with cell cycle checkpoint via interaction with the Rad9-Rad1-Hus1 (9-1-1) complex. The 9-1-1 complex facilitates cell cycle checkpoint activity and coordinates this activity with ongoing DNA repair. The interdomain connector (IDC, residues 295-350) between the catalytic domain and the 8-oxoguanine recognition domain of hMYH is a critical element that maintains interactions with the 9-1-1 complex. We report the first crystal structure of a eukaryotic MutY protein, a fragment of hMYH (residues 65-350) that consists of the catalytic domain and the IDC. Our structure reveals that the IDC adopts a stabilized conformation projecting away from the catalytic domain to form a docking scaffold for 9-1-1. We further examined the role of the IDC using Schizosaccharomyces pombe MYH as model system. In vitro studies of S. pombe MYH identified residues I261 and E262 of the IDC (equivalent to V315 and E316 of the hMYH IDC) as critical for maintaining the MYH/9-1-1 interaction. We determined that the eukaryotic IDC is also required for DNA damage selection and robust enzymatic activity. Our studies also provide the first evidence that disruption of the MYH/9-1-1 interaction diminishes the repair of oxidative DNA damage in vivo. Thus, preserving the MYH/9-1-1 interaction contributes significantly to minimizing the mutagenic potential of oxidative DNA damage.     

Calmodulin kinase II chimeras used to investigate the structural requirements for smooth muscle myosin light chain kinase autoinhibition and calmodulin-dependent activation

Segments of the autoregulatory domain of MK, a catalytically active fragment of the monomeric smooth muscle myosin light chain kinase (smMLCK) (residues 472-972), were replaced with their counterparts from a homologous but multimeric enzyme, calmodulin-dependent protein kinase II (CaM KII). Chimeric proteins in which both the autoregulatory and oligomerization domains of CaM KII (residues 281-478) were substituted for residues 781-972 of smMLCK, MK(CK281-478), or only the autoregulatory domain of CaM KII (residues 281-315) was exchanged for residues 781-813 of smMLCK, MK(CK281-315), exhibited significant enzymatic activity in the absence of Ca(2+)/CaM. In contrast, both MK and a chimeric protein in which the C-terminal half of the autoregulatory domain of smMLCK was replaced with CaM KII residues 301-315, MK(CK301-315), were inactive in the absence of Ca(2+)/CaM. These results indicate that the sequence of the N-terminal half of the autoregulatory domain of smMLCK is important for complete autoinhibition of its enzymatic activity. All proteins bound to Ca(2+)/CaM, and the chimeric proteins MK(CK281-478) and MK(CK281-315) were activated by Ca(2+)/CaM with activation constants (K(CaM)) and maximal enzymatic activities comparable to those of the wild-type MK enzyme. This demonstrates that the entire autoregulatory domain of CaM KII can replace that of smMLCK in its ability to promote efficient CaM-dependent activation of the smMLCK enzyme. However, the inability of the chimeric protein MK(CK301-315) to be activated by Ca(2+)/CaM suggests that replacement of only the C-terminal half of the autoregulatory domain of smMLCK, while still retaining the ability to bind Ca(2+)/CaM, also substitutes residues that prevent activation of the enzyme by Ca(2+)/CaM.     

Spontaneous development of protective anti-T cell receptor autoimmunity targeted against a natural EAE-regulatory idiotope located within the 39-59 region of the TCR-V beta 8.2 chain.

