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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The importance of conserved amino acids in the amino and carboxyl non-Gly-X-Y domains of Caenorhabditis elegans cuticle collagens was examined by analyzing site-directed mutations of the sqt-1 and rol-6 collagen genes in transgenic animals. Altered collagen genes on transgenic arrays were shown to produce appropriate phenotypes by injecting in vivo cloned mutant alleles. Equivalent alterations in sqt-1 and rol-6 generally produced the same phenotypes, indicating that conserved amino acids in these two collagens have similar functions. Serine substitutions for either of two conserved carboxyl domain cysteines produced LRol phenotypes. Substitution for both cysteines in sqt-1 also resulted in an LRol phenotype, demonstrating that disulfide bonding is important for normal function but not required for assembly. Arg-1 or Arg-4 to Cys mutations in homology block A (HBA; consensus, 1-RXRRQ-5; in the amino non-Gly-X-Y domain) caused RRol phenotypes, while the same alteration at Arg-3 had no effect, indicating that Arg-3 is functionally different from Arg-1 and Arg-4. Substitutions of Arg-4 with Ser, Leu, or Glu also produced the RRol phenotype, while Lys substitutions for Arg-1 or Arg-4 did not generate any abnormal phenotypes. His substitutions for Arg-1 or Arg-4 caused somewhat less severe RRol phenotypes. Therefore, strong positively charged residues, Arg or Lys, are required at positions 1 and 4 for normal function. The conserved pattern of arginines in HBA matches the cleavage sites of the subtilisin-like endoproteinases. HBA may be a cleavage site for a subtilisin-like protease, and cleavage may be important for cuticle collagen processing.
Mol Cell Biol 1994 Apr
PMID:In vitro mutagenesis of Caenorhabditis elegans cuticle collagens identifies a potential subtilisin-like protease cleavage site and demonstrates that carboxyl domain disulfide bonding is required for normal function but not assembly. 813 71

With a view to exploring its use as a metal-binding factor in transgenic plants we prepared the alpha-domain of metallothionein by reconstitution of rabbit apometallothionein and proteolysis of MT-1 and MT-2 with subtilisin. The isolated alpha-domains were characterised by UV and CD spectroscopy Double-Stranded. DNA encoding the alpha-domain (106 bp) of the human MT-IA was constructed from chemically synthesized oligomers by repair synthesis and enzymatic ligation, cloned into pUC19 and sequenced. A expression construct containing the cloned alpha-domain was introduced into tobacco cells on a disarmed Agrobacterium tumefaciens Ti-plasmid. Transformed tobacco cells were selected and regenerated on medium containing cadmium and kanamycin. The growth of roots and shoots of transformants was unaffected by up to 100 microM cadmium, whereas control plants showed severe inhibition of root and shoot growth, and chlorosis of leaves on medium containing only 10 microM cadmium. Southern hybridization confirmed the presence of the transgene in the transformed plant tissues. The concentration of human alpha-domain peptides in transgenic tobacco leaves was determined by the Cd/hemoglobin saturation assay and polarography using the rabbit alpha-domain as standard. The results indicate that the alpha-domain, one of two domains in MT molecules, is not only stable in vitro, but is also expressed efficiently and functions independently in transgenic plant cells.
Mol Gen Genet 1994 Mar
PMID:Alpha-domain of human metallothionein IA can bind to metals in transgenic tobacco plants. 815 17

