Strain V8 Research Articles

Differential responses of Ceratitis capitata to infection by the entomopathogenic fungus Purpureocillium lilacinum.

The medfly Ceratitis capitata is one of the most damaging fruit pests with quarantine significance due to its extremely wide host range. The use of entomopathogenic fungi constitutes a promising approach with potential applications in integrated pest management. Furthermore, developing insect control methods can involve the use of fungal machinery to cause metabolic disruption, which may increase its effectiveness by impairing insect development. Insect species, including C. capitata, relies on reproduction potential, nutrient reserves, metabolic activities, and immune response for survival. Accordingly, the purpose of this study was to investigate the impacts of the entomopathogenic fungus Purpureocillium lilacinum on C. capitata pre-mortality. The medfly V8 strain was subjected to laboratory bioassays, which consisted on determining the virulence of P. lilacinum on the medfly. Purpureocillium lilacinum was applied on abdominal topical of 5-day-old males and females. Following the fungal inoculation, we have confirmed (i) a significant increase in tissue sugar content, (ii) a significant decrease in carbohydrase activities, digestive glycosyl hydrolase, and proteinase activities in whole midguts of treated flies, (iii) the antimicrobial peptides (AMPs) genes expression profile was significantly influenced by fly gender, fly status (virgin, mature, and mated), and time after infection, but infection itself had no discernible impact on the AMPs for the genes that were examined. This study provides the first insight into how P. lilacinum could affect C. capitata physiological mechanisms and provides the foundation for considering P. lilacinum as a novel, promising biocontrol agent.

Implications of SiO2 nanoparticles for in vitro biodegradation of low-density polyethylene with potential isolates of Bacillus, Pseudomonas, and their synergistic effect on Vigna mungo growth

In the natural environment, low-density polyethylene (LDPE) contamination is one of the major sources of pollution and creates the hazardous threat. Biodegradation is an eco-friendly approach to control such type of pollution and maintain the sustainable environment. The presence of nanoparticles influences the growth ability of polymer-degrading microbes. In the present study, SiO2 nanoparticles were employed to scrutinize their effect on polymer biodegradation efficiency. LDPE containing minimal salt medium supplemented with SiO2 nanoparticles (20 nm) at concentration of 0.01% w/v. Out of five bacterial strains, Bacillus sp. V8 and Pseudomonas sp. C 2 5 were found to be the most potential strains for polymer degradation. SiO2 nanoparticles improved growth profiling by shifting in lag phase, and it was also found effective to increase the biodegradation efficiency of bacterial strains by means of λ-max shifts, and Fourier transform infrared analysis revealed the formation or alteration of the chemical structure of the degraded polymer. The values of λ-max were shifted 217–227, 211–226, 209–224, 210–225, and 214–224 in the presence of Bacillus sp. strain V8, Pseudomonas sp. strain C 2 5, Pseudomonas sp. strain V1, Acinetobacter sp. strain V4, and Paracoccus sp. strain B1 4-, respectively. Fourier transform infrared analysis showed absorption frequencies of bacteria-treated polymers corresponding to 3049, 3275, 3527.8, 2196, and 3034 and confirmed the C–H stretching, O–H stretching, and C≡H stretching, respectively. The study signifies the bacteria–nanoparticles interactions that significantly influence the competence of polymer degradation.

Draft Genome Sequence of Acidihalobacter ferrooxidans DSM 14175 (Strain V8), a New Iron- and Sulfur-Oxidizing, Halotolerant, Acidophilic Species.

ABSTRACTThe use of halotolerant acidophiles for bioleaching provides a biotechnical approach for the extraction of metals from regions where high salinity exists in the ores and source water. Here, we describe the first draft genome of a new species of a halotolerant and iron- and sulfur-oxidizing acidophile, Acidihalobacter ferrooxidans DSM-14175 (strain V8).

