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At least 199 records · Page 11Linked to original sources
PubMed2005

Derivitization of the C-terminus of ubiquitin and ubiquitin-like proteins using intein chemistry: methods and uses.

Here we describe a general method to synthesize and use a panel of reagents with selectivity for deubiquitinating enzymes exhibiting specificity for each ubiquitin-like protein. A substrate (Ubl-AMC), a reversible inhibitor (Ubl-aldehyde), and an active-site-directed irreversible inhibitor (Ubl-vinylsulfone) are described for each Ubl. Because of space constraints, we give details for only the Nedd8 derivatives, but these methods have been used in our laboratory to produce these derivatives of Ubiquitin, Nedd8, Sumo-1, Sumo-2, and Isg15. These reagents are useful in defining the specificity of DUBs, as well as in purifying and identifying these important proteins. The reagents are selective and useful in crude extracts. The reactivity of these reagents reveals differences in both the S1 and S1' sites of deubiquitinating enzymes. Only active enzymes are efficiently detected with these reagents. Published results indicate that specificity is not strictly defined by the evolutionary relationships of these DUBs.

Base Sequence↗
PubMed2005

An intein-based genetic selection allows the construction of a high-quality library of binary patterned de novo protein sequences.

Combinatorial libraries of synthetic DNA are increasingly being used to identify and evolve proteins with novel folds and functions. An effective strategy for maximizing the diversity of these libraries relies on the assembly of large genes from smaller fragments of synthetic DNA. To optimize library assembly and screening, it is desirable to remove from the synthetic libraries any sequences that contain unintended frameshifts or stop codons. Although genetic selection systems can be used to accomplish this task, the tendency of individual segments to yield misfolded or aggregated products can decrease the effectiveness of these selections. Furthermore, individual protein domains may misfold when removed from their native context. We report the development and characterization of an in vivo system to preselect sequences that encode uninterrupted gene segments regardless of the foldedness of the encoded polypeptide. In this system, the inserted synthetic gene segment is separated from an intein/thymidylate synthase (TS) reporter domain by a polyasparagine linker, thereby permitting the TS reporter to fold and function independently of the folding and function of the segment-encoded polypeptide. TS-deficient Escherichia coli host cells survive on selective medium only if the insert is uninterrupted and in-frame, thereby allowing selection and amplification of desired sequences. We demonstrate that this system can be used as a highly effective preselection tool for the production of large, diverse and high-quality libraries of de novo protein sequences.

Amino Acid Sequence↗
PubMed2003

Occurrence, horizontal transfer and degeneration of VDE intein family in Saccharomycete yeasts.

VDE is a homing endonuclease gene originally discovered as an intervening element in VMA1s of Saccharomyces cerevisiae. There have been two independent subfamilies of VDE, one from S. cerevisiae strain X2180-1A and the other from Saccharomyces sp. DH1-1A in the host VMA1 gene, and they share the identity of 96.3%. In order to search the occurrence, intra/interspecies transfer and molecular degeneration of VDE, complete sequences of VMA1 in 10 strains of S. cerevisiae, eight species of saccharomycete yeasts, Candida glabrata and Kluyveromyces lactis were determined. We found that six of 10 S. cerevisiae strains contain VDEs 99.7-100% identical to that of the strain X2180-1A, one has no VDE, whereas the other three harbour VDEs 100% identical to that of the strain DH1-1A. S. carlsbergensis has two VMA1s, one being 99.8% identical to that of the strain X2180-1A with VDE 100% identical to that of the strain DH1-1A and the other containing the same VMA1 in S. pastorianus with no VDE. This and other evidence indicates that intra/interspecies transmissions of VDEs have occurred among saccharomycete yeasts. Phylogenetic analyses of VMA1 and VDE suggest that the S. cerevisiae VDEs had branched earlier than other VDEs from an ancestral VDE and had invaded into the host loci as relatively late events. The two VDEs seemed to degenerate in individual host loci, retaining their splicing capacity intact. The degeneration of the endonuclease domains was distinct and, if compared, its apparent rate was much faster than that of the protein-splicing domains.

