5 research outputs found
Structural and biophysical characterization of photoswitchable peptides and their complexes
Networks of biomacromolecular interaction form the core of internal cellular logic where
a wide range of input signals get analysed and results in a genetically programmed output.
This description holds not only for healthy cells but is true also for those virally infected
when a virus implements new biomacromolecular elements and modifies existing networks.
Those hostile interactions are crucial for the HIV virus replication cycle. Due to the scale-free
nature of biomacromolecular interaction networks they are prone to malfunction if a hub
(node with the highest interactions) is damaged. Perhaps the best example of such a situation
can be observed in the apoptotic machinery. Overexpression, deletion or mutations
(depending on the context) in apoptotic proteins can lead to various diseases, most
prominently cancer. Therefore current research efforts concentrate on designing inhibitors of
malfunctioning interactions. Occasionally interaction between the two binding partners relies
on a well-defined secondary structures element. This allows to engineer this element and use
it to bias the given interaction equilibrium towards dissociation. One such approach
concentrates on α-helices that are crosslinked with azobenzene derivatives. This permits not
only to increase the helical content of a peptide, which translates to increased affinity
(potency), but transplants a photo-controllability. Photoswitchable peptides created in this
way can be controlled externally; their affinity towards targets can be effectively photomodulated.
The first chapter of the thesis concentrates on the development and evaluation of photocontrollable
peptides derived from the arginine rich region of the HIV type I Rev protein.
Rev, which is an early product generated from the HIV genome, binds to RRE containing
mRNA and removes it from the nucleus, allowing expression of unspliced or singly spliced
mRNA into different protein products. As a consequence the RRE-Rev interaction controls
the infection cycle of the HIV type 1, as these later products are necessary for virus particle
assembly. The α-helical peptide derived from Rev was shown to retain most of the affinity to
RRE. Rev-RRE serves as a prototypical protein-RNA interaction; a model for the
development of the first photo-controllable peptide that targets RNA. This research presents
two approaches for the peptide production: chemical synthesis and biosynthesis
(overexpression of the Rev peptide gene). Initially the water soluble azobenzene derivative
was used bearing two negatively charged sulphonate substituents on the aromatic rings.
Surprisingly, the first generation of the peptides had substantially decreased affinity to RRE
VI
as compared to the wild type Rev peptide. By applying computational chemistry techniques a
possible rationalisation was found: sulphonates interact strongly with crucial arginines on the
peptide making them unavailable to engage the phosphate backbone; additionally the peptide
was forced into non-helical conformation prior to binding. This hypothesis was validated by
crosslinking Rev peptides with azobenzene derivative deprived of the sulphonates. The
second generation of Rev peptides binds RRE with high affinity and the interaction in
question can be controlled effectively by light.
The second chapter of the thesis focuses on the structural characterization of a photocontrollable
Bak peptide bound to Bcl-xL – a prominent member of the Bcl-2 family of
proteins. The Bcl-2 family of proteins includes the major regulators and effectors of the
intrinsic apoptosis pathway. Cancers are frequently formed when activation of the apoptosis
mechanism is compromised either by misregulated expression of prosurvival family members
or, more frequently, by damage to the regulatory pathways that trigger intrinsic apoptosis.
Short peptides derived from the pro-apoptotic members of the Bcl-2 family can activate
mechanisms that ultimately lead to cell death. The recent development of photocontrolled
peptides that are able to change their conformation and activity upon irradiation with an
external light source has provided new tools to target cells for apoptosis induction with
temporal and spatial control. In this thesis the first NMR solution structure of a
photoswitchable peptide derived from the proapoptotic protein Bak in complex with the
antiapoptotic protein Bcl-xL is reported. This structure provides insight into the molecular
mechanism, by which the increased affinity of such photopeptides compared to their native
forms, is achieved, and offers a rationale for the large differences in the binding affinities
between the helical and nonhelical states
Design of photocontrolled RNA-binding peptidomimetics
The first examples of photocontrolled RNA binding peptides are described. The large number of positively charged sides chains in the Rev response element (RRE) of an HIV type I targeting α-helix imposes constraints on the choice of azobenzene-derived crosslinker
NMR Solution Structure of a Photoswitchable Apoptosis Activating Bak Peptide Bound to Bcl-x(L)
The Bcl-2 family of proteins includes the major regulators and effectors of the intrinsic apoptosis pathway. Cancers are frequently formed when activation of the apoptosis mechanism is compromised either by misregulated expression of prosurvival family members or, more frequently, by damage to the regulatory pathways that trigger intrinsic apoptosis. Short peptides derived from the pro-apoptotic members of the Bcl-2 family can activate mechanisms that ultimately lead to cell death. The recent development of photocontrolled peptides that are able to change their conformation and activity upon irradiation with an external light source has provided new tools to target cells for apoptosis induction with temporal and spatial control. Here, we report the first NMR solution structure of a photoswitchable peptide derived from the proapoptotic protein Bak in complex with the antiapoptotic protein Bcl-xL. This structure provides insight into the molecular mechanism, by which the increased affinity of such photopeptides compared to their native forms is achieved, and offers a rationale for the large differences in the binding affinities between the helical and nonhelical states
NMR Solution Structure of a Photoswitchable Apoptosis Activating Bak Peptide Bound to Bcl-x<sub>L</sub>
The Bcl-2 family of proteins includes the major regulators
and
effectors of the intrinsic apoptosis pathway. Cancers are frequently
formed when activation of the apoptosis mechanism is compromised either
by misregulated expression of prosurvival family members or, more
frequently, by damage to the regulatory pathways that trigger intrinsic
apoptosis. Short peptides derived from the pro-apoptotic members of
the Bcl-2 family can activate mechanisms that ultimately lead to cell
death. The recent development of photocontrolled peptides that are
able to change their conformation and activity upon irradiation with
an external light source has provided new tools to target cells for
apoptosis induction with temporal and spatial control. Here, we report
the first NMR solution structure of a photoswitchable peptide derived
from the proapoptotic protein Bak in complex with the antiapoptotic
protein Bcl-x<sub>L</sub>. This structure provides insight into the
molecular mechanism, by which the increased affinity of such photopeptides
compared to their native forms is achieved, and offers a rationale
for the large differences in the binding affinities between the helical
and nonhelical states
1H, 13C and 15N chemical shift assignments of unliganded Bcl-xL and its complex with a photoresponsive Bak-derived peptide
Here we report the 1H, 13C and 15N resonance assignments of free Bcl-xL and of Bcl-xL in complex with an azobenzene-modified peptide derived from the BH3 domain of the pro-apoptotic Bak. The spectra suggest predominantly folded proteins; chemical shift difference analysis provides a detailed view of the reorganization occurring on peptide binding