Reductive cleavage and regeneration of the disulfide bonds inStreptomyces subtilisin inhibitor (SSI) as studied by the carbonyl13C NMR resonances of cysteinyl residues

Summary Four enhanced carbonyl carbon resonances were observed whenStreptomyces subtilisin inhibitor (SSI) was labeled by incorporating specifically labeled [1-¹³C]Cys. The¹³C signals were assigned by the¹⁵N,¹³C double-labeling method along with site-specific mutagenesis. Changes in the spectrum of the labeled protein ([C]SSI) were induced by reducing the disulfide bonds with various amounts of dithiothreitol (DTT). The results indicate that, in the absence of denaturant, the Cys⁷¹-Cys¹⁰¹ disulfide bond of each SSI subunit can be reduced selectively. This disulfide bond, which is in the vicinity of the reactive site scissile bond Met⁷³-Val⁷⁴, is more accessible to solvent than the other disulfide bond. Cys³⁵-Cys⁵⁰, which is embedded in the interior of SSI. This half-reduced SSI had 65% of the inhibitory activity of native SSI and maintained a conformation similar to that of the fully oxidized SSI. Reoxidation of the half reduced-folded SSI by air regenerates fully active SSI which is indistinguishable with intact SSI by NMR. In the presence of 3 M guanidine hydrochloride (GuHCl), however, both disulfide bonds of each SSI subunit were readily reduced by DTT. The fully reduced-unfolded SSI spontaneously refolded into a native-like structure (fully reduced-folded state), as evidenced by the Cys carbonyl carbon chemical shifts, upon removing GuHCl and DTT from the reaction mixture. The time course of disulfide bond regeneration from this state by air oxidation was monitored by following the NMR spectral changes and the results indicated that the disulfide bond between Cys⁷¹ and Cys¹⁰¹ regenerates at a much faster rate than that between Cys³⁵ and Cys⁵⁰.Nomenclature of the various states of SSI that are observed in the present study Fully oxidized-folded native or intact (without GuHCl or DTT) - half reduced-folded (Cys⁷¹-Cys¹⁰¹ reduced; DTT without GuHCl) - inversely half reduced-folded (Cys³⁵-Cys⁵⁰ reduced; a reoxidation intermediate from fully reduced-folded state) - fully reduced-unfolded (reduced by DTT in the presence of GuHCl) - fully reduced-folded (an intermediate state obtained by removing DTT and GuHCl from the fully reduced-unfolded SSI reaction mixture)     

Relationship between utilization of dual translational initiation signals and protein processing in Streptomyces

Summary Two sets of the Shine-Dalgarno sequence and the initiation codon (ATG) for translation of a gene encoding the protein SSI (Streptomyces subtilisin inhibitor) were studied in vivo by site-directed mutagenesis. The result shows that each ATG can function as an initiator of translation in either Streptomyces lividans 66 or Escherichia coli. The choice of initiation codon seems dependent on the host strain and is closely related to the processing mechanism of pre-SSI protein. The upstream ATG is presumed to be utilized preferentially giving two cleavage sites in pre-SSI in S. albogriseolus S-3253, the original SSI producer strain.Abbreviations SD Shine-Dalgarno - SSI Streptomyces subtilisin inhibitor     

A Novel Proteinaceous Kex 2 Proteinase Inhibitor,Kexstatin, from Streptomyces platensis Q268

We found a novel proteinaceous Kex 2 proteinase inhibitor, named kexstatin, in the culture supernatant of Streptomyces platensis Q268. The purified kexstatin was homogeneous by SDS–PAGE and the molecular weight was estimated to be 13,000. The N-terminal amino acid sequence of kexstatin has high similarity to Streptomyces subtilisin inhibitor (SSI), suggesting that kexstatin belongs to the SSI family. Kexstatin was a strong inhibitor of Kex 2 proteinase and subtilisin but not thermolysin, trypsin, or chymotrypsin. The IC₅₀ value of kexstatin against 1μg of Kex 2 proteinase was 1.4μg.     

