Infoscience

Thesis

Switch peptides: in situ nucleation of conformational transitions relevant to protein misfolding diseases

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.

    Thèse École polytechnique fédérale de Lausanne EPFL, n° 3295 (2005)
    Section de chimie et génie chimique
    Faculté des sciences de base
    Institut des sciences et ingénierie chimiques
    Laboratoire de chimie biomimétique et des peptides
    Jury: Annette Beck-Sickinger, Michaël Grätzel, Eric Grouzmann, Hilal Lashuel

    Public defense: 2005-7-22

    Reference

    Record created on 2005-07-12, modified on 2016-08-08

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