Conjugated fluorescent polymer sensor for proteolytic activity detection with designed specificity

AbstractA portable fluorescence assay for direct endopeptidase activity detection has been developed with the use of a cyclophane‐based conductive conjugated fluorescent polymer and peptide substrates. The substrates, carrying internal quenching amino acid, were designed to be cleaved in a sequence‐specific manner by a protease of interest. Intact substrates were incapable of quenching the fluorescence of the polymer due to steric constraints. Upon specific cleavage, the quencher became exposed and could interact with the ring structure of the fluorescent polymer, disrupting the conjugation and quenching the fluorescence along the polymer chain. The approach was developed using a model Glu‐C endopeptidase from Staphylococcus aureus strain V8, detected in the picomolar to micromolar concentration range. The developed assay was tested for the detection of endopeptidase activity of botulinum toxin. The feasibility study showed that botulinum neurotoxin (BoNT)‐A could be detected down to picomolar concentration, with limit of detection of 5 pg, or 33 amol in 5 µL of sample, and a total assay time under 2 h. The assay exhibited high specificity and no cross‐reactivity with BoNT‐B was detected. Following this proof‐of‐concept work, the assay can be further optimized and expanded to differentiate between various botulinum toxin serotypes in their active proteolytic form, or modified for detection of proteases with other specificities. © 2015 Society of Chemical Industry

Production, Purification and Characterization of Levan Polymer from Bacillus lentus V8 Strain

Production, Purification and Characterization of Levan Polymer from Bacillus lentus V8 Strain

Synthesis and evaluation of novel photoreactive α-amino acid analog carrying acidic and cleavable functions

A novel photoreactive α-amino acid bearing an acidic residue and a cleavable diazirine was developed. To mimic common acidic α-amino acids, the residue was designed to be N-acylsulfonamide that possesses an acidic proton and is able to dissociate under the physiological conditions. The inhibition assay of its biotin-tagged derivative with glutamyl endopeptidase from Staphylococcus aureus V8 strain revealed a Ki app value of 162 μM, which is slightly higher than the K m value of a common substrate. Upon UV irradiation, this derivative specifically photolabeled glutamyl endopeptidase, l-glutamate dehydrogenase, glutamic oxalacetic transaminase, and l-glutamine synthetase, all the enzymes exhibit high affinity toward acidic α-amino acids. In addition, N-acylsulfonamide group functioned as a cleavable linker in mild basic solution after a brief N-alkylation. Either the multifunctional nature or the simple structure of this acidic α-amino acid surrogate would be useful as versatile photoreactive building block.

An Escherichia coli expression system for glutamyl endopeptidases optimized by complete suppression of autodegradation

V8 protease (GluV8), a member of the glutamyl endopeptidase I family isolated from the V8 strain of Staphylococcus aureus, is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. We recently developed an Escherichia coli expression system for the production of GluV8 based on a technique that suppresses the autoproteolysis—the use of the prosequence of its homologue (GluSE) from Staphylococcus epidermidis as a chimeric form or the introduction of four substitutions in the prosequence of GluV8. In the current study, we refined this technique through five amino acid substitutions within the prosequence of GluV8 for complete suppression of the autodegradation. As a result, the recovery of GluV8 proform was enhanced to 20 fg/cell, which was comparable to the level of a constitutive inactive form of GluV8, indicating complete suppression of the autoproteolysis. This mutated propeptide was also effective for the expression of the mature sequence of the glutamyl endopeptidase from Staphylococcus warneri. The recombinant proteins were successfully converted to their active forms through a common cleavage mechanism mediated by thermolysin in vitro. This strategy may shed light on the way for the expression of the proteases that have been scarcely produced in E. coli to date.

