Current
Koksch group research projects  |
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| Peptides
and proteins carry out most of the vital functions in complex
biological systems including the human body. The shape and function
of these biooligomers is decisively influenced by the physical
and chemical properties of the amino acid building blocks. Misfolded
proteins and disturbed protein-protein interactions are the
cause of a host of devastating diseases. Our laboratory aims
to understand and influence peptide structure. To control and
inhibit folding and aggregation processes effectively, a fundamental
understanding of the interplay between primary structure and
folding stability under the influence of environmental conditions
is required. The incorporation of particular amino acids in
specifically designed positions will not only improve the therapeutic
efficacy of peptide-based drugs. Extending the spectra of building
blocks which can be used for peptide and protein engineering
beyond the natural amino acids also extends the scope of peptides
and proteins to areas such as materials science. Highly functionalized
amino acid residues serve as valuable tools to be used as biophysical
probes for detailed studies of structure-function relationships
or for the construction of tailor-made biomolecules. Especially,
the incorporation of fluorine was shown to have dramatic effects
on protein stability and the physical properties of protein-based
materials.
Since joining the faculty at the Free University of Berlin in
2004, we have created a research program aimed at understanding
and modulating protein structure and function by development
of small peptide model systems. Based on fundamental insights
fueled by innovative chemical approaches, a variety of applications
are being pursued.
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C. Jaeckel and B. Koksch. Fluorine in peptide design and protein
engineering. Eur. J. Org. Chem. 2005,
21, 4482-4503
K. Pagel, T. Vagt, B. Koksch. Directing polypeptide’s
secondary structure at will - from a-helix to amyloids and reverse?
Org. Biomol. Chem. 2005, 3,
3843-3850
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Application
of the unique properties of fluorinated amino acids for peptide
and protein modification
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| We
have developed a peptide model to systematically study the complex
molecular interactions of fluoroalkyl groups within native polypeptides
regarding space filling, lipophilicity, and hydrogen bonding.
Differences in even one single fluoro substituent in the side
chain of one amino acid can be detected using this model system.
Using these in vitro investigations we determined how fluorinated
amino acids influence the folding of native polypeptide motifs
and established the key influence of the fluorine-flourine interactions
on peptide and protein folding. Currently we are extending this
model to a phage display system. Screening of a high number
of different protein sequences will enable selection of the
preferred binders of different fluorinated amino acids. These
studies pave the way for the use of the beneficial properties
of fluorinated amino acids for a deliberate de novo design of
biologically relevant peptide drugs and fluorinated protein-based
materials. |
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| Fig.
1. Using
a screening system based on a 41 amino acid residue alpha-helical
coiled coil peptide we have demonstrated that the electrostatic
consequences of alkyl fluorination can have a much stronger
influence on hydrophobic interactions in a protein than the
increase in molecular volume upon stepwise fluorination. |
| C.
Jaeckel, M. Salwiczek, B. Koksch. Fluorine in a native protein
environment – How space filling and polarity of fluoroalkyl
groups affect protein folding. Angew. Chem. 2006,
118, 4305-4309, Angew. Chem. Int. Ed. 2006,
45, 4198-4203
C. Jaeckel, W. Seufert, S. Thust, B. Koksch. Evaluation of the
molecular interactions of fluorinated amino acids with native
polypeptides. ChemBioChem. 2004, 5,
717-720 |
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| Access
to modified peptides requires the key building blocks. Therefore,
our lab is developing routine methods for the synthesis of non-natural
amino acids with a particular focus on fluorinated amino acids.
Strategies to incorporate non-natural building blocks into peptides
and proteins are being explored including enzymatic methods. |
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| Fig.
2. A general model of the CPY-catalyzed peptide synthesis
using Calpha-alkylated alanine methyl ester and different
nucleophilic amino components. R1: CH3;
R2: H, CH3, CH(CH3), CH2CH(CH3)2;
x: 2, 3; y: 0, 1, 3. N-protected Ala methyl ester derivates bearing
a methyl-, difluoromethyl-, or trifluoromethyl group, respectively,
instead of the alpha-proton, were accepted as substrates by CPY
and could, therefore, be coupled directly to various nucleophiles.