Stoichiometric complexation of streptomyces subtilisin inhibitor and subtilisin

Subtilisin (Sbt) andStreptomyces subtilisin inhibitor (SSI) were analyzed either alone or together using sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). With all ratios of Sbt to SSI tested, the proteins formed a stoichiometric complex, and migrated abnormally at the top of the gel. Electroblotting and amino acid sequence analysis of the complex band showed both Sbt and SSI present at approximately equal molar ratios. When excess Sbt was present, it migrated as a free but still folded form slightly above the band corresponding to the complex. When excess SSI was present, it migrated as several species with molecular weights smaller than the intact form; in fact, the sequences of some of these species indicated that they lacked different amounts of N-terminal and possibly C-terminal residues.     

Role of catalytic residues in the formation of a tetrahedral adduct in the acylation reaction of bovine β-trypsin: A molecular orbital study

In the acylation reaction of serine proteases the effect of amino acid residues on the geometrical change of the catalytic site from Michaelis to tetrahedral state was studied by using ab initio molecular orbital calculations. Amino acid residues in the catalytic site and the peptide substrate were calculated as a quantum mechanical region, and all the other amino acid residues and the calcium ion were included in the calculation as the electrostatic effects. The effects of Asp102, Asp194, N-terminus and the oxyanion binding site are large. The oxyanion binding site directly stabilizes the tetrahedral substrate. Asp102 stabilizes the enzyme intermediate, interacting with the protonated His57 residue. In order to elucidate the roles of Asp102 and the oxyanion binding site, energy decomposition analyses were done for the intermolecular interactions. The contribution of Asp102 and the oxyanion binding site to the decrease of energy in the geometrical change is due to the electrostatic effect. The energies of the proton shuttle from Ser195 O^γ to the leaving group of the substrate were calculated for amide and ester substrate models.     

Evolutionary dynamics of mating system shifts in Arabidopsis lyrata

Outcrossing is the prevalent mode of reproduction in plants and animals despite its substantial costs, while selfing and mixed mating occur at much lower frequency. Comparative research on plants has demonstrated the lability of self‐incompatibility, but there is little information about the transition on a within‐species level from self‐incompatibility to predominant selfing. We studied variation in mating system among 18 populations of Arabidopsis lyrata within a phylogenetic context to shed light on the evolution of selfing. Realized and potential mating systems were assessed by genetic analysis with microsatellite markers and hand‐self‐pollinations on 30 plants from each population. The fraction of self‐incompatible plants in a population was highly correlated with the outcrossing rate, showing that the spread of self‐compatibility is accompanied by or soon followed by an increase in the rate of selfing. The four predominantly selfing populations (outcrossing rates < 0.25) fell into more than one phylogenetic cluster, suggesting that the transition to selfing occurred more than once independently. Hence, A. lyrata offers an opportunity for the comparative analysis of outcrossing as a predominant mode of reproduction in plants and of the causes of the shift to selfing.     

Temperature dependent development and temperature thresholds of Rhyncaphytoptus ficifoliae Keifer (Diptilomiopidae)

The effects of temperature on developmental rate of Rhyncaphytoptus ficifoliae Keifer (Diptilomiopidae) were determined at six constant temperatures (17, 20, 25, 30, 33 and 36 °C) on fig leaves. The total developmental time of females decreased as temperature increased from 17 (21.62 days) to 33 °C (6.02 days), and then increased at 36 °C (6.47 days). Using the ordinary and Ikemoto and Takai (2000) linear models the estimated lower temperature thresholds (T_min) for total developmental time of females were 10.78 and 10.37 °C and the constant temperatures (k) were 140.25 and 144.78°-days (DD), respectively. Data also were fitted to SSI nonlinear temperature-dependent model. The estimated T_L, intrinsic optimum temperature (T_Ф) and T_h for total immature stages of females by SSI model were 11.11, 23.72 and 37.98 °C, respectively. With use of the obtained data from rearing R. ficifoliae under constant temperatures in laboratory and temperature data in Khorramabad region in 2017, the real developmental rate of this mite in natural conditions was described. The highest (100%) and lowest values (51.67%) of survival rate for immature stages were found at 25 and 36 °C, respectively. The presented information could be used to predict the population dynamics of main pest R. ficifoliae for an effective management.     