Characterization of the glutamyl endopeptidase from Staphylococcus aureus expressed in Escherichia coli

V8 protease, a member of the glutamyl endopeptidase I family, of Staphylococcus aureus V8 strain (GluV8) is widely used for proteome analysis because of its unique substrate specificity and resistance to detergents. In this study, an Escherichia coli expression system for GluV8, as well as its homologue from Staphylococcus epidermidis (GluSE), was developed, and the roles of the prosegments and two specific amino acid residues, Val69 and Ser237, were investigated. C-terminal His(6)-tagged proGluSE was successfully expressed from the full-length sequence as a soluble form. By contrast, GluV8 was poorly expressed by the system as a result of autodegradation; however, it was efficiently obtained by swapping its preprosegment with that of GluSE, or by the substitution of four residues in the GluV8 prosequence with those of GluSE. The purified proGluV8 was converted to the mature form in vitro by thermolysin treatment. The prosegment was essential for the suppression of proteolytic activity, as well as for the correct folding of GluV8, indicating its role as an intramolecular chaperone. Furthermore, the four amino acid residues at the C-terminus of the prosegment were sufficient for both of these roles. In vitro mutagenesis revealed that Ser237 was essential for proteolytic activity, and that Val69 was indispensable for the precise cleavage by thermolysin and was involved in the proteolytic reaction itself. This is the first study to express quantitatively GluV8 in E. coli, and to demonstrate explicitly the intramolecular chaperone activity of the prosegment of glutamyl endopeptidase I.

SarA is a repressor of hla (alpha-hemolysin) transcription in Staphylococcus aureus: its apparent role as an activator of hla in the prototype strain NCTC 8325 depends on reduced expression of sarS.

In most Staphylococcus aureus strains, inactivation of sarA increases hla transcription, indicating that sarA is a repressor. However, in S. aureus NCTC 8325 and its derivatives, used for most studies of hla regulation, inactivation of sarA resulted in decreased hla transcription. The disparate phenotype of strain NCTC 8325 seems to be associated with its rsbU mutation, which leads to sigma(B) deficiency. This has now been verified by the demonstration that sarA repressed hla transcription in an rsbU+ derivative of strain 8325-4 (SH1000). That sarA could act as a repressor of hla in an 8325-4 background was confirmed by the observation that inactivation of sarA in an agr sarS rot triple mutant dramatically increased hla transcription to wild-type levels. However, the apparent role of sarA as an activator of hla in 8325-4 was not a result of the rsbU mutation alone, as inactivation of sarA in another rsbU mutant, strain V8, led to increased hla transcription. Northern blot analysis revealed much higher levels of sarS mRNA in strain V8 than in 8325-4, which was likely due to the mutation in the sarS activator, tcaR, in 8325-4, which was not found in strain V8. On the other hand, the relative increase in sarS transcription upon the inactivation of sarA was 15-fold higher in 8325-4 than in strain V8. Because of this, inactivation of sarA in 8325-4 means a net increase in repressor activity, whereas in strain V8, inactivation of sarA means a net decrease in repressor activity and, therefore, enhanced hla transcription.

Salivary Lactoferrin Is Recognized by the Human Herpesvirus-8

Human herpesvirus-8 (HHV-8) is commonly detected in all epidemiologic forms of Kaposi's sarcoma. Despite the broad cellular tropism of HHV-8, studies on mucosal shedding of HHV-8 have shown that infectious particles are restricted to saliva isolated from the oropharynx. We used biotinylated purified HHV-8 particles in a direct binding assay to whole clarified human salivary samples isolated from HHV-8-infected and uninfected individuals. We found that the major binding activity was carried out by a protein of 78-kDa size, which was further characterized as human lactoferrin (hLf) using 2-D electrophoresis and MALDI-ToF analysis. Preliminary comparison of HHV-8 binding activity of 76 salivary samples from HHV-8-infected and uninfected individuals showed that 7.8% of the uninfected population exhibited a form of Lf not recognized by HHV-8. Deglycosylation of hLf by PNGase F did not reduce HHV-8 binding activity, whereas endoproteinase cleavage of native hLf generated a non-glycosylated 8-kDa peptide recognized by HHV-8 particles and was located at the position Ala606-Tyr679 in the native hLf amino acid sequence, corresponding to the C-terminal region of the glycoprotein. This work identify the lactoferrin in saliva as a ligand for HHV-8 and suggests that this glycoprotein could be used as a carrier for the viral particles.

