Feuillet bêta | EPFL Graph Search

Les feuillets β ou feuillets β plissés est la deuxième forme de structure secondaire régulière observée dans les protéines, avec une fréquence de présence plus faible que les hélices α. Les feuillets β sont constitués de brins bêta (brins β) reliés latéralement par au moins deux ou trois liaisons hydrogène entre des atomes du squelette carboné de la chaine polypeptidique pour former un plan plissé (comme un accordéon), généralement tordu. Un brin β est une partie de la chaine polypeptidique formé en moyenne de trois à dix acides aminés dont la chaine carbonée principale est étendue. L'association de nombreux feuillets β est observée dans l'agrégation de protéines, dans de nombreuses maladies humaines, notamment les amyloses telles que la maladie d'Alzheimer. Découverte La première structure des feuillets β est proposée par William Astbury dans les années 1930, après des observations indiquant des changements très importants des clichés diffraction de rayon X de fibres de l

Amyloid ß-derived switch-peptides as tool to study conformational changes relevant in degenerative diseases

Marie-Stéphanie Camus

The rapid growing number of patients diagnosed with a neurodegenerative disease and more particularly with Alzheimer's disease (AD) has stimulated intensive research in determining and understanding biological phenomena causing such devastating diseases and hence allowing for the elaboration of adapted therapeutic treatments. These diseases are also commonly called "conformational" diseases because they result from the misfolding of a protein leading to the formation of self-associated β-sheets, which in turn give rise to the formation of oligomers, protofibrils as well as insoluble fibrils characterizing the plaques found in the brain of affected patients. Consequently, the investigation of such proteins, in particular of Amyloid β (Aβ) in the case of AD, is a limited and difficult task to achieve, which often leads to contradictory results. To overcome these difficulties and to be able to study the key steps of conformational transitions and misfolding of such peptides and proteins, our research group has developed a new tool, called switch-peptides, enabling to block (Soff state) and trigger (Son state) peptide folding at will (Figure). The introduction of a switch element S built from Ser, Thr or Cys residues disrupts the regular polypeptide chain by the insertion of an ester and a flexible C-C bond resulting in a conformational disconnection of P1 and P2 (Figure), i.e. in an unordered (random coil), non-folded conformation. Each S element is protected by a protecting group Y (Soff state) that can be cleaved independently by adding a base, an enzyme or by light, depending on the chemical nature of Y. The cleavage of the different protecting groups Y triggers a spontaneous O to N acyl migration, re-establishing the regular amide backbone of the peptide chain, hence enabling the peptide to fold "in statu nascendi" and to adopt a well-defined secondary structure. The present thesis explores the potential of this novel concept for the example of conformational transitions relevant in amyloid β misfolding. In the first part we investigate the chemical stability of the S element in aqueous media, exposing a number of switch-peptides to various experimental conditions. Most notably, the ester bond proved to be stable at acidic as well as physiological conditions for several hours, opening a broad range of biological applications. The second part of the work is dedicated to the study of conformational transitions of switch-peptides derived from Aβ(1-42). By incorporating one or several switch elements disposing orthogonal protecting groups Y, the impact of different fragments of the peptide as nucleation site for the process of β-sheet formation, self-assembly and aggregation has been revealed as monitored by CD, TEM studies and ThT (pathway B, Figure). For the first time, the orthogonal triggering of the two switch elements, i.e. S26 and S37 allowed to delineate the important role of the C-terminal part of Aβ in the early step of misfolding. Subsequently, one of the nucleation sites for aggregation, i.e. segment Aβ(14-24) was excised from the native sequence and transformed to a switch-peptide applying the host-guest technique. Detailed CD studies were applied for investigating conformational transitions of type random-coil (Soff) to β-sheet structure (Son), serving as proof of concept for the use of Aβ-derived nucleation sites as guest sequence in combination with β-sheet promoting host peptides for the screening of potential inhibitors of fibril formation as early molecular event in the context of AD. This has been demonstrated in applying the elaborated host-guest peptides to evaluate the β-sheet breaking potential of pseudo-proline (ψPro)-containing switch-peptides derived from Aβ (pathway C, Figure). Preliminary results indicate that the in situ formation of kink-conformations may exert a β-sheet destabilizing effect, confirming previous observations from the Soto group. Finally, the use of switch-peptides as β-sheet and fibril breaking molecules by in situ α-helix nucleation (pathway A, Figure) has been explored. To this end, the potentially β-sheet forming segment Aβ(14-24) was linked via S element to a helix nucleating peptidomimetic ("N-Cap"). In the Soff state (pH ≤ 4), CD and TEM studies point to the onset of a β-sheet, fibril forming structure. In triggering O,N-acyl migration (pH ≈ 7), a so far unprecedented transition of type β-sheet to α-helix is observed, paralleled by a drastic increase in solubility and a complete disappearance of fibrils. The reversibility of β-sheet and fibril formation by α-helix nucleation in situ represents a most interesting observation and deserves further exploration as potential tool in the study of folding processes. In conclusion, the concept of "switch-peptides" has been successfully applied to biologically relevant molecular events of utmost therapeutic interest.