Peptide yields between 20% and 75% were obtained depending on
the nucleophilic amino acid derivative and the enantiomer of the
fluoroalkyl amino acid. |
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| Fig
3. Yields (%) of chymotrypsin-catalyzed peptide synthesis
using substrate mimetics of Calpha,alpha-dialkyl alanine
derivates. Condition: 0.1 M HEPES, 0.2 M NaCl, 0.02 M CaCl2,
pH 8.0, 10% DMSO, 25 °C. [acyl donor] 4 mM, [acyl acceptor]
20 mM, [chymotrypsin] 4.8 × 10-5M; all errors less than
5%. |
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| We
introduced a methodology for the incorporation of sterically demanding
Calpha-fluoroalkyl aminoacids into the P1
position of peptides catalyzed by the commercially available protease
trypsin and chymotrypsin. |
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Synthesis
of Calpha,alpha-Dialkyl Amino Acid-4-guanindinophenyl
Esters.
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Fig.
4. a R1: CH3, CF2H,
CF3; R2: CH2C6H5,
CH2CH(CH3)2; R3:
CH3. (i) DIC, HOAt, THF; (ii) n-butyllithium, THF;
(iii) TBTU, DIEA, DMF; (iv) TFA, ultrasound.
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R.
Smits, B. Koksch. How Calpha-fluorinated amino acids
interact with enzymes: Studies concerning proteolytic stability,
enzymatic resolution and peptides coupling. Curr. Topics in
Med. Chem. 2006
B. Koksch, T. Michel, P. Kaptain, P. Quaedflieg, Q.B.
Broxterman. Enzymatic resolution of Calpha-fluoroalkyl
amino acids. Tetrahedron: Asymmetry. 2004,
15, 1401-1407
S. Thust, B. Koksch. Discovery of carboxypeptidase Y
as catalyst for the incorporation of sterically demanding a-fluoroalkyl
amino acids into peptides. Tetrahedron Lett. 2004,
45, 1163-1165
S. Thust, B. Koksch. Protease catalyzed peptide synthesis
for the site-specific incorporation of alpha-fluoroalkyl amino
acids into peptides. J. Org. Chem. 2003,
68, 2290-2296 |
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Peptide
model systems to study peptide folding and aggregation
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A
special property of proteins is the ability to adopt more than
one stable conformation. This feature is, e.g., associated with
many neurodegenerative diseases such as Alzheimer's, Huntington's
disease as well as prion-based diseases. Here, the change in
protein secondary and tertiary structures are the cause of so-called
plaque formation. When deposited in nerve tissue these insoluble
protein aggregates can cause severe neurodegenerative diseases.
The formation of fibrils from monomeric and oligomeric peptide
building blocks is not yet understood although it has been studied
intensely. Our lab is currently addressing some key questions
of fundamental biochemical and biophysical importance such as
(1) How can complex folding mechanisms that commence with the
conversion of alpha-helices to beta-sheets result in amyloids?
(2) Which intrinsic factors enable peptide and proteins to force
their own secondary structure onto unfolded peptides and proteins?
(3) Can the aggregation of beta-sheets be inhibited? (4) How
can the ability of proteins to reversible change their conformation
as a reaction to environmental conditions be used for the generation
of bionanomaterials?
To address these issues a model system has been developed by
our lab that enables a systematic evaluation of the influence
of mutations within the primary structure of a peptide on the
stability of the secondary structure of a-helices and b-sheets.