Purification, cloning and characterization of variant LukE-LukD with strong leukocidal activity of staphylococcal bi-component leukotoxin family.

Staphylococcus aureus produces bi-component leukotoxins composed of non-associated soluble proteins, S and F. Neither S nor F component alone is cytotoxic, but components together are active. These include Panton-Valentine leukocidin (PVL), gamma-hemolysin, LukE-LukD and others. Purification of leukotoxin from Staphylococcus aureus V8 strain (ATCC 27733) which does not have PVL genes, identified an F component with 100% identical to that of PVL in the first twenty-five N-terminal amino acids. Molecular cloning of this toxin obtained 2,595 nucleotides sequences containing two novel open reading frames for S and F. Deduced amino acid sequences of the S and F were respectively 91 and 94% identical to those of LukE and LukD. These were named variant of LukE-LukD (LukEv-LukDv). The activity of the recombinant LukEv-LukDv to rabbit leukocytes was similar to that of recombinant PVL. LukEv-LukDv was hemolytic to rabbit red blood cells although the activity was only 8% of gamma-hemolysin, but PVL was not. These activities were quite different from the LukE-LukD which was reported no hemolytic and poorly cytotoxic to leukocytes compared to PVL. The lukEv-IukDv was found in 87% of clinical isolates of Staphylococcus aureus but lukE-lukD was not detected. These data demonstrate the existence of variant LukE-LukD in V8 strain (ATCC 27733).

Genetic and biochemical characterization of glutamyl endopeptidase of Staphylococcus warneri M

A Staphylococcus warneri strain M, newly isolated from processed seafood (smoked Watasenia scintillans), produced an extracellular protease. The protease, designated to as m-PROM (the mature form of PROM), selectively cleaved the carbonyl side of glutamic acid residues in β-casein. Sequence of N-terminal 27 amino acids of m-PROM, RANVILPNNDRHQINDTTLGHYAPVTF, was found to be similar to those of other glutamyl endopeptidases, V8 protease ( Staphylococcus aureus strain V8) and SPase ( S. aureus ATCC 12600). To determine the complete primary structure and precursor of PROM, its gene ( proM) was cloned and sequenced. The gene proM was found to encode for a protein of 316 amino acids. The amino acid residues from 64 to 90 completely coincided with the N-terminal 27 amino acids of the m-PROM, suggesting that the N-terminal 63 amino acids region of p-PROM (the precursor form of PROM) might be processed posttranslationally. Moreover, the whole amino acid sequence deduced from the primary structure of proM shows significant similarity to those of other glutamyl endopeptidases, V8 protease and SPase. These results suggested that PROM belongs to the glutamyl endopeptidase class. PROM, however, differs from V8 and SPase proteases in the processing site and the C-terminal region.

Phage conversion of Panton-Valentine leukocidin in Staphylococcus aureus: molecular analysis of a PVL-converting phage, φSLT

Staphylococcal Panton-Valentine leukocidin (PVL) is an important virulence factor, which causes leukocytolysis and tissue necrosis. Our previous report on the existence of the PVL genes (lukS-PV and lukF-PV) on the genome of prophage φPVL in the Staphylococcus aureus strain V8 suggested the horizontal transmission of PVL genes by temperate bacteriophage among S. aureus (Kaneko, et al., 1998. Gene 215, 57–67). Here, we demonstrated the phage conversion of S. aureus leading to the production of PVL by discovery of a novel PVL-carrying phage, φSLT (Staphylococcal Leukocytolytic Toxin) from a clinical isolate of S. aureus. φSLT was able to lysogenize several clinical isolates of PVL-negative S. aureus strains as well as strain RN4220 at the conserved 29-bp sequence (attB) and all the lysogenized S. aureus strains had the ability to produce PVL. φSLT had an elongated head of about 100×50 nm and a flexible tail of 400 nm long, that was quite different from φPVL which had an isometric hexagonal head of about 60 nm diameter. The linear double-stranded φSLT genome comprised 42,942 bp with 29-bp attachment core sequences and contained 62 open reading frames. Only 6.4 kbp region containing lysis cassette, PVL genes, attP, integrase, and orf204 of φSLT was identical to that of φPVL, while other regions were different from those of φPVL. Thus, it can be concluded that PVL genes are carried by different temperate phages, which have the same attachment site.