The cDNA coding for vitellogenin of the mosquito Aedes aegypti was cloned and sequenced. An immunological analysis of expressed deletions from the 5'-end of the vitellogenin cDNA clones using vitellogenin subunit-specific antibodies showed that the small vitellogenin subunit is located at the N terminus and the large one at the carboxy-portion of the pre-provitellogenin. The position of the cleavage between the vitellogenin subunits in the pre-provitellogenin was identified by locating the N terminus of the large subunit. The cleavage site has a consensus RXRR for the subtilisin-processing endoprotease. Mosquito vitellogenin is highly hydrophilic with 17 putative N-linked glycosylation sites and 13 potential tyrosine sulfation sites. In contrast to known invertebrate vitellogenins, mosquito vitellogenin contains three polyserine domains that are similar to those of phosvitins in vertebrate vitellogenins. These polyserine domains, originally presumed to be vertebrate-specific, have several phosphorylation consensus sites in their sequences. Unlike other known vitellogenins, mosquito vitellogenin is rich in aromatic amino acid residues, tyrosine and phenylalanine, and in this respect is similar to insect serum proteins, arylphorins. This similarity suggests that mosquito vitellogenin may supply aromatic amino acids to the cuticle of rapidly developing embryos.
J Mol Biol 1994 Apr 15
PMID:Analysis of mosquito vitellogenin cDNA. Similarity with vertebrate phosvitins and arthropod serum proteins. 815 43

Beryllium fluoride (BeFx) has been widely used as a phosphate analogue in nucleotide-binding proteins. It was found to bind tightly to F- but not G-actin (Combeau C., and Carlier M. F. (1988) J. Biol. Chem. 263, 17429-17436) and to affect the three-dimensional structure of filaments by stabilizing the subdomain 2 region of the actin promoter (Orlova, A., and Egelman, E. H. (1992) J. Mol. Biol. 227, 1043-1053). In this work we examined the BeFx-induced structural and functional changes in G- and F-actin by using proteolysis, chemical modifications, ATPase, and in vitro motility assays. The results of proteolysis studies show that BeFx binds also to MgADP-G-actin and renders its subdomain 2 region more similar to that in MgATP-G-actin. This is manifested in enhanced subtilisin and decreased tryptic digestions in subdomain 2 of G-actin. BeFx had a strong effect on the proteolysis of MgADP-F-actin: both the tryptic and subtilisin digestions in subdomain 2 were completely inhibited. Significant protection against proteolysis in this region was observed even at 1:14 molar ratios of BeFx to actin indicating cooperative effects on the structure of the actin filament. A similar although milder effect of phosphate on the proteolysis of F-actin suggests that BeFx acts as a phosphate analogue in this system. BeFx also induces changes in the subdomain 1 region of F-actin. This is revealed via reduced rates of Cys-374 alkylation with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin and an increased subtilisin cleavage near the C terminus of actin in the presence of BeFx. The BeFx-induced structural changes in actin have little effect on its interactions with myosin. BeFx inhibits only slightly the actin-activated ATPase activity of S1 by decreasing Vmax without affecting KM. Additionally, the binding of BeFx to actin does not change the sliding velocity of actin filaments in the in vitro motility assays. The BeFx-induced specific and distinct changes in G- and F-actin point to the dynamic nature of actin structure and the local differences between monomeric and polymeric forms of actin.
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PMID:Dynamic properties of actin. Structural changes induced by beryllium fluoride. 816 84

