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Item does not contain fulltext Consensus engineering, which is replacing amino acids by the most frequently occurring one at their positions in a multiple sequence alignment (MSA), is a known strategy to increase the stability of a protein. The application of this concept to the entire sequence of an enzyme, however, has been tried only a few times mainly because of the problems determining the consensus in highly variable regions. We show that this problem can be solved by replacing such problematic regions by the corresponding sequence of the natural homologue closest to the consensus. When one or a few sub-families are overrepresented in the MSA the consensus sequence is a biased representation of the sequence space. We examine the influence of this bias by constructing three consensus sequences using different MSAs of sucrose phosphorylase (SP). Each consensus enzyme contained about 70 mutations compared to its closest natural homologue and folded correctly and displayed activity on sucrose. Correlation analysis revealed that the family's co-evolution network was kept intact, which is one of the main advantages of full-length consensus design. The consensus enzymes displayed an "average" thermostability, that is, one that is higher than some but not all known representatives. We cautiously present practical rules for the design of consensus sequences, but warn that the measure of success depends on which natural enzyme is used as point of comparison. Biotechnol. Bioeng. 2013;110: 2563-2572. (c) 2013 Wiley Periodicals, Inc.

Cell surface mucins configure the cell surface by presenting extended protein backbones that are heavily O-glycosylated. The glycopeptide structures establish physicochemical properties at the cell surface that enable and block the formation of biologically important molecular complexes. Some mucins, such as MUC1, associate with receptor tyrosine kinases and other cell surface receptors, and engage in signal transduction in order to communicate information regarding conditions at the cell surface to the nucleus. In that context, the MUC1 cytoplasmic tail (MUC1CT) receives phosphorylation signals from receptor tyrosine kinases and serine/threonine kinases, which enables its association with different signaling complexes that conduct these signals to the nucleus and perhaps other subcellular organelles. We have detected the MUC1CT at promoters of over 500 genes, in association with several different transcription factors, and have shown that promoter occupancy can vary under different growth factor conditions. However, the full biochemical nature of the nuclear forms of MUC1 and its function at these promoter regions remain undefined. I will present evidence that nuclear forms of the MUC1CT include extracellular and cytoplasmic tail domains. In addition, I will discuss evidence for a hypothesis that the MUC1CT possesses a novel catalytic function that enables remodeling of the transcription factor occupancy of promoters, and thereby engages in regulation of gene expression.

Contains fulltext : 154902.pdf (Publisher’s version ) (Open Access) During 11-12 August 2014, a Protein Bioinformatics and Community Resources Retreat was held at the Wellcome Trust Genome Campus in Hinxton, UK. This meeting brought together the principal investigators of several specialized protein resources (such as CAZy, TCDB and MEROPS) as well as those from protein databases from the large Bioinformatics centres (including UniProt and RefSeq). The retreat was divided into five sessions: (1) key challenges, (2) the databases represented, (3) best practices for maintenance and curation, (4) information flow to and from large data centers and (5) communication and funding. An important outcome of this meeting was the creation of a Specialist Protein Resource Network that we believe will improve coordination of the activities of its member resources. We invite further protein database resources to join the network and continue the dialogue.

Netherlands Bioinformatics Centre (NBIC) Netherlands Genomics Initiative (NGI), BSIK European Commission Dutch Top Institute Pharma The GPCRDB is a Molecular Class-Specific Information System (MCSIS) that collects, combines, validates and disseminates large amounts of heterogeneous data on G protein-coupled receptors (GPCRs). the GPCRDB contains experimental data on sequences, ligand-binding constants, mutations and oligomers, as well as many different types of computationally derived data such as multiple sequence alignments and homology models. the GPCRDB provides access to the data via a number of different access methods. It offers visualization and analysis tools, and a number of query systems. the data is updated automatically on a monthly basis. the GPCRDB can be found online at http://www.gpcr.org/7tm/. Radboud Univ Nijmegen Med Ctr, NCMLS, CMBI, NL-6525 GA Nijmegen, Netherlands MSD, Dept Mol Design & Informat, NL-5340 BH Oss, Netherlands Universidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04023062 São Paulo, Brazil Universidade Federal de São Paulo, Escola Paulista Med, Dept Biophys, BR-04023062 São Paulo, Brazil European Commission: LHSG-CT-2004-512092 Dutch Top Institute Pharma: D1-105 Web of Science

