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The oxidation of Met-residues sets off a fatal structural change in the human prion protein
Figure 1. Oxidation of methionine residues and conformation of the prion protein. In spite of the huge amount of literature on prion proteins available to date little is known about the initial event leading to its misfolding. The moderately hydrophobic canonical amino acid methionine usually stabilizes α-helices in proteins. However its oxidized form, methionine-sulfoxide is hydrophilic and has a higher preference for β-sheets. If the oxidative stress within the cell is sufficiently high to oxidize certain methionine side chains within the prion protein, at least one portion of protein's α-helices is converted into β-sheet structures.
Figure 2. Chemical model to study the nature of α-helix to β-sheet conversion in prion protein structure. By expanding the genetic code of the AUG triplet with extremely hydrophobic norleucine and extermely hydrophilic metoxinine it is possible to arrest physiologically and pathologically relevant conformational states of the prion protein. In this way we have a kind of Yin and Yang states reflecting two extreme conditions: One prion that mimics the stable, reduced form and one in which all methionine side chains are oxidized. The norleucine variant resulted in an α-helix rich protein that lacks the in vitro aggregation of the parent protein. In contrast, the methoxinine variant resulted in a β-sheet rich protein with strong aggregation tendency.
These extreme conformational states are usually not amenable in animal models nor with peptide fragments. This experimental approach might be highly relevant to study neurodegenerative as well as other aging-related diseases (i.e. Alzheimer and Parkinson).
Wolschner, C., Giese, A., Kretzschmar, H., Huber, R., Moroder, L. and Budisa, N. (2009). Design of anti- and pro-aggregation variants to assess the effects of methionine oxidation in human prion protein. Proc. Natl. Acad. Sci. USA. Early Edition (April 2009), doi10.1073/PNAS.0902688106.
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Our long-term goal is the in vivo expression of intrinsically colored proteins without the need for further posttranslational modification or chemical functionalization by externally added reagents. Biocompatible azaindoles/azatryptophans as optical probes represent almost ideal isosteric substitutes for natural tryptophan in cellular proteins. To overcome the limits of the traditionally used 7-azaindole/7-azatryptophan, we have substituted the single tryptophan residue in human annexin A5 by 4- and 5-azatryptophan in Trp-auxotrophic Escherichia coli cells. Both cells and proteins with these fluorophores possess intrinsic blue fluorescence detectable upon routine UV irradiations. We identified 4-azaindole as a superior optical probe due to its pronounced Stokes-shift of ~130 nm, its significantly higher quantum yield in aqueous buffers and its enhanced quenching resistance. 4-Azaindoles`s intracellular metabolic transformation into 4-azatryptophan coupled with high yield incorporation into proteins is the most straightforward method for the conversion of naturally colorless proteins and cells into their blue counterparts from amino acid precursors.
Lepthien, S., Hoesl, MG., Merkel, L. and Budisa, N. (2008). Azatryptophans endow proteins with intrinsic blue fluorescence.Proc. Natl. Acad. Sci. USA. [epub ahead of print].
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Nonproteinogenic amino acids that either occur naturally or are synthesized chemically are becoming important tools in modern drug discovery. In this context, fluorinated amino acids have great potential in the development of novel pharmaceuticals and drugs. To assess whether different fluorinated aromatic amino acid analogues of phenylalanine, tyrosine, and tryptophan are potentially interesting as therapeutic drugs, we examined their cytostatic and cytotoxic effects on the growth of the human breast cancer cell line MCF-7. Of all the tested analogues L-4-fluorotryptophan, L-6-fluorotryptophan and L-p-fluorophenylalanine effectively and irreversibly inhibited cell growth with IC50 values in the low micromolar range (3-15 µM). Additionally, using L-4-[14C]fluorotryptophan, and L-6-[14C]fluorotryptophan, we discovered that the cellular uptake of these fluorinated amino acids occurs through active transport with a 70-fold excess of intracellular over extracellular concentrations. We identified system L as the responsible amino acid transporter. Our findings fully support the idea that fluorinated aromatic amino acid analogues are promising chemotherapeutics with the potential for use in combination with classical cancer therapy, and as new cytotoxic drugs for certain tumor types such as melanoma.
Giese, C., Lepthien, S., Metzner, L., Brandsch, M., Budisa, N, and Lilie, H. (2008). Intracellular uptake and inhibitory activity of aromatic fluorinated amino acids in human breast cancer cells. ChemMedChem 3 (9), 1449-1456.
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Non-canonical amino acids (NAA) as building blocks for peptides and proteins during ribosomal translation represent a nearly infinite supply of novel functions. Thereby, the specific selection, activation and tRNA-charging of amino acids by aminoacyl-tRNA synthetases (aaRS) in the aminoacylation reaction are essential steps. In most cases, the aminoacylation of NAA is a good indication that the related amino acid will participate in the ribosomal translation as well. However, testing translational capacity of amino acid analogs has technical limitations. Therefore, a rapid and reliable in silico test for NAA recognition by aaRS would be of advantage in experimental setup design. We chose trytophanyl-tRNA synthetase from Escherichia coli as model system for docking studies with various tryptophan analogs using the FlexX-Pharm strategy. We were able to calculate relative binding energies of Trp analogues in TrpRS which correlate well with their translational activities in E. coli. In particular, FlexX-Pharm predicted the binding sites of fluoro-, amino-, hydroxyl- and aza-containing Trp analogs within 1.5 Å of Trp in the homology model ofE. coli TrpRS. Therefore, the approach of utilizing ligand docking prior to NAA incorporation experiments, might provide a straightforward means for determining of NAAs that can be efficiently incorporated into a protein.
Azim, M. K. and Budisa, N. (2008). Docking of Tryptophan analogues to Tryptophanyl-tRNA Synthetase; implication for non-natural amino acid incorporation. Biol. Chem. [epub ahead of print].
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Bulky amino acids in positions two and three of proteins protect not only methionine but also noncanonical azidohomoalanine, introduced by the auxotrophy-based method, from being excised by the enzymes responsible for N-terminal Met excision. Subsequent bioorthogonal transformation enables new specific functionalization of target proteins. We validated this general concept by designing an N-terminal glycoconjugated barstar capable of lectin binding without loss of its original biological activity.
Merkel, L., Beckmann, H. S. G., Wittmann, V. and Budisa, N. (2008). Efficient N-terminal Glycoconjugation of Proteins by the N-End Rule. ChemBioChem 9 (8), 1220-1224.
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Steiner, T., Hess, P., Bae, J. H, Moroder, L. and Budisa, N. (2008). Synthetic Biology of Proteins: Tuning GFP´s Folding and Stability with Fluoroproline. PLoSONE 3, e1680.
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