Formation of the cystine between cysteine 225 and cysteine 462 from ribonucleoside diphosphate reductase is kinetically competent.

Participation of the formation of the cystine between cysteine 225 and cysteine 462 in the R1 protein to the enzymatic mechanism of aerobic ribonucleoside diphosphate reductase from Escherichia coli has been examined by use of rapid quenching and site-directed immunochemistry. Prereduced ribonucleotide reductase in the presence of ATP was mixed with CDP in a quench flow apparatus. The reaction was terminated with a solution of acetic acid and N-ethylmaleimide. The protein was precipitated and digested with chymotrypsin and the proteinase from Staphylococcus aureus strain V8 in the presence of N-ethylmaleimide to yield the peptide SS[S-(N-ethylsuccinimid-2-yl)cysteinyl]VLIE containing cysteine 225 and the mixed disulfide between the peptide SSCVLIE and the peptide IALCTL containing cysteine 462. These two peptides were retrieved together from the digest by immunoadsorption. The affinity-purified peptides were modified at their amino termini with the fluorescent reagent 6-aminoquinolyl-N-hydroxysuccimidyl carbamate and submitted to high-pressure liquid chromatography. The areas of the respective peaks of fluorescence corresponding to the S-(N-ethylsuccimidyl) peptide, and the mixed disulfide were used to determine the percentage of the cystine that had formed during each interval. The rate constant for the formation of the cystine following the association of free, fully reduced ribonucleotide reductase with the reactant CDP was 8 s(-)(1). Because only 50% of the active sites participated in this pre-steady-state reaction, the maximum steady-state rate consistent with the involvement of this cystine in the enzymatic reaction would be 4 s(-1). Since the turnover number of the enzyme under the same conditions in a steady state assay was only 1 s(-)(1), the formation of the cystine between these two cysteines is kinetically competent.

TsTX-IV, a short chain four-disulfide-bridged neurotoxin from Tityus serrulatus venom which acts on Ca 2+-activated K + channels

The primary structure of TsTX-IV, a neurotoxin isolated from Tityus serrulatus scorpion venom, is reported. Its amino acid sequence was determined by automated Edman sequential degradation of the reduced and carboxymethylated toxin and of relevant peptides obtained by digestion with Staphylococcus aureus strain V8 protease or trypsin and cleavage by CNBr. The complete sequence showed 41 amino acid residues, which account for an estimated molecular weight of 4520, and eight half-cystine residues which cross-link the toxin molecule with four disulfide bonds. The molecular weight determined by mass spectrometry was 4518. Comparison of this sequence with those from other scorpion toxins showed a resemblance with toxins which act on different types of K + channels. TsTx-IV was able to block Ca 2+-activated K + channels of high conductance. TsTX-IV is the first four-disulfide-bridged short toxin from T. serrulatus so far completely sequenced.

Proteolytic degradation of barley ribulose-1,5-bisphosphate carboxylase/oxygenase and recognition of the fragments by monoclonal antibodies

Limited proteolysis of barley ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCO) was effected by treatments with trypsin and Staphylococcus aureus strain V8 protease. Treatment of native RuBPCO with proteases resulted in the degradation of the large subunit (LS) of the enzyme. Trypsin cleaved three fragments from the LS but the S. aureus strain V8 protease cleaved only one. The small subunit (SS) was not affected. In the presence of 0.5 % sodium dodecyl sulfate, RuBPCO degraded into several fragments; some of them were fairly stable. Monoclonal antibodies (Mabs) against barley RuBPCO were applied in immunoblotting analysis to distinguish which of the fragments were recognized. All the Mabs recognized the fragments with molecular masses close to those of the LS. Differences among Mabs were observed in the fragments with low molecular mass.

