Beate Koksch

Multivalency as a Chemical Organization and Action Principle

Multivalent interactions can be applied universally for a targeted strengthening of an interaction between different interfaces or molecules. The binding partners form cooperative, multiple receptor-ligand interactions that are based on individually weak, noncovalent bonds and are thus generally reversible. Hence, multi- and polyvalent interactions play a decisive role in biological systems for recognition, adhesion, and signal processes. The scientific and practical realization of this principle will be demonstrated by the development of simple artificