Genome Analysis (B070)
Im Neuenheimer Feld 580
Synthetic Biology have become key
research areas in the quest for understanding
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
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
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
Molecules produced this way could overcome several of the limitations that are currently still
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,
biological systems could be pieced together that - in the very long run
- may lead to the assembly of an archetypical
model of a cell.
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,
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'
For all projects, there are immediate practical utilities next to the
mere improvement of the basic knowledge about molecular features.
Life: synthesis of an enzymatically active mirror-image DNA-ligase made
of D-amino acids
the synthesis of a functional DNA-ligase
in the D-enantiomeric conformation, which is an exact mirror-image of
natural enzyme, exhibiting DNA ligation activity on chirally inverted
acids in L-conformation, but not acting on natural substrates and with
co-factors. Starting from the known structure of the Paramecium
chlorella virus 1 DNA-ligase and the homologous but shorter DNA-ligase
Haemophilus influenza, we designed and synthesized chemically peptides,
could then be assembled into a full-length molecule yielding a
protein. The structure and the activity of the mirror-image ligase were
characterized, documenting its enantiospecific functionality.
al. (2021) Trends
Biochem. Sci., in press.
al. (2019) CELL Chem.
Biol. 26, 645-651.
of a fully synthetic mirror-image biological system (MirrorBio)
The basic motivation behind this collaborative project is
biological processes to an extent that will permit their re-creation.
ability would be documented best, if functioning molecular systems
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
into artificial molecular systems. Also, artificial experimental
complement current Systems Biology,
evaluating biological models experimentally. Similar to what is ongoing
insight into cellular functioning will be gained by an iterative
information resulting from experimental and theoretical analyses.
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
Plückthun, University of Zürich, Switzerland
Taipale, University of Helsinki, Finland
Weidmann et al. (2016) Org. Lett. 18, 164-167.
Olea et al. (2015) Chem. Biol. 22, 1437-1441.
Hauser et al. (2006) Nucleic Acids Res. 34, 5101-5111.
sequence and stability information for directing nanobody stability
Variable domains of camelid
heavy-chain antibodies, commonly named nanobodies, have high
potential. In view of their broad range of applications in research,
diagnostics and therapy, engineering their stability is of particular
Towards these ends, we analyzed the sequences and thermostabilities of
purified nanobody binders. From this data, potentially stabilizing
variations were identified and studied experimentally. Some improved
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
conformational stability and aggregation behavior, explaining the
effect on individual molecules. Other potentially stabilizing
actually led to thermal destabilization of the proteins. The reasons
contradiction between prediction and experiment were investigated. The
illustrate the potential and limitations of engineering nanobody
thermostability from a medium-throughput data set and indicate
a species-specificity of nanobody architecture.
Kunz et al. (2019) Protein Eng. Des. Sel. 32,
al. (2018) Sci. Rep. 8,
Kunz et al. (2015) BBA-Gen. Subjects 1861, 2196-2205.
Figure legend: Mechanism
nanobody stabilisation by N-terminal
mutations (Q1E and Q5V). (A) The thermodynamic stability of nanobody
its N-terminally mutated variant was measured in guanidinium chloride
equilibrium unfolding experiments. Unfolded protein was measured by
tryptophan fluorescence. Red lines represent fitted curves. (B)
of thermostabilisation by single and double mutations in nanobody NbD4,
measured by differential scanning fluorimetry in triplicate.
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
melting nanobody NbD1 and its N-terminally mutated variant; in panel C,
concentration of 32.7 μM was used; in panel D, concentration was
13.1 μM. Aggregation is indicated by a reduced amplitude of the
transitions in the fluorescence traces and can be quantified by
values of both concentration sets. Heating rate: 0.5 °C/min.
Revised CHARMM force field parameters for iron-containing co-factors of
Photosystem II is a complex
protein–cofactor machinery that splits water molecules into molecular
protons, and electrons. All-atom molecular dynamics simulations have
potential to contribute to our general understanding of how photosystem
To perform reliable all-atom simulations, we need accurate force field
parameters for the cofactor molecules. We present here CHARMM bonded
non-bonded parameters for the iron-containing cofactors of photosystem
include a six-coordinated heme moiety coordinated by two histidine
a non-heme iron complex coordinated by bicarbonate and four histidines.
force field parameters presented here give water interaction energies
geometries in good agreement with the quantum mechanical target data.
Adam et al. (2018) J. Comput. Chem. 39, 7-20.
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.