Coupling between trans/cis proline isomerization and protein stability in staphylococcal nuclease.

The nucleases A produced by two strains of Staphylococcus aureus, which have different stabilities, differ only in the identity of the single amino acid at residue 124. The nuclease from the Foggi strain of S. aureus (by convention nuclease WT), which contains His124, is 1.9 kcal.mol-1 less stable (at pH 5.5 and 20 degrees C) than the nuclease from the V8 strain (by convention nuclease H124L), which contains Leu124. In addition, the population of the trans conformer at the Lys116-Pro117 peptide bond, as observed by NMR spectroscopy, is different for the two variants: about 15% for nuclease WT and 9% for nuclease H124L. In order to improve our understanding of the origin of these differences, we compared the properties of WT and H124L with those of the H124A and H124I variants. We discovered a correlation between effects of different residues at this position on protein stability and on stabilization of the cis configuration of the Lys116-Pro117 peptide bond. In terms of free energy, approximately 17% of the increase in protein stability manifests itself as stabilization of the cis configuration at Lys116-Pro117. This result implies that the differences in stability arise mainly from structural differences between the cis configurational isomers at Pro117 of the different variants at residue 124. We solved the X-ray structure of the cis form of the most stable variant, H124L, and compared it with the published high-resolution X-ray structure of the cis form of the most stable variant, WT (Hynes TR, Fox RO, 1991, Proteins Struct Funct Genet 10:92-105). The two structures are identical within experimental error, except for the side chain at residue 124, which is exposed in the models of both variants. Thus, the increased stability and changes in the trans/cis equilibrium of the Lys116-Pro117 peptide bond observed in H124L relative to WT are due to subtle structural changes that are not observed by current structure determination technique. Residue 124 is located in a helix. However, the stability changes are too large and follow the wrong order of stability to be explained simply by differences in helical propensity. A second site of conformational heterogeneity in native nuclease is found at the His46-Pro47 peptide bond, which is approximately 80% trans in both WT and H124L. Because proline to glycine substitutions at either residue 47 or 117 remove the structural heterogeneity at that position and increase protein stability, we determined the X-ray structures of H124L + P117G and H124L + P47G + P117G and the kinetic parameters of H124L, H124L + P47G, H124L + P117G, and H124L + P47G + P117G. The individual P117G and P47G mutations cause decreases in nuclease activity, with kcat affected more than Km, and their effects are additive. The P117G mutation in nuclease H124L leads to the same local conformational rearrangement described for the P117G mutant of WT (Hynes TR, Hodel A, Fox RO, 1994, Biochemistry 33:5021-5030). In both P117G mutants, the loop formed by residues 112-117 is located closer to the adjacent loop formed by residues 77-85, and residues 115-118 adopt a type I' beta-turn conformation with the Lys116-Gly117 peptide bond in the trans configuration, as compared with the parent protein in which these residues have a typeVIa beta-turn conformation with the Lys116-Pro117 peptide bond in the cis configuration. Addition of the P47G mutation appears not to cause any additional structural changes. However, the electron density for part of the loop containing this peptide bond was not strong enough to be interpreted.

Effect of Limited Proteolysis on the Enzymatic Phosphorylation of Soy Protein

The effects of limited proteolysis on the enzymatic phosphorylation of commercial soy protein isolate were evaluated for several proteases. Proteases were chosen on the basis of their ability to hydrolyze peptide bonds between specific amino acids and included trypsin (basic), chymotrypsin (aromatic), endoproteinase Glu-C from Staphylococcus aureus, strain V8 (acidic), and Pronase E (nonspecific). Hydrolyzed soy isolate was phosphorylated with the catalytic subunit of bovine cardiac muscle protein kinase, and phosphorylation was followed by incorporation of 32P into the protein. Phosphorylation decreased with increased proteolysis for all proteases. Decreased phosphorylation was most pronounced for Pronase E-hydrolyzed protein and least pronounced for trypsin-hydrolyzed soy isolate. The solubility−pH profile of the trypsin-hydrolyzed, phosphorylated soy isolate was compared to those of both the untreated and the hydrolyzed profiles. There was no significant difference in solubility among the profiles over the pH 3−6 range. Keywords: Polypeptide; modification; functionality; hydrolysis; interaction

Engineered disulfide bonds in staphylococcal nuclease: effects on the stability and conformation of the folded protein.