An empirical function was used to calculate free energy change (delta G) of complex formation between the following inhibitors and enzymes: Kunitz inhibitor (BPTI) with trypsin, trypsinogen and kallikrein; turkey ovomucoid 3rd domain (OMTKY3) with alpha-chymotrypsin and the Streptomyces griseus protease B; the potato chymotrypsin inhibitor with the protease B; and the barely chymotrypsin inhibitor and eglin-c with subtilisin and thermitase. Using X-ray coordinates of the nine complexes, we estimated the contributions that hydrophobic effect, electrostatic interactions and side-chain conformational entropy make towards the stability of the complexes. The calculated delta G values showed good agreement with the experimentally measured ones, the only exception being the kallikrein/BPTI complex whose X-ray structure was solved at an exceptionally low pH. In complexes with different enzymes, the same inhibitor residues contributed identically towards complex formation (delta G(residue) Spearman rank correlation coefficient 0.7 to 1.0). The most productive enzyme-contacting residues in OMTKY3, eglin-c, and the chymotrypsin inhibitors were found in analogous positions on their respective binding loops; thus, our calculations identified a functional (energetic) motif that parallels the well-known structural similarity of the binding loops. The delta G values calculated for BPTI complexed with trypsin (-21.7 kcal) and trypsinogen (-23.4 kcal) were similar and close to the experimental delta G value of the trypsin/BPTI complex (-18.1 kcal), lending support to the suggestion that the 10(7) difference in the observed stabilities (KA) of these two complexes reflects the energetic cost of conformational changes induced in trypsinogen during the pre-equilibrium stages of complex formation. In almost all of the complexes studied, the stabilization free energy contributed by the inhibitors was larger than that donated by the enzymes. In the trypsin-BPTI complex, the calculated delta G contribution of the amino group from the BPTI residue Lys15 (9.7 kcal) was somewhat higher than that arrived at in experiments with semisynthetic inhibitor analogs (7.5 kcal). In OMTKY3, different binding loop residues are known to affect differently the binding (delta delta G) to alpha-chymotrypsin and protease B; a good qualitative agreement was found between the calculated delta G(residue) estimates and the experimental delta delta G data (correlation coefficient 0.7). Large variations were observed in local surface complementarity and related interfacial volume in the two OMTKY3 complexes (by 20 to 60% for some side-chains).(ABSTRACT TRUNCATED AT 400 WORDS)
J Mol Biol 1993 Dec 05
PMID:Affinity and specificity of serine endopeptidase-protein inhibitor interactions. Empirical free energy calculations based on X-ray crystallographic structures. 825 66

Dichelobacter nodosus, a Gram-negative obligate anaerobe and the causative organism of ovine footrot, secretes a family of extracellular serine proteases with pI's in the range of 5.2 to 5.6 and a serine basic protease with a pI of approximately 9.5. The primary structure of acidic protease V5 (pI approximately 5.2) from D. nodosus virulent strain 198 was determined by direct amino acid sequencing. This protease consists of a single polypeptide chain of 347 amino acids, contains two disulfide bonds and has a M(r) of 35960. Comparison of the D. nodosus acidic protease V5 sequence with that of other serine proteases showed that it is a member of the subtilisin family of proteases with strong conservation of identity around the catalytic residues. The sequence of protease V5 showed 64% identity to D. nodosus basic protease (pI approximately 9.5) and 53% identity to the extracellular serine protease of Xanthomonas campestris, a plant pathogen but only 25-35% identity to other proteases of the subtilisin family. The D. nodosus proteases are similar in length to X. campestris protease (but some 70 residues shorter than the subtilisins) and they share two conserved disulfide bonds with the X. campestris protease, a feature not observed for other members of the subtilisin family.
Biochem Mol Biol Int 1993 Apr
PMID:Amino acid sequence of extracellular acidic protease V5 of Dichelobacter nodosus, the causative organism of ovine footrot. 833 22

Long-range coulombic interaction energies between surface-charges in barnase and subtilisin have been determined to provide data for calibrating theoretical methods. The pKa of His18 in barnase can be measured accurately by titrating the fluorescence of Trp94 that is significantly quenched on protonation of His18. The pKa of His64, the active site base of subtilisin, has previously been shown to be measured accurately from the pH dependence of kcat/Km for the hydrolysis of substrates. The titration curves of both histidine residues fit the theoretical equations for the ionization of single groups with great precision; the Hill constants for wild-type and mutant enzymes are all close to 1.0. The coulombic interaction energies of distant charged side-chains with the protonated form of His18 and His64 have been measured from changes in pKa of these residues on mutation of those charged side-chains. The interaction energies between single charges on the surfaces of the proteins at low ionic strength are small, some 0.3-0.5 kcal mol-1 at a distance of 12 A, and fall gradually with distance to 0.05-0.3 kcal mol-1 at 20 A. Multiple mutations are frequently additive. Effects are larger in subtilisin than in barnase, possibly related to the degree of solvent exposure of the charge. These data have been used to benchmark the finite-difference method of calculating electrostatic interactions as implemented in the program DelPhi. There is reasonable agreement between the calculated and measured results as a function of both position and ionic strength.
J Mol Biol 1993 Jul 20
PMID:Long-range surface charge-charge interactions in proteins. Comparison of experimental results with calculations from a theoretical method. 834 24

