Functional Genome Analysis  (B070)
Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580
D-69120 Heidelberg, Germany.
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  Synthetic Biology


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Systems and Synthetic Biology have become key research areas in the quest for understanding living cells. What is missing is an experimental system in which the knowledge gained from molecular analyses and modelling could be reproduced in an artificial setting that is void of the risk of contamination from natural sources. We work at setting up a self-replicating molecular system that forms the basis toward the establishment of an artificial biology that is entirely independent from Nature but identical in terms of biophysical and biochemical parameters. Utilizing enantiomeric L-nucleotides and D-amino acids rather than natural components, we use chemical synthesis to produce L-form nucleic acids and D-form proteins. We take advantage of these molecules in order to obtain basic molecular activities in a form that is a mirror-image to Nature. All parts - including co-factors such as ATP - have to be enantiomeric to their natural counterparts and need to be produced synthetically.

The objective is to set up a self-replicating system, in particular aiming at enzymatically catalysed D-protein production. Various enzymes and other factors are required to achieve this goal. While still some way off, such a procedure could offer a major advance in medicine. Molecules produced this way could overcome several of the limitations that are currently still inherent to therapeutic proteins, such as protecting them from degradation or reducing their immunogenicity. Also, once protein production would not be based on chemical synthesis anymore but driven by enzymatic processes, many pieces of a mirror-image molecular puzzle could be produced with relative ease. Thereby, more complex artificial biological systems could be pieced together that - in the very long run - may lead to the assembly of an archetypical model of a cell.
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In addition, we work at the identification of factors that affect protein stability, whether in natural or enantiomeric conformation. An emphasis is the improvement of binder molecules, such as nanobodies. We pursue the production of molecules, which exhibit superior performance, by modulating parameters such as aggregation. This is complemented by modelling so as to define and predict structural features that are critical for molecules' activity.

For all projects, there are immediate practical utilities next to the mere improvement of the basic knowledge about molecular features.

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Copying Life: synthesis of an enzymatically active mirror-image DNA-ligase made of D-amino acids

We report the synthesis of a functional DNA-ligase in the D-enantiomeric conformation, which is an exact mirror-image of the natural enzyme, exhibiting DNA ligation activity on chirally inverted nucleic acids in L-conformation, but not acting on natural substrates and with natural co-factors. Starting from the known structure of the Paramecium bursaria chlorella virus 1 DNA-ligase and the homologous but shorter DNA-ligase of Haemophilus influenza, we designed and synthesized chemically peptides, which could then be assembled into a full-length molecule yielding a functional protein. The structure and the activity of the mirror-image ligase were characterized, documenting its enantiospecific functionality.

Weidmann et al. (2019) CELL Chem. Biol. 26, 645-651. pdf icon


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Establishment of a fully synthetic mirror-image biological system (MirrorBio)

EraSynbio logo        

The basic motivation behind this collaborative project is the ambition to understand biological processes to an extent that will permit their re-creation. This ability would be documented best, if functioning molecular systems could be established in a mirror-image, enantiomeric version, since this would be entirely independent of any natural compounds. Instead, the molecular components are generated by initially only chemical processes for the production of synthetic biomolecules and their assembly into artificial molecular systems. Also, artificial experimental systems will complement current Systems Biology, evaluating biological models experimentally. Similar to what is ongoing in physics, insight into cellular functioning will be gained by an iterative processing of information resulting from experimental and theoretical analyses. Eventually, this may lead to an archetypical model of a cell.

The project is performed as an EraSynBio consortium with partners from three other institutions:

  • Philip E. Dawson, The Scripps Research Institute, USA
  • Andreas Plückthun, University of Zürich, Switzerland
  • Jussi Taipale, University of Helsinki, Finland
logo-Uni-Zurich            logo-The-Scripps-Research-Institute            logo-Helsinki-University

Weidmann et al. (2016) Org. Lett. 18, 164-167. pdf icon
Olea et al. (2015) Chem. Biol. 22, 1437-1441. pdf icon
Hauser et al. (2006) Nucleic Acids Res. 34, 5101-5111. pdf icon

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Exploiting sequence and stability information for directing nanobody stability engineering

Variable domains of camelid heavy-chain antibodies, commonly named nanobodies, have high biotechnological potential. In view of their broad range of applications in research, diagnostics and therapy, engineering their stability is of particular interest. Towards these ends, we analyzed the sequences and thermostabilities of 78 purified nanobody binders. From this data, potentially stabilizing amino acid variations were identified and studied experimentally. Some improved the stability of nanobodies by up to 6.1°C, with an average of 2.3°C across eight modified nanobodies. The stabilizing mechanism involves an improvement of both conformational stability and aggregation behavior, explaining the different effect on individual molecules. Other potentially stabilizing variations actually led to thermal destabilization of the proteins. The reasons for this contradiction between prediction and experiment were investigated. The results illustrate the potential and limitations of engineering nanobody thermostability from a medium-throughput data set and indicate a species-specificity of nanobody architecture.
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Kunz et al. (2019) Protein Eng. Des. Sel., in press. (doi: 10.1093/protein/gzz017).
Kunz et al. (2018) Sci. Rep. 8, 7934. pdf icon..
Kunz et al. (2015) BBA-Gen. Subjects 1861, 2196-2205. pdf icon

Figure legend: Mechanism of nanobody stabilisation by N-terminal mutations (Q1E and Q5V). (A) The thermodynamic stability of nanobody NbD1 and its N-terminally mutated variant was measured in guanidinium chloride dependent equilibrium unfolding experiments. Unfolded protein was measured by intrinsic tryptophan fluorescence. Red lines represent fitted curves. (B) Additive effect of thermostabilisation by single and double mutations in nanobody NbD4, measured by differential scanning fluorimetry in triplicate. Improvements by mutations Q5V (0.9 °C) and Q1E (2.3 °C) match the stabilisation in the double mutant (3.1 °C). (C, D) Tryptophan fluorescence ratio (350 nm/330 nm) for melting nanobody NbD1 and its N-terminally mutated variant; in panel C, a concentration of 32.7 μM was used; in panel D, concentration was reduced to 13.1 μM. Aggregation is indicated by a reduced amplitude of the unfolding transitions in the fluorescence traces and can be quantified by comparing Tm values of both concentration sets. Heating rate: 0.5 °C/min.

Figure of nanobody stability measurements
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Cover-JCompChem-2018  
Structural Modelling:
Revised CHARMM force field parameters for iron-containing co-factors of photosystem II

Photosystem II is a complex protein–cofactor machinery that splits water molecules into molecular oxygen, protons, and electrons. All-atom molecular dynamics simulations have the potential to contribute to our general understanding of how photosystem II works. To perform reliable all-atom simulations, we need accurate force field parameters for the cofactor molecules. We present here CHARMM bonded and non-bonded parameters for the iron-containing cofactors of photosystem II that include a six-coordinated heme moiety coordinated by two histidine groups, and a non-heme iron complex coordinated by bicarbonate and four histidines. The force field parameters presented here give water interaction energies and geometries in good agreement with the quantum mechanical target data.
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Adam et al. (2018) J. Comput. Chem. 39, 7-20. pdf icon
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Figure legend: Cover image of the Journal of Computational Chemistry, volume 39, issue 1, on 5 January 2018. It presents artwork that is based on the results reported in the above mentioned publication.
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