Aspects of conformational transitions, folding, and misfolding of peptides and proteins have moved into the center of interest in various domains of research at the interface of chemistry, biology, and medicine because of their impact on neurodegenerative diseases.[1] For example, recent research suggests that conformational transitions of soluble amyloid b precursor molecules into aggregated, b-sheet-type forms play a key role in the deposition of cerebral amyloid plaques

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The use of peptidomimetics and topological templates has become an important tool in protein design and mimicry." -4 1 By introducing these elements in the design process, the synthetic chemist aims to unravel the complex interplay between protein structure and function. In order to avoid the well-known protein folding problem, we have introduced the concept of template assembled synthetic proteins (TASP); here, templates serve as built-in devices for directing the intramolecular assembly of covalently attached peptide blocks into characteristic folding t o p~l o g i e s .~~ So far, problems with the synthesis have prevented the development of the full potential of this app r~a c h . '~] According to the TASP approach, the functional part of a protein, for example the antigen binding site of an antibody or the ligand binding site of a receptor, is detached from the rest of the molecule and assembled on a topological template, which mimics the loop supporting structural framework of the native protein (Fig. 1). Here, we elaborate the methodologies for the synthesis of this novel generation of functional TASP compounds.The approach comprises two key elements: 1) peptide sequences (loops) containing C-and N-terminal functional-

-Sheet-based assemblies have attracted a considerable amount of attention from researchers of various disciplines because of their association with a variety of diseases and their emerging potential in material sciences and biotechnology. [1] Protein misfolding and self-assembly into highly ordered b-sheet-rich fibrillar assemblies that are known as amyloid fibrils are common features of a growing class of systemic and neurodegenerative diseases, which include Alzheimer s, Parkinson s, and Huntington s diseases, senile systemic amyloidoses, as well as type II diabetes. [2] Although there is strong evidence that implicates amyloid formation in the pathogenesis of these diseases, the precise mechanisms of amyloid formation and clearance in vivo, as well as the structural basis of amyloid toxicity, remain unknown. The lack of tools to monitor and/or control the initial structural transitions associated with protein misfolding, amyloid formation, and/or dissociation is the main cause of this gap in knowledge. Significant efforts have been devoted to study proteins and small peptides that self-assemble into amyloidlike fibrillar structures as model systems to investigate amyloid formation or to generate materials with interesting physical properties. However, knowledge of the mechanical and structural dynamics within b-sheet assemblies such as amyloid fibrils remains limited. Early assumptions that bsheet assemblies, which include amyloid fibrils, occupy a global minimum of free energy that is lower than that of the native state [3] led to a greater emphasis on the understanding and inhibition of amyloid formation, rather than that of amyloid dissociation and clearance. Despite the stability of bsheet-rich amyloid fibrils against proteases, acids, and chemical denaturants, increasing evidence from human [4] and in vitro studies indicates that a dynamic structure exists within amyloid fibrils and suggests that the process of amyloid formation is reversible. [5a,b] These findings, along with the fact that strategies aimed at the destabilization of amyloid fibrils and/or the acceleration of their clearance seem to reverse the disease phenotype, [6][7] suggest that a detailed understanding of the stability and dynamic behavior of amyloid fibrils is of critical importance to the development of therapeutic strategies for amyloid diseases.Our research group has previously shown [8][9][10] that the incorporation of molecular switches into polypeptides, based on an in situ intramolecular O!N acyl group migration, [11] allows for the controlled induction or reversal of secondary structural transitions [12a,b,c] and self-assembly of small peptide chains. Herein we describe a switch peptide that is designed to disrupt amyloid-like b-sheet assemblies through the controlled induced transition from a b-sheet to an a-helix structure (Figure 1). The experimental results illustrate the potential of switch peptides as a tool to investigate the structural dynamics of amyloid fibrils, to provide an insight into the structural basis o...