KIR genes and HLA class I ligands in Gaucher disease

Gaucher disease (GD) is caused by reduced activity of the lysosomal enzyme glucocerebrosidase, which leads to a buildup of glucocerebroside within the cells and chronic stimulation of the immune system. GD is associated with clinical variability even in the same family, which suggests the influence of modifier genes. Natural killer (NK) cells play an important role in the immune response, and their number is decreased in GD. Killer-cell immunoglobulin-like receptors (KIR) regulate the activity of NK cells through an interaction with specific human leukocyte antigen (HLA) class I molecules on target cells.     

Identification of random amplified polymorphic DNA (RAPD) markers for self-incompatibility alleles in Corylus avellana L.

 Random amplified polymorphic DNA (RAPD) markers were identified for self-incompatibility (SI) alleles that will allow marker-assisted selection of desired S-alleles in hazelnut (Corylus avellana L.). DNA was extracted from young leaves collected from field-planted parents and 26 progeny of the cross OSU 23.017 (S₁S₁₂)×VR6-28 (S₂S₂₆) (OSU23×VR6). Screening of 10-base oligonucleotide RAPD primers was performed using bulked segregant analysis. DNA samples from 6 trees each were pooled into four ‘bulks’, one for each of the following: S₁ S₂, S₁ S₂₆ ^, S₂ S₁₂, and S₁₂ S₂₆. ‘Super bulks’ of 12 trees each for S₁, S₂, S₁₂, and S₂₆ were then created for each allele by combining the appropriate bulks. The DNA from these four super bulks and from the parents was used as a template in the PCR assays. A total of 250 primers were screened, and one RAPD marker each was identified for alleles S₂ (OPI07₇₅₀) and S₁ (OPJ14₁₇₀₀). OPJ14₁₇₀₀ was identified in 13 of 14 S₁ individuals of the cross OSU23×VR6 used in bulking and yielded a false positive in 1 non-S₁ individual. This same marker was not effective outside the original cross, identifying 4 of 5 S₁ progeny in another cross, ‘Willamette’×VR6-28 (‘Will’×VR6), but yielded false positives in 4 of 9 non-S₁ individuals from the cross ‘Casina’×VR6-28 (‘Cas’×VR6). OPI07₇₅₀ served as an excellent marker for the S₂ allele and was linked closely to this allele, identifying 12 of 13 S₂ individuals in the OSU23×VR6 population with no false positives. OPI07₇₅₀ was found in 4 of 4 S₂ individuals from ‘Will’×VR and 7 of 7 S₂ individuals of ‘Cas’×VR6 with no false positives, as well as 10 of 10 S₂ individuals of the cross OSU 296.082 (S₁S₈)×VR8-32 (S₂S₂₆), with only 1 false positive individual out of 21 progeny. OPI07₇₅₀ was also present in 5 of 5 cultivars carrying the S₂ allele, with no false-positive bands in non-S₂ cultivars, and correctly identified all but 2 S₂ individuals in 57 additional selections in the breeding program. In the OSU23×VR6 population, the recombination rate between the marker OPJ14₁₇₀₀ and the S₁ allele was 7.6% and between the OPI07₇₅₀ marker and the S₂ allele was 3.8%. RAPD marker bands were excised from gels, cloned, and sequenced to enable the production of longer primers (18 or 24 bp) that were used to obtain sequence characterized amplified regions (SCARs). Both the S₁ and S₂ markers were successfully cloned and 18 bp primers yielded the sole OPJ14₁₇₀₀ product, while 24-bp primers yielded OPI07₇₅₀ as well as an additional smaller product (700 bp) that was not polymorphic but was present in all of the S-genotypes examined. Received: 10 January 1998 / Accepted: 26 January 1998