This model system serves well to follow the interconversion
of certain secondary structures depending on factors such as
pH, ionic strength, metal ions, oxidative stress, temperatures,
chaperones or solvents. These peptides respond to environmental
changes by adopting different secondary structures. Without
any restrictions such as linkers, non-natural building blocks
in the peptide sequence, important conclusions can be drawn
on how complementary interactions and cooperative processes
determine structure formation of peptides and proteins under
native conditions. For the first time, it is possible to study
such important and complex issues as the role of electrostatic
interactions as well as of metal ions in aggregation processes
in the context of all other environmental conditions at the
molecular level by applying high resolution analytical methods. |
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| Fig.
5. |
K.
Pagel, S.C. Wagner, K. Samedov, H. v. Berlepsch, C. Böttcher,
B. Koksch. Random coils, b-sheet ribbons and a-helical fibers
- One peptide adopting three different secondary structures at
will,J. Am. Chem. Soc. 2006, 128(7),
2196-2197
K. Pagel, T. Kohajda, B. Koksch. From a-helix to b-sheet –
a reversible metal ion induced peptide secondary structure switch,
Org. Biomol. Chem. 2005, 3,
2500-2502
K. Pagel, B. Seiwert, K. Seeger, S. Berger, A. Mark, B. Koksch.
Advanced approaches for the characterization of a de dovo designed
antiparallel coiled coil peptide,Org. Biomol. Chem. 2005,
3, 1189-1194 |
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Organization
of nanoparticles and lipid vesicles through interaction with alpha-helical
coiled coil-based peptides
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| To
study the interaction of peptides and proteins with biological
membranes is one of the most challenging endeavors in bioorganic
and biophysical chemistry. The complexity of these systems induces
a considerable diversity of parameters that contribute to binding
and induction of conformational changes. Besides the fundamental
biological and biochemical questions related to peptide-membrane
interaction, there has been a growing interest in biotechnological
applications of short peptide sequences. Most prominent examples
are antimicrobial peptides and carrier peptides. The latter molecules
have been developed as transport vehicles for genes, drugs, or
other molecules across cellular membranes. Our lab has recently
introduced a simple peptide model system that enables a systematic
study of (1) the role of a membrane environment on peptide/protein
folding, (2) the impact of peptide secondary structure on peptide-membrane
as well as on membrane-membrane interaction, and (3) the dynamics
of protein-membrane interactions depending on environmental conditions
like pH, ionic strength, temperature, etc., on a molecular level.
These results lay the foundation for a systematic application
of the alpha-helical coiled coil folding motif in any kind of
membrane active events on a molecular level. |
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Fig.
6.
Using a de novo designed peptide model system we systematically
study (i) the role of a membrane environment on peptide folding,
(ii) the impact of different domains of an alpha-helical coiled
coil heptad repeat on the interaction with membranes, and (iii)
the dynamics of peptide-membrane interactions depending on environmental
conditions. |
T.
Vagt, O. Zschörnig, D. Huster, B. Koksch. Membrane binding
and structure of de novo designed a-helical cationic coiled
coil, Peptides. ChemPhysChem. 2006,
6, 1361-1371 |
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We
are now developing a peptide-based system that will allow organization
of functionalized nanoparticles such as cadmium sulfide, silver
and gold in defined networks. In comparison to already known
DNA-nanoparticle-conjugates, peptides provide many advantages
for the synthesis of bionanomaterials. The anionic nature of
a DNA-molecule allows for only a few defined and rigid interactions
with particles. Peptides, however, provide a huge diversity
of functionalities that allow a fine-tuning of the structural
and electostatic properties of the resulting materials. Our
group is now combining the intrinsic properties of peptides
with their peptide models to generate nanomaterials that can
be reversibly switched between different states of structural
organization.
Successful work on these topics requires the combination of
several scientific disciplines such as organic chemistry, biochemistry,
biophysics, inorganic chemistry, material sciences, analytics,
and theory. We are collaborating with several groups at the
Free University Berlin, Max-Planck-Institutes and on the national
and international level.
Our work has been reported on by international journals such
as Chem&EngNews,
Chemistry
World and Chemical Education. |
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