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Item does not contain fulltext The hydrophobic S1' subsite is one of the major determinants of the substrate specificity of thermolysin and related M4 family proteases. In the thermolysin-like protease (TLP) produced by Bacillus stearothermophilus (TLP-ste), the hydrophobic S1' subsite is mainly formed by Phe130, Phe133, Val139 and Leu202. In the present study, we have examined the effects of replacing Leu202 by smaller (Gly, Ala, Val) and larger (Phe, Tyr) hydrophobic residues. The mutational effects showed that the wild-type S1' pocket is optimal for binding leucine side chains. Reduction of the size of residue 202 resulted in a higher efficiency towards substrates with Phe in the P1' position. Rather unexpectedly, the Leu202-->Phe and Leu202-->Tyr mutations, which were expected to decrease the size of the S1' subsite, resulted in a large increase in activity towards dipeptide substrates with Phe in the P1' position. This is probably due to the fact that 202Phe and 202Tyr adopt a second possible rotamer that opens up the subsite compared to Leu202, and also favours interactions with the substrate. To validate these results, we constructed variants of thermolysin with changes in the S1' subsite. Thermolysin and TLP-ste variants with identical S1' subsites were highly similar in terms of their preference for Phe vs. Leu in the P1' position.

Chorion is the major component of silkmoth eggshell. More than 95% of its dry mass consists of the A and B families of low molecular weight structural proteins, which have remarkable mechanical and chemical properties, and protect the oocyte and the del eloping embryo from the environment. We present data from negative staining, Congo red binding, X-ray diffraction, Fourier transform-Raman, attenuated total reflectance infrared spectroscopy and modelling studies of a synthetic peptide analogue of a part of the central domain of the B family of silkmoth chorion proteins, indicating that this peptide folds and self-assembles, forming amyloid-like fibrils, These results support further our proposal, based on experimental data from a synthetic peptide analogue of the central domain of the A family of chorion proteins, that silkmoth chorion is a natural, protective amyloid [Iconomidou et a.,, FEBS Lett, 479 (2000) 141-145].: (C) 2001 Federation of European Biochemical Societies, Published by Elsevier Science B.V. All rights reserved.

Chorion is the major component of silkmoth eggshell. More than 95%, of its dry mass consists of proteins that have remarkable mechanical and chemical properties protecting the oocyte and the developing embryo from a wide range of environmental hazards. We present data from electron microscopy (negative staining and shadowing), X-ray diffraction and modeling studies of synthetic peptide analogues of silkmoth chorion proteins indicating that chorion is a natural amyloid, The folding and self-assembly models of chorion peptides strongly support the beta-sheet helix model of amyloid fibrils proposed recently by Blake and Serpell [Structure 4 (1996) 989-998], (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

In recent years, many efforts have been made to isolate enzymes from extremophilic organisms in the hope to unravel the structural basis for hyperstability and to obtain hyperstable biocatalysts. Here we show how a moderately stable enzyme (a thermolysin-like protease from Bacillus stearothermophilus, TLP-ste) can be made hyperstable by a limited number of mutations. The mutational strategy included replacing residues in TLP-ste by residues found at equivalent positions in naturally occurring, more thermostable variants, as well as rationally designed mutations. Thus, an extremely stable 8-fold mutant enzyme was obtained that was able to function at 100 degrees C and in the presence of denaturing agents. This 8-fold mutant contained a relatively large number of mutations,whose stabilizing effect is generally considered to result from a reduction of the entropy of the unfolded state ("rigidifying" mutations such as Gly --> Ala, Ala --> Pro, and the introduction of a disulfide bridge). Remarkably, whereas hyperstable enzymes isolated from natural sources often have reduced activity at low temperatures, the 8-fold mutant displayed wild-type-like activity at 37 degrees C.