EPFL2008

Switch peptides

Richard Alan Mimna

Aspects of conformational transitions, folding, and misfolding of peptides and proteins have recently taken center stage in various domains at the interface of chemistry, biology, and medicine because of their impact on protein misfolding diseases. Due to the intimate relationship between sequence, secondary structure, and physicochemical properties, studies on oligo- and polypeptides exhibiting high propensity for β-sheet formation are severely limited by their intrinsic tendency toward self-association and irreversible aggregation. Consequently, β-sheet and fibril-forming processes related to protein misfolding diseases remain elusive, as delineation of structure nucleation or inhibition is a formidable task, often yielding contradictory observations. With the aim of surmounting these experimental difficulties and of inducing conformational transitions in situ, the present thesis elaborates the novel concept of switch-peptides (see figure). Modular switch-peptides are synthesized comprising three distinct elements: a conformation induction unit (σ), a switch-element (S), and a peptide (P). Of central importance to the switch-peptide concept, the switch-element S dissects the native polyamide backbone of the peptide P by an ester and a flexible C-C bond, thereby preventing the peptide from folding and separating the conformational impact of σ. The triggering of the switch-element by removal of the protecting group Y reestablishes the native polyamide backbone via a spontaneous O → N acyl migration reaction, whereupon peptide folding can then proceed. Detailed herein is the development of the key elements of the concept, σ and S, and their subsequent application to various model peptides and peptides of biological interest (P). In particular, two synthetic routes to S-elements were demonstrated, i.e. the stepwise solid phase synthesis of depsipeptides using a minimal protection strategy and the preparation of protected depsidipeptides for use as building blocks in solution or solid-phase synthesis. Kinetic investigations of their acyl rearrangements reveal half-lives that range from less than one minute to several hours. The possibility to tailor the half-lives by manipulation of experimental conditions is also elaborated. Orthogonal protection strategies including photolytically and enzymatically labile groups were developed. This enabled the incorporation of multiple S-elements into a peptide that could be triggered sequentially. In this way, the mutual conformational impact of peptide segments as well as their role in folding and self-assembly was explored. The design and synthesis of novel, helix-nucleating N-cap compounds (a type of σ element) is described and applied for the in situ nucleation of helical conformations according to the figure. For the first time, a detailed study of a wide variety of original N-caps is realized in which their helix inducing abilities are quantified by CD spectroscopy. When applied to model β-sheet-forming switch-peptides (random coil → β-sheet), the use of an N-cap is shown to overcome the intrinsic β-sheet propensity and instead induce a conformational transition to an α-helix. Furthermore, an hitherto unobserved conformational transition of type β-sheet → α-helix in an amyloid beta-derived switch-peptide is demonstrated and shown by electron microscopy to inhibit its fibrillization. Through a series of designed switch-peptides, the S-element is shown to indeed conformationally decouple segments σ and P and, upon triggering, to induce conformational transitions relevant to protein misfolding. The results obtained firmly establish the capability of employing the switch-peptide concept as a tool to study folding events during the dynamic process of structure onset and evolution. With the methodologies elaborated herein, the stage is set for its further application to the study of structure-function relationships, prodrug design, the design of diagnostic systems for evaluating inhibitors of secondary structure, and to β-breaker peptides.