Subtilisin BPN' is an extracellular serine protease from Bacillus amyloliquefaciens that requires an N-terminal 77 amino acid pro-sequence for correct folding of the catalytic domain. We have expressed an inactive, stable pro-subtilisin variant in Escherichia coli and show that it has structural properties similar to native subtilisin in terms of its near- and far-UV circular dichroism spectra, its compactness, and its capacity to bind calcium ions stoichiometrically. Unlike subtilisin, the pro-subtilisin variant unfolds reversibly with guanidinium chloride, and unfolding occurs via a folding intermediate. This intermediate is similar to the metastable intermediate state recently found for folding of subtilisin in the absence of the pro-sequence. The intermediate state has native-like secondary but little tertiary structure, and has a compactness between that of the native and unfolded state. Pro-subtilisin folds from the intermediate to the folded state in a single co-operative transition mediated by the pro-sequence. The isolated pro-sequence does not appear from its circular dichroism and 1H-NMR spectrum to have enough intrinsic stabilizing interactions to fold autonomously. However, the difference circular dichroism spectra of the pro-subtilisin variant and native subtilisin suggest that it is folded in the context of the pro-subtilisin molecule. The inability of the pro-subtilisin variant to bind a polypeptide inhibitor supports further the hypothesis that the pro-sequence interacts with subtilisin in the region where the active site is exposed. Our results suggest that the interactions provided by the pro-sequence are important only late on the folding pathway of pro-subtilisin and stabilize the transition state for folding. Kinetic analysis of the refolding reaction in the presence and absence of the pro-sequence reveal this stabilization to be in excess of 7.5 kcal/mol; folding is accelerated more than five orders of magnitude.
J Mol Biol 1993 Sep 20
PMID:Folding of subtilisin BPN': role of the pro-sequence. 837 4

A method using cytochrome c as the substrate of proteinases trypsin, chymotrypsin and subtilisin based on peroxidase effect of the formed haem octapeptide, which is much higher than that of cytochrome c, is described. The octapeptide is degraded by excess hydrogen peroxide and the competition between oxidation of guaiacol catalyzed by the octapeptide and octapeptide degradation leads under experimental conditions to rapid production of a stable colour. Absorption at 476 nm is proportional to proteolytic activity. The method was standardized using the Anson test for proteolytic activity with haemoglobin as a substrate.
Biochem Mol Biol Int 1993 Jun
PMID:Proteolytic activity assay based on enhanced peroxidase effect of hydrolyzed cytochrome c. 839 22

The problem of why serine protease inhibitors having substrate-like structure around the reactive site are not degraded by the cognate protease has prompted us to investigate the structural requirements in Streptomyces subtilisin inhibitor (SSI) for its inhibitory action. We removed the disulfide bridge between Cys71 and Cys101 near the reactive site by oligonucleotide-directed mutagenesis, replacing both Cys residues with Ser residues. Inhibitory activity of the mutated SSI toward subtilisin BPN' was initially potent, but decreased remarkably with increasing incubation time after mixing (temporary inhibition), due to degradation of the mutated SSI by subtilisin via a specific intermediate with a nick at the reactive site (Met73-Val74). Binding affinity of subtilisin for the mutated SSI was reduced by more than 10(3)-fold, and the mutated SSI showed a 20 deg.C decrease in melting temperature, which probably mainly reflects the destruction of the region of alpha-helix containing Cys101. These results imply that the susceptibility of the mutated SSI to protease, and the irreversibility of the peptide bond cleavage at the reactive site, result from increased flexibility around the reactive site in the complex of the disulfide-bond-removed SSI with the protease, demonstrating the requirement of conformational rigidity around the reactive site of the inhibitor for its native inhibitory action.
J Mol Biol 1993 Mar 20
PMID:Requirement for a disulfide bridge near the reactive site of protease inhibitor SSI (Streptomyces subtilisin inhibitor) for its inhibitory action. 846 55


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