The active site of thermolysin-like proteases (TLPs) is located at the bottom of a cleft between the N- and C-terminal domains. Crystallographic studies have shown that the active-site cleft is more closed in ligand-binding TLPs than in ligand-free TLPs. Accordingly, it has been proposed that TLPs undergo a hinge-bending motion during catalysis resulting in “closure” and “opening” of the active-site cleft. Two hinge regions have been proposed. One is located around a conserved glycine 78; the second involves residues 135 and 136. The importance of conserved glycine residues in these hinge regions was studied experimentally by analyzing the effects of Gly → Ala mutations on catalytic activity. Eight such mutations were made in the TLP of Bacillus stearothermophilus (TLP-ste) and their effects on activity toward casein and various peptide substrates were determined. Only the Gly78Ala, Gly136Ala, and Gly135Ala + Gly136Ala mutants decreased catalytic activity significantly. These mutants displayed a reduction in kcat/Km for 3-(2-furylacryloyl)-l-glycyl-l-leucine amide of 73%, 62%, and 96%, respectively. Comparisons of effects on kcat/Km for various substrates with effects on the Ki for phosphoramidon suggested that the mutation at position 78 primarily had an effect on substrate binding, whereas the mutations at positions 135 and 136 primarily influence kcat. The apparent importance of conserved glycine residues in proposed hinge-bending regions for TLP activity supports the idea that hinge-bending is an essential part of catalysis.

Autolytic degradation of Bacillus subtilis thermolysin-like proteinase (TLP-sub) is responsible for the irreversible inactivation of the enzyme at elevated temperatures. Previously, we reported five autolysis sites in B. subtilis neutral protease (Van den Burg et al., 1990, Biochem. J. 272:93-97). In an attemp to render the enzyme less susceptible to autolytic breakdown, one of the sites, located between Leu156 and Ile157, was modified by substituting the residue C-terminal of the fission (P-1' residue), Ile157, by Asp. The introduction of the Asp, which is unfavoured at the P-1' position in substrates for TLPs, resulted in an altered autolysis pattern. The peptides formed in wild-type TLP-sub by chain cleavage N-terminal of Ile157, were absent in the constructed mutant (I157D). However, an autolysis product was detected with I157D that was not seen in case of the wild-type. The N-terminal sequence of this peptide was determined and it was concluded that in the mutant a peptide accumulated as the result of chain cleavage of the Gly148-Val149 bond. Structural aspects and effects of the mutation on the activity have been analyzed.

Using genetic techniques the contribution of surface loops to the thermal stability of Bacillus subtilis neutral protease (NP-sub) was studied. Mutations were designed to make the surface of NP-sub more similar to the surface of more thermostable neutral proteases such as thermolysin (TLN). The mutations included the replacement of an irregular loop by a shorter variant and the introduction of a ten-residue beta-hairpin. In general, these drastic mutations had little effect on the production and activity of NP-sub, indicating the feasibility of major structural rearrangements at the surface of proteins. In the most stable mutant, exhibiting an increase in thermal stability of 1.1 degrees C, similar to 10% of the surface of NP-sub was modified. Several NP-sub variants carrying multiple mutations were constructed. Non-additive effects on thermal stability were observed, which were interpreted on the basis of a model for thermal inactivation, that emphasizes the importance of local unfolding processes for thermal stability.

The thermostability of neutral proteases has been shown to depend on autolysis which presumably occurs in flexible regions of the protein. In an attempt to rigidify such a region in the neutral protease of Bacillus stearothermophilus, residues in the solvent-exposed 63-69 loop were replaced by proline. The mutations caused large positive (Ser-65-->Pro, Ala-69-->Pro) or negative (Thr-63-->Pro, Tyr-66-->Pro) changes in thermostability, which were explained on the basis of molecular modelling of the mutant proteins. The data show that the introduction of prolines at carefully selected positions in the protein can be a powerful method for stabilization.

A 10 residue beta-hairpin, which is characteristic of thermostable Bacillus neutral proteases, was engineered into the thermolabile neutral protease of Bacillus subtilis. The recipient enzyme remained fully active after introduction of the loop. However, the mutant protein exhibited autocatalytic nicking and a 0.4-degrees-C decrease in thermostability. Two additional point mutations designed to improve the interactions between the enzyme surface and the introduced beta-hairpin resulted in reduced nicking and increased thermostability. After the introduction of both additional mutations in the loop-containing mutant, nicking was largely prevented and an increase in thermostability of 1.1-degrees-C was achieved.

Using site-directed mutagenesis, Ala166 in the neutral protease of Bacillus stearothermophilus was changed into Ser. Model building and molecular dynamics simulations of the mutant enzyme indicated that the Ser hydroxyl group fits well in a cavity which contains a water molecule in the wild-type enzyme. The Ala166 --> Ser mutation was expected to exert a stabilizing effect because of the gain in entropy resulting from the release of a water molecular from the folded protein to the solvent. In addition, the hydrogen-bonding network around residue 166 was improved upon the mutation. As a result of this mutation the thermostability of the neutral protease was increased by 1.2 +/- 0.1-degrees-C.