Prolegomena zum experimentellen Engineering des genetischen Codes durch Erweiterung seines Aminosäurerepertoires

Budiša, Nediljko

doi:10.1002/ange.200300646

Cited by 76 publications

(24 citation statements)

References 269 publications

(346 reference statements)

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“…[1][2][3][4] In particular, the introduction of non-natural amino acids in a multisite fashion allows systematic engineering of the overall physical behavior of recombinant proteins. Multisite incorporation is easy to implement and yields protein products in amounts sufficient for detailed physical characterization.…”

Section: Introductionmentioning

confidence: 99%

Stabilization of bzip Peptides through Incorporation of Fluorinated Aliphatic Residues

2006

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Two fluorinated amino acids, 5,5,5-trifluoroisoleucine (5TFI) and (2S,3R)-4,4,4-trifluorovaline (4TFV), which have been shown to serve as isoleucine surrogates in protein synthesis in Escherichia coli, have been incorporated in vivo into basic leucine zipper (bzip) peptides derived from GCN4. The extents of residue-specific incorporation of 5TFI and 4TFV were 90 and 88 %, respectively, of the encoded isoleucine residues, as evidenced by MALDI mass spectrometry and amino acid analysis. Both circular dichroism and equilibrium sedimentation studies of the fluorinated bzip peptides indicated preservation of secondary and higher-order protein structure. Thermal-denaturation experiments showed an increase of 27 degrees C in melting temperature when isoleucine was replaced by 5TFI. However, the T(m) of the peptide containing 4TFV was increased by only 4 degrees C over that of the peptide containing valine. Similar trends were observed in chemical denaturation studies in which DeltaDeltaG(unfold) in water was determined to be 2.1 or 0.3 kcal mol(-1) upon incorporation of 5TFI or 4TFV, respectively. When the fluorinated peptides were tested for DNA binding, both their affinity and specificity were similar to those of the respective hydrogenated peptides. These results suggest that fluorinated amino acids, even when introduced into the same positions, can have markedly different effects on the physical properties of proteins, while having little impact on secondary and higher-order structure.

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Section: Introductionmentioning

confidence: 99%

Stabilization of bzip Peptides through Incorporation of Fluorinated Aliphatic Residues

2006

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“…First, a certain class of amino acids was excluded from the code through selection since they might exhibit adverse effects on the folding and structural integrity of target proteins [35]. Second, the explanation for the exclusion of other amino acid types that do not violate these rules is most probably historical, such as their unavailability or insufficient evolutionary time to produce them metabolically [36]. Therefore, we generally anticipate that future research on the expanded amino acid repertoire for protein building and folding will, at least in part, not be possible without parallel work on protein de novo design.…”

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confidence: 99%

Efforts towards the Design of ‘Teflon’ Proteins: In vivo Translation with Trifluorinated Leucine and Methionine Analogues

Budiša

Pipitone

Siwanowicz

et al. 2004

Chemistry & Biodiversity

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In vivo incorporation of monofluorinated noncanonical amino acids into recombinant proteins has been well-established for decades. Proteins fluorinated in this way proved to be useful tools for many practical applications. In contrast, trifluorinated amino acids have been incorporated in only a few peptides and relatively small proteins by using expression systems in living cells. A novel class of proteins with a fluorous core can be envisaged only if full replacement of the core-building hydrophobic and aliphatic amino acids such as leucine or methionine with the related analogues trifluoromethionine and trifluoroleucine would be feasible. However, our systematic efforts to introduce these amino acids in larger proteins (over 10 Da) that contain different structural motifs clearly show that only partial substitutions are possible. The reasons are high toxicity of these substances and difficulties to accommodate them into the compact cores of natural proteins without adverse effects on their structural integrity. Therefore, engineering of such three dimensional 'Teflon'-like structures would require, besides an expansion of the amino acid repertoire of the genetic code, a de novo protein design as well.

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“…Their transmission to eukaryotic cells is not a trivial task. The use of stop codon suppression is additionally limited by read-through efficiency (usually~40 %) [43] and permissiveness of the particular sequence positions (i.e., not all positions in the sequence are equally well suited for labeling). Last but not least, evolved enzymes for amino acid recognition and cognate tRNA have poor catalytic performance and selectivity.…”

Section: Methodological Issuesmentioning

confidence: 99%

“…Last but not least, evolved enzymes for amino acid recognition and cognate tRNA have poor catalytic performance and selectivity. [43] The other approach, known as selective pressure incorporation (SPI) [43] relies on the relaxed substrate specificity of aminoacyl-tRNA synthetases as the crucial enzymes in the interpretation of the genetic code. Thereby, the activation and transfer onto cognate tRNAs of a variety of structurally and chemically similar substrate analogues is possible without the necessity for extensive host cell engineering.…”

Section: Methodological Issuesmentioning

confidence: 99%

Blue Fluorescent Amino Acids as In Vivo Building Blocks for Proteins

et al. 2010

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In vivo expression of colored proteins without post-translational modification or chemical functionalization is highly desired for protein studies and cell biology. Cell-permeable tryptophan analogues, such as azatryptophans, have proved to be almost ideal isosteric substitutes for natural tryptophan in cellular proteins. Their unique spectral features, such as markedly red-shifted fluorescence, are transmitted into protein structures upon incorporation. Among the azaindoles under study (2-, 4-, 5-, 6-, and 7-azaindole) 4-azaindole has exhibited the largest Stokes shift (approximately 130 nm) in steady-state fluorescence measurements. It is also highly biocompatible and as 4-azatryptophan it can be translated into target protein sequences. However, its quantum yield and fluorescence intensity are still significantly lower when compared with natural indole/tryptophan. Since azatryptophans are hydrophilic, their presence in the hydrophobic core of proteins could be harmful. In order to overcome these limitations we have performed nitrogen methylation of azaindoles and generated mono- and dimethylated azaindoles. Some of these methyl derivatives retain the pronounced red shift present in the parent 4-azaindole, but with much higher fluorescence intensity (reaching the level of indole/tryptophan). Therefore, the blue fluorescence of azaindole-containing proteins could be further enhanced by the use of methylated analogues. Further substitution of any azaindole ring with either endo- or exocyclic nitrogen will not yield a spectral fluorescence maximum shift beyond 450 nm under steady-state conditions in the physiological milieu. However, green fluorescence is a special feature of tautomeric species of azaindoles in various nonaqueous solvents. Thus, the design or evolution of the protein interior combined with the incorporation of these azaindoles might lead to the generation of specific chromophore microenvironments that facilitate tautomeric or protonated/deprotoned states associated with green fluorescence.

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Prolegomena zum experimentellen Engineering des genetischen Codes durch Erweiterung seines Aminosäurerepertoires

Cited by 76 publications

References 269 publications

Stabilization of bzip Peptides through Incorporation of Fluorinated Aliphatic Residues

Stabilization of bzip Peptides through Incorporation of Fluorinated Aliphatic Residues

Efforts towards the Design of ‘Teflon’ Proteins: In vivo Translation with Trifluorinated Leucine and Methionine Analogues

Blue Fluorescent Amino Acids as In Vivo Building Blocks for Proteins

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