The study of conformational transitions of peptides has obtained considerable attention recently because of their importance as a molecular key event in a variety of degenerative diseases. However, the study of peptide selfassembly into β‐sheets and amyloid β (Aβ) fibrils is strongly hampered by their difficult synthetic access and low solubility. We have recently developed a new concept termed “switch‐peptides” that allows the controlled onset of polypeptide folding and misfolding at physiologic conditions. As a major feature, the folding process is initiated by chemically or enzyme triggered O,N‐acyl migration in flexible and soluble folding precursors containing Ser‐ or Thr‐derived switch (S)‐elements. The elaborated methodologies are exemplified for the in situ conversion of NPY‐ and Cyclosporine A‐derived prodrugs, as well as for the onset and reversal of α and β conformational transitions in Aβ peptides. In combining orthogonally addressable switch‐elements, the consecutive switching on of S‐elements gives new insights into the role of individual peptide segments (“hot spots”) in early processes of polypeptide self‐assembly and fibrillogenesis. Finally, the well‐known secondary structure disrupting effect of pseudoprolines (ΨPro) is explored for its use as a building block (S‐element) in switch‐peptides. To this end, synthetic strategies are described, allowing for the preparation of Ψ Pro‐containing folding precursors, exhibiting flexible random‐coil conformations devoid of fibril forming propensity. The onset of β‐sheet and fibril formation by restoring the native peptide chain in a single step classify Ψ Pro‐units as the most powerful tool for inhibiting peptide self‐assembly, and complement the present methodologies of the switch‐concept for the study of fibrillogenesis. © 2007 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 88: 239–252, 2007. This article was originally published online as an accepted preprint. The ‘Published Online’ date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Background: Nicotine is the main culprit for dependence on tobacco-containing products, which in turn are a major etiologic factor for cardiovascular diseases and cancer. This publication describes a vaccine, which elicits antibodies against nicotine. The antibodies in the blood stream intercept the nicotine molecule on its way to its receptors and greatly diminish the nicotine influx to the brain shortly after smoking. Methods: The nicotine molecule is chemically linked to cholera toxin B as a carrier protein in order to induce antibodies. The potential to elicit antibodies after subcutaneous as well as intranasal immunization is evaluated. In order to simulate realistic conditions, nicotine pumps delivering the nicotine equivalent of 5 packages of cigarettes for 4 weeks are implanted into the mice 1 week prior to vaccination. The protective effect of the vaccine is measured 5 weeks after vaccination by comparing the influx of radiolabeled nicotine in the brains of vaccinated and non-vaccinated animals 5 min after challenge with the nicotine equivalent of 2 cigarettes. Results: The polyclonal antibodies induced by the vaccine show a mean avidity of 1.8 × 10⁷ l/Mol. Subcutaneous immunization elicits high antibody levels of the IgG class, and significant IgA antibody levels in the saliva of vaccinated mice can be found after intranasal vaccination. The protective effect also in the animals with implanted nicotine pumps is significant: less than 10% of radiolabeled nicotine found in the brains of non-vaccinated animals can be found in the brains of vaccinated animals. Conclusions: These data provide credible evidence that a vaccine can break the vicious circle between smoking and instant gratification by intercepting the nicotine molecule. Astonishingly, there is no sign of exhaustion of specific antibodies even under extreme conditions, which makes it highly unlikely that a smoker can overcome the protective effect of the vaccine by smoking more. Finally, the high titers of specific antibodies after 1 year let us hope that booster vaccinations are probably only necessary in intervals of years.

Tissue transglutaminase (TGase) has been implicated in a number of cellular processes and disease states, where the enzymatic actions of TGase may serve in both, cell survival and apoptosis. To date, the precise functional properties of TGase in cell survival or cell death mechanisms still remain elusive. TGasemediated cross-linking has been reported to account for the formation of insoluble lesions in conformational diseases. We report here that TGase induces intramolecular cross-linking of ␤-amyloid peptide (A␤), resulting in structural changes of monomeric A␤. Using high resolution mass spectrometry (MS) of cross-linked A␤ peptides, we observed a shift in mass, which is, presumably associated with the loss of NH 3 due to enzymatic transamidation activity and hence intramolecular peptide cross-linking. We have observed that a large population of A␤ monomers contained an 0.984 Da increase in mass at a glutamine residue, indicating that glutamine 15 serves as an indispensable substrate in TGase-mediated deamidation to glutamate 15. We provide strong analytical evidence on TGasemediated A␤ peptide dimerization, through covalent intermolecular cross-linking and hence the formation of A␤ 1-40 dimers. Our in depth analyses indicate that TGase-induced post-translational modifications of A␤ peptide may serve as an important seed for aggregation.

Protein design aims to mimic some of the structural and functional properties of native proteins. The complexity of the folding mechanism, i.e. the pathway by which a linear polypeptide chain finds its unique 3D-structure, represents one of the most intriguing hurdles in this rapidly growing field. In order to bypass this well-known protein-folding problem, some years ago we proposed the construction of non-native chain architectures with a high propensity for folding. According to this concept, termed TASP (template-assembled synthetic proteins), topological templates are used as a built-in device for directing covalently attached peptide blocks to a predetermined packing arrangement, resulting in branched chain architectures. Recent progress in the synthetic methodology for assembling peptides now allows us to access the full potential of the TASP concept. In this article, we discuss the state of the art of template-based protein de novo design, with special emphasis on progress in peptide synthesis and template design and show that some fundamental questions in protein assembly, structure and function can be approached by designing protein mimetics of reduced structural and functional complexity.