The development of experimental autoimmune encephalomyelitis (EAE) in Lewis rats is mediated by V beta 8.2+ T cells specific for myelin basic protein. One consequence of this biased expression of V beta 8.2 is the spontaneous development of regulatory T cells and antibodies against residues 39-59 of the V beta 8.2 sequence. Moreover, a synthetic V beta 8.2-39-59 peptide could induce protection against and speed recovery from EAE. T cells and antibodies specific for V beta 8.2-39-59 could transfer protection from EAE. Recently, we reported that the protective T cell epitope is subsumed within the V beta 8-44-54 sequence. We now report that protection induced by V beta 8-44-54 lasted at least 102 days and produced "split tolerance," enhancing anti-myelin basic protein antibody titers but reducing anti-myelin basic protein T cell frequency. The shorter V beta 8-44-54 peptide induced a distinct set of antibodies that did not cross-react with the longer V beta 8.2-39-59 peptide, although both specificities could stain V beta 8.2+ T cells and were equally protective against EAE. However, the V beta 8.2-39-59 peptide, but not the V beta 8-44-54 peptide, would appear to represent the natural idiotope: antibodies to V beta 8.2-39-59 that develop spontaneously during EAE could be boosted to higher titers only by the V beta 8.2-39-59, but not by other TCR peptides from the V beta 8.2 sequence, including V beta 8-44-54 that contains the functional T cell epitope. These results suggest that natural processing of the TCR V beta-chain favors the formation of a peptide that resembles the V beta 8.2-39-59 sequence. The B cell epitope present on the V beta 8-44-54 sequence was evident only in the absence of residues 39-43 and 55-59, suggesting that the two peptides possess distinct conformations. However, the V beta 8-44-54 B cell epitope is most likely expressed on the V beta 8.2+ T cells, either as a low affinity determinant on the intact TCR alpha/beta heterodimer or as a cryptic epitope bound in the cleft of surface MHC molecules.     

NMR structure of the calflagin Tb24 flagellar calcium binding protein of Trypanosoma brucei

Flagellar calcium binding proteins are expressed in a variety of trypanosomes and are potential drug targets for Chagas disease and African sleeping sickness. The flagellar calcium binding protein calflagin of Trypanosoma brucei (called Tb24) is a myristoylated and palmitoylated EF‐hand protein that is targeted to the inner leaflet of the flagellar membrane. The Tb24 protein may also interact with proteins on the membrane surface that may be different from those bound to flagellar calcium binding proteins (FCaBPs) in T. cruzi. We report here the NMR structure of Tb24 that contains four EF‐hand motifs bundled in a compact arrangement, similar to the overall fold of T. cruzi FCaBP (RMSD = 1.0 Å). A cluster of basic residues (K22, K25, K31, R36, and R38) located on a surface near the N‐terminal myristoyl group may be important for membrane binding. Non‐conserved residues on the surface of a hydrophobic groove formed by EF2 (P91, Q95, D103, and V108) and EF4 (C194, T198, K199, Q202, and V203) may serve as a target protein binding site and could have implications for membrane target recognition.     

Hyperproduction of a recombinant fusion protein of Staphylococcus aureus V8 protease in Escherichia coli and its processing by OmpT protease to release an active V8 protease derivative

The expression of a recombinant fusion protein including Staphylococcus aureus V8 protease was studied by using Escherichia coli as the host strain. When the mature V8 protease was expressed as a fusion protein with a truncated E. coli \-galactosidase (\-gal97S4D), we could not obtain a sufficient amount of the enzyme because of the toxicity resulting from the expressed protease activity. Synthesis of V8 protease was increased by constructing a sandwich-type fusion protein consisting of \-gal197S4D, a V8 protease derivative with the 56 C-terminal amino acids deleted (V856) and a truncated aminoglycoside-3'-phosphotransferase. This fusion protein was successfully produced as inactive inclusion bodies. To release the V856 protease from the fusion protein, we developed a novel processing method using an endogeneous E. coli OmpT protease, which can recognize the dibasic amino acid residues located in the linker peptides of the fusion protein. After solubilizing the inclusion bodies with urea, the V856 protein was automatically released from the fusion protein by the OmpT protease, which was coprecipitated with the inclusion bodies. The V856 protease thus obtained showed the same enzymatic activity as that of the native V8 protease. We demonstrate in this study that the N-terminal prepro sequence and the C-terminal repeated sequence of this enzyme are not necessary for its enzymatic activity and protein folding.