Four Models for Peptide Engineering

Peptide and protein dysfunction are a major aspect for many diseases, but also various peptide based drugs and innovative biomaterials are highly esteemed. Many studies concentrate on peptides due to their versatility, their specificity on the other hand and the complex structural patterns. After cancer, the so called amyloidosis are the second largest research field in the globalized world. The misfolding of functional peptides and proteins is one intrinsic pathologic pattern for many diseases like Alzheimer’s and Parkinson’s disease or diabetes type two. On the other hand uses nature the highly stable and homogenous morphology of amyloids. One famous example is spider silk, but also hormones in the endocrine system are stored in amyloid-like structures. In addition, a range of studies concentrate on applications which are based on functional amyloids. Nevertheless, the collective disadvantages of natural amyloid prone peptides are the difficult isolation and handling. Hence, model peptides with defined design can be used to address certain questions and analytical approaches. In this manuscript presented are two different kinds of amyloid-forming model peptides. The first one is a de novo designed model peptide with a recognition motif for enzymatic phosphorylation. The goal is to understand the impact of the phosphate group and control the phosphorylation process. The second peptide is the critical hydrophobic sequence part of the diabetes type two derived peptide IAPP. With the help of the short peptide sequence, the structure of oligomers during the initial growing phase and the following processes of amyloid formation can be investigated. Thus, insights into so far sparsely examined beginnings of the pathologic event can be described. To transfer the knowledge on model peptides for in vivo studies, a light sensitive caging linker was designed. The optimized synthesis method for the, on natural flavour-based, coumarin linker and the caging process of different model peptides is described in detail. One major problem for peptide applications in vivo is the low bioavailability due to proteolytic digestion. Thus, aim of the fourth project is an idealized protease substrate peptide to study the impact of fluorinated side chains on proteolytic stability. Here presented results are part of a series of comparable studies regarding different kinds of side chain fluorination. Objective is to understand which requirements have to be addressed to inhibit proteolytic digestion of a peptide. As a result, a data bank with this knowledge will be generated as a toolbox for peptide design. One example is the design of metabolic resistant peptide drugs

Inhibition of peptide aggregation by means of enzymatic phosphorylation

As is the case in numerous natural processes, enzymatic phosphorylation can be used in the laboratory to influence the conformational populations of proteins. In nature, this information is used for signal transduction or energy transfer, but has also been shown to play an important role in many diseases like tauopathies or diabetes. With the goal of determining the effect of phosphorylation on amyloid fibril formation, we designed a model peptide which combines structural characteristics of α-helical coiled-coils and β-sheets in one sequence. This peptide undergoes a conformational transition from soluble structures into insoluble amyloid fibrils over time and under physiological conditions and contains a recognition motif for PKA (cAMP- dependent protein kinase) that enables enzymatic phosphorylation. We have analyzed the pathway of amyloid formation and the influence of enzymatic phosphorylation on the different states along the conformational transition from random-coil to β-sheet-rich oligomers to protofilaments and on to insoluble amyloid fibrils, and we found a remarkable directing effect from β -sheet-rich structures to unfolded structures in the initial growth phase, in which small oligomers and protofilaments prevail if the peptide is phosphorylated

The protofilament architecture of a de novo designed coiled coil-based amyloidogenic peptide

International audienceAmyloid fibrils are polymers formed by proteins under specific conditions and in many cases they are related to pathogenesis, such as Parkinson's and Alzheimer's diseases. Their hallmark is the presence of a β-sheet structure. High resolution structural data on these systems as well as information gathered from multiple complementary analytical techniques is needed, from both a fundamental and a pharmaceutical perspective. Here, a previously reported de novo designed, pH-switchable coiled coil-based peptide that undergoes structural transitions resulting in fibril formation under physiological conditions has been exhaustively characterized by transmission electron microscopy (TEM), cryo-TEM, atomic force microscopy (AFM), wide-angle X-ray scattering (WAXS) and solid-state NMR (ssNMR). Overall, a unique 2-dimensional carpet-like assembly composed of large coexisiting ribbon-like, tubular and funnel-like structures with a clearly resolved protofilament substructure is observed. Whereas electron microscopy and scattering data point somewhat more to a hairpin model of β-fibrils, ssNMR data obtained from samples with selectively labelled peptides are in agreement with both, hairpin structures and linear arrangements

Position-dependent impact of hexafluoroleucine and trifluoroisoleucine on protease digestion

Rapid digestion by proteases limits the application of peptides as therapeutics. One strategy to increase the proteolytic stability of peptides is the modification with fluorinated amino acids. This study presents a systematic investigation of the effects of fluorinated leucine and isoleucine derivatives on the proteolytic stability of a peptide that was designed to comprise substrate specificities of different proteases. Therefore, leucine, isoleucine, and their side-chain fluorinated variants were site-specifically incorporated at different positions of this peptide resulting in a library of 13 distinct peptides. The stability of these peptides towards proteolysis by α-chymotrypsin, pepsin, proteinase K, and elastase was studied, and this process was followed by an FL-RP-HPLC assay in combination with mass spectrometry. In a few cases, we observed an exceptional increase in proteolytic stability upon introduction of the fluorine substituents. The opposite phenomenon was observed in other cases, and this may be explained by specific interactions of fluorinated residues with the respective enzyme binding sites. Noteworthy is that 5,5,5-trifluoroisoleucine is able to significantly protect peptides from proteolysis by all enzymes included in this study when positioned N-terminal to the cleavage site. These results provide valuable information for the application of fluorinated amino acids in the design of proteolytically stable peptide-based pharmaceuticals