EPFL2005

Self-Assembly of ß-Amyloid Peptides in Cells and on Solid Supports

Enrico Condemi

Neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease and Huntington's disease share common mechanisms characterized by protein misfolding and aggregation, including formation of plaques or inclusion bodies. The rapid growing number of patients has motivated intensive research to understand biological phenomena involved in the pathology and hence developing therapeutic strategies. Here, we will focus on β-amyloid peptide (Aβ) involved in Alzheimer's disease. In the first part of this thesis, tissue transglutaminase (tTGase) has been investigated due to its implication in a number of cellular processes and disease states, where the enzymatic actions of tTGase may serve in both, cell survival and apoptosis. To date, the precise functional properties of tTGase in cell survival or cell death mechanisms still remain elusive. tTGase mediated cross-linking has been reported to account for the formation of insoluble lesions in conformational diseases. We report here that, tTGase induces intramolecular cross-linking of Aβ peptide, resulting in structural changes of monomeric Aβ. Using high resolution mass spectrometry (MS) of cross-linked Aβ peptides, we observed a shift in mass, which is, presumably associated with the loss of NH3 due to enzymatic transamidation activity and hence intramolecular peptide cross-linking. We have observed that, a large population of Aβ monomers contained an increase in mass of 0.984 Da at a glutamine residue, indicating that glutamine Q15 serves as an indispensable substrate in TGase mediated deamidation to glutamate E15. We provide strong analytical evidence on tTGase mediated Aβ peptide dimerization, through covalent intermolecular cross-linking and hence the formation of Aβ1-40 dimers. Our in depth analyses indicate that, tTGase induced post-translational modifications of Aβ peptide may serve as an important seed for aggregation. In the second part of this work, we explore the idea of using synthetic peptides that can switch conformation in a controlled way as a sensor for β-breakers of Aβ. The concept of switch-peptides allows the controlled onset of polypeptides folding and misfolding at physiological pH by in situ O,N-acyl migration. The synthesized sequence consisted in the core sequence responsible for Aβ aggregation, flanked by two β-sheet-inducing template through ester bonds. The triggering of the acyl migration allows the native sequence to be recovered from the ester bonds and structural change from flexible unordered conformation to β-sheets and subsequently fibrils formation were observed in solution. In a more biological environment made of lipid vesicles, the conformation of the peptide has been shown to act comparably to wild type Aβ peptide by circular dichroism (CD), namely increased negative charges over the surface led to increased helical propensity followed by a transition to β-sheets and accelerated fibrils formation. Thereafter, the conformation of the cysteine analogue of the switch-peptide covalently linked to gold surface was characterized by Fourier-transform infrared spectroscopy (FTIR). In monolayer of switch-peptide tethered to a planar gold surface, the peptide already adopts most likely β-turn conformation and no changes in the infrared spectra were detected after the triggering of the switch. On gold nanoparticles, the switch-peptide folds spontaneously into β-sheet conformation. These unwanted foldings limit the efficiency of the sensor and has driven us to an alternative configuration, where gold nanoparticles were coated with a 95:5 mixture of two peptide sequences able to stabilize the nanoparticles and to act as recognition sequence, respectively. In this configuration, the switch-peptide is added in the gold nanoparticles solution and the aggregation triggered. Here, we want to take advantage of a particular feature of gold nanoparticles that consists in a change of solution color from red to blue as the nanoparticles come closer to each other during aggregation. Preliminary results have shown that a discrimination between aggregated and soluble samples is possible. However, further improvements are necessary before a potential biosensor could be expected.

EPFL2011