Amino Acid Substitution↗
PubMed2002

Intein-mediated synthesis of geranylgeranylated Rab7 protein in vitro.

Production of recombinant proteins is an important prerequisite for biotechnology and life sciences in general. However, there is a paucity of methods for production of posttranslationally modified recombinant proteins or proteins with non-native functional groups, such as fluorophores, spin labels, and so forth. In this work we have used a combination of organic synthesis and in vitro protein ligation to construct monoprenylated Rab7 GTPase. The protein was prepared from a recombinant N-terminal portion and a peptide mimicking the C terminus of Rab7. For construction of a synthetic six-amino-acid-long fluorescent monoprenylated peptide, we used a block condensation strategy. Ligation was achieved with a yield of >70%. The resulting protein was purified from the unligated peptide by a combination of organic extraction and phase partitioning and refolding. The refolded monoprenylated semisynthetic Rab7 protein (Rab7GG) formed a stable complex with its natural chaperone REP-1 (Rab escort protein 1) and could serve as an acceptor of the second prenyl group in the enzymatic prenylation reaction. Using fluorescence spectroscopy, we characterized the interaction of the Rab7GG:REP-1 complex with Rab geranylgeranyl transferase and came to the conclusion that it functioned as a genuine intermediate of the prenylation reaction. Thus, we present the first example of the in vitro generation of a semisynthetic lipidated protein using the native chemical ligation method.

Alkyl and Aryl Transferases↗
PubMed1998

Amino acid residues in both the protein splicing and endonuclease domains of the PI-SceI intein mediate DNA binding.

A structure-based model describing the interaction of the two-domain PI-SceI endonuclease with its 31-base pair DNA substrate suggests that the endonuclease domain (domain II) contacts the cleavage site region of the substrate, while the protein splicing domain (domain I) interacts with a distal region that is sufficient for high affinity binding. To support this model, alanine-scanning mutagenesis was used to assemble a set of 49 PI-SceI mutant proteins that were purified and assayed for their DNA binding and cleavage properties. Fourteen mutant proteins were 4- to >500-fold less active than wild-type PI-SceI in cleavage assays, and one mutant (T225A) was 3-fold more active. Alanine substitution at two positions in domain I reduces overall binding >60-fold by perturbing the interaction of PI-SceI with the minimal binding region. Conversely, mutations in domain II have little effect on binding, reduce binding to the cleavage site region only, or affect binding to both regions. Interestingly, substitutions at Lys301, which is part of the endonucleolytic active site, eliminate binding to the cleavage site region but permit contact with the minimal binding region. This experimental evidence demonstrates that the protein splicing domain as well as the endonuclease domain is involved in binding of a DNA substrate with the requisite length.

Amino Acids↗
PubMed1999

PI-PfuI and PI-PfuII, intein-coded homing endonucleases from Pyrococcus furiosus. II. Characterization Of the binding and cleavage abilities by site-directed mutagenesis.

PI- Pfu I and PI- Pfu II from Pyrococcus furiosus are homing endonucleases, as shown in the accompanying paper. These two endonucleases are produced by protein splicing from the precursor protein including ribonucleotide reductase (RNR). We show here that both enzymes specifically interact with their substrate DNA and distort the DNA strands by 73 degrees and 67 degrees, respectively. They have two copies of the amino acid sequence motif LAGLIDADG, which is present in the majority of homing endonucleases and provides some of the catalytic residues necessary for DNA cleavage activity. Site-specific mutagenesis studies showed that two acidic residues in the motifs, Asp149 and Glu250 in PI- Pfu I, and Asp156 and Asp249 in PI- Pfu II, were critical for catalysis. The third residues of the active site triads, as predicted from the structure of PI- Sce I, were Asn225 in PI- Pfu I and Lys224 in PI- Pfu II. Substitution of Asn225 in PI- Pfu I by Ala did not affect catalysis. The cleavage activity of PI- Pfu II was 50-fold decreased by the substitution of Ala for Lys224. The binding affinity of the mutant protein for the substrate DNA also decreased 6-fold. The Lys in PI- Pfu II may play a direct or indirect role in catalysis of the endonuclease activity.

Amino Acid Motifs↗