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Photochemical Regulation of an Artificial Hydrolase by a Backbone Incorporated Tertiary Structure Switch
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II. (Lars Baltzer)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II. (Adolf Gogoll)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Organic Chemistry II.
2009 (English)In: Chemistry - A European Journal, ISSN 0947-6539, E-ISSN 1521-3765, Vol. 15, no 2, 501-505 p.Article in journal (Refereed) Published
Abstract [en]

A stilbene chromophore has been incorporated into the turn region of a 42 amino acid peptide, linking two helical peptide sections. Spatial proximity between these sections, as well as aggregation into dimers, is required to facilitate the catalytic function of this artificial hydrolase. Photomodulation of the hydrolase activity results in an increase of the activity of 42 % upon switching from the trans to the cis isomer of the chromophore. This is rationalized by a change in the aggregation state of the peptidomimetic, which is supported by diffusion coefficients obtained from PFG-NMR experiments. The results show that incorporation of a small, relatively flexible chromophore into a large peptide is capable of inducing a considerable change in tertiary structure and thus, functionality.

Place, publisher, year, edition, pages
2009. Vol. 15, no 2, 501-505 p.
Keyword [en]
aggregation, chromophores, enzyme catalysis, peptidomimetics
National Category
Organic Chemistry
Research subject
Organic Chemistry
Identifiers
URN: urn:nbn:se:uu:diva-99821DOI: 10.1002/chem.200801808ISI: 000262491500025OAI: oai:DiVA.org:uu-99821DiVA: diva2:208822
Available from: 2009-03-31 Created: 2009-03-20 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Expanding the Amino Acid Alphabet by Design: Enhanced and Controlled Catalytic Activity in Folded Polypeptide Catalysts
Open this publication in new window or tab >>Expanding the Amino Acid Alphabet by Design: Enhanced and Controlled Catalytic Activity in Folded Polypeptide Catalysts
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis addresses structure and reactivity of polypeptide catalysts in reactions that mimic the hydrolysis of RNA and DNA. A designed helix-loop-helix motif was used as a scaffold where the amino acid residues were systematically varied. The reactivity of a previously reported catalyst, HNI, was evaluated and the catalytic residues of the active site, Arg and His, were replaced in a stepwise manner by the artificial amino acid Gcp. Gcp has a guanidinocarbonyl pyrrole side chain, i.e. a side chain that mimics that of Arg but with a lower pKa. Gcp was used to replace both histidine and arginine in the polypeptide catalysts and was able to bind phosphate as well as carry out general base catalysis.

The parent polypeptide HNI was shown to catalyse phosphoryl transfer reactions of phosphodiesters in an active site with two His and two Arg residues. The performance of the active site was improved by the introduction of two Tyr residues to form the catalyst HJ1 designed to provide nucleophilic catalysis in the hydrolysis of DNA model substrates.

To improve the catalytic activity beyond that of HJI, Gcp was introduced to replace Arg and His residues in the HN1 scaffold. The designed catalyst JL3 was capable of a 150-fold rate enhancement compared to HNI in the reaction of the substrate HPNP, representing the first step in RNA hydrolysis. Mechanistic studies of JL3 catalysis suggested that the pKa value of the Gcp residue in the folded polypeptides was around 5. In combination with the observation of a solvent kinetic isotope effect of 1.7 the Gcp residue was proposed to provide general base catalysis and transition state stabilisation in the reaction of uridine 3′-2,2,2-trichloroethylphosphate, a realistic RNA model with a leaving group pKa of 12.5. The JL3 polypeptide catalyst followed saturation kinetics with a kcat/KM of 1.08 x 10-3 M-1s-1.

The introduction of a designed photoswitchable amino acid in a catalytic polypeptide allowed the activity of the polypeptide in the reaction with an activated ester to be under photochemical control. Photoisomerization of this switch altered the structure of the polypeptide and affected the catalytic activity of the polypeptide catalyst.

The chemical synthesis of designed molecules expands the amino acid alphabet and makes it possible to downsize enzymatic functions. It opens up possibilities for the production of novel biocatalysts that can catalyse natural as well as non-natural reactions.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 62 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 630
National Category
Other Basic Medicine
Research subject
Organic Chemistry
Identifiers
urn:nbn:se:uu:diva-100324 (URN)978-91-554-7485-0 (ISBN)
Public defence
2009-05-08, B42, BMC, Husargatan 3, Uppsala, 10:15 (English)
Opponent
Supervisors
Available from: 2009-04-17 Created: 2009-03-30 Last updated: 2009-04-20Bibliographically approved
2. Photoswitchable Peptidomimetics: Synthesis and Photomodulation of Functional Peptides
Open this publication in new window or tab >>Photoswitchable Peptidomimetics: Synthesis and Photomodulation of Functional Peptides
2008 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The secondary structure of peptides is of pivotal importance for their biological function. The introduction of photoswitchable moieties into the backbones of peptides provides a unique way of regulating their conformation using an external stimulus, i.e., light. This thesis addresses the design, synthesis, and conformational analysis of photoswitchable peptidomimetics (PSPM). Examples of photomodulation of their functional properties are given.

PSPM were prepared by incorporation of stilbene and thioaurone chromophores (switches) into dipeptides. Synthetic schemes for preparing these chromophores have been developed. Their suitability for incorporation into peptidomimetics has been demonstrated, and the resulting PSPM have been subjected to photoisomerization as well as computational and spectroscopic conformational analysis. The chromophore’s potential as a β-hairpin inducer was particularly interesting.

To investigate the factors that govern the formation of β-hairpins, a series of decapeptides were prepared. Turn regions consisting of amino acids or chromophores were combined with antiparallel peptide strands with hydrophobic side chains. Linear tryptophan zipper peptidomimetics and cyclic peptidomimetics with a second, hairpin-inducing turn region were particularly promising. Comparison between switches revealed that the more flexible stilbene is a better choice for upholding the β-hairpin conformation than the thioaurone.

The catalytic properties of an artificial hydrolase with a helix-loop-helix structure can be improved by introducing a stilbene photoswitch into the loop region. Photoisomerization regulates the catalytic activity of this peptidomimetic, and provides a means to control its aggregation state.

The activity of the enzyme Mycobacterium tuberculosis ribonucleotide reductase was realized by incorporating a stilbene moiety into a linear peptide. Here, one photoisomer proved to be an inhibitor at nM concentrations. A significantly lower effect was observed for the other isomer.

Finally, the decomposition of thioaurones, mainly to thioflavonols and thiaindenes, under conditions used for solid-phase peptide synthesis has been mapped. These findings are expected to have implications for future use of this chromophore.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2008. 70 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 565
Keyword
β-hairpin, peptidomimetics, stilbene, thioaurone, photochemistry
National Category
Organic Chemistry
Identifiers
urn:nbn:se:uu:diva-9345 (URN)978-91-554-7323-5 (ISBN)
Public defence
2008-11-27, B7:101a, BMC, Husargatan 3, Uppsala, 10:15
Opponent
Supervisors
Available from: 2008-11-04 Created: 2008-11-04 Last updated: 2010-12-15Bibliographically approved

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Lindgren, N. Johan V.Gogoll, Adolf

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