Efforts to enhance the stability of proteins by introducing engineered disulfide bonds have resulted in mixed success. Most approaches to the prediction of the energetic consequences of disulfide bond formation in proteins have considered only the destabilizing effects of cross-links on the unfolded state (chain entropy model) [Pace, C. N., Grimsley, G. R., Thomson, J. A., & Barnett, B. J. (1988) J. Biol. Chem. 263, 11820-11825: Doig, A. J., & Williams, D. H. (1991) J. Mol. Biol. 217, 389-398]. It seems clear, however, that disulfide bridges also can influence the stability of the native state. In order to assess the importance of the latter effect, we have studied four variants of staphylococcal nuclease (V8 strain) each containing one potential disulfide bridge created by changing two wild-type residues to cysteines by site-directed mutagenesis. In each case, one of the introduced cysteines was within the type VIa beta turn containing cis Pro117, and the other was located in the adjacent extended loop containing Gly79. In all four cases, the overall loop size was kept nearly constant (the number of residues in the loop between the two cysteines varied from 37 to 42) so as to minimize differences from chain entropy effects. The objective was to create variants in which a change in the reduction state of the disulfide would be coupled to a change in the position of the equilibrium between the cis and trans forms of the Xxx116-Pro117 peptide bond in the folded state of the protein. The position of this equilibrium, which can be detected by NMR spectroscopy, has been shown previously to correlate with the stability of the native protein. Its determination provides a measure of strain in the folded state. The thermal stabilities and free energies for unfolding by elevated temperature and guanidinium chloride were measured for each of the four mutants under conditions in which the introduced cysteines were cross-linked (oxidized) and unlinked (reduced). In addition, reduction potentials were determined for each mutant. Formation of the different disulfide bridges was found to induce varying levels of folded state strain. The stabilization energy of a given disulfide bridge could be predicted from the measured perturbation energy for the peptide bond isomerization, provided that energetic effects on the unfolded state were calculated according to the chain entropy model. Undiagnosed strain in native states of proteins may explain the variability observed in the stabilization provided by engineered disulfide bridges.

Topological disposition of tyrosine 486 in anion exchanger from human erythrocytes.

The location with respect to the plasma membrane of tyrosine 486 in the native anion exchanger of human erythrocytes has been determined by site-directed immunochemistry. Intact erythrocytes and inside-out vesicles were [125I]radioiodinated by lactoperoxidase in the same vessel. After the erythrocytes and inside-out vesicles had been separated by differential centrifugation, the modified polypeptide of the anion exchanger was isolated from each sample and digested with the proteinase from Staphylococcus aureus strain V8 and trypsin to generate the peptide YIVGR. An immunoadsorbent that was specific for the carboxy-terminal sequence -IVGR was used to purify the peptide YIVGR, which contains tyrosine 486 of the anion exchanger, from the products of the digestion. The [125I]radioiodinated peptides isolated by the immunoadsorbent were submitted to high-pressure liquid chromatography, and their respective mobilities were compared to those of synthetic peptides that had been iodinated at tyrosine. By applying this technique, the peptide containing tyrosine 486 was unambiguously identified, and the incorporation of [125I]iodine into this residue in anion exchanger could be monitored. When inside-out vesicles and intact cells were [125I]radioiodinated in the same suspension, tyrosine 486 was modified to at least a 6-fold greater specific radioactivity in the inside-out vesicles than it was in the intact cells. This amino acid, therefore, was assigned to the cytoplasmic surface of native anion exchanger. It follows that the polypeptide of anion exchanger spans the membrane three times before it reaches the extracellular region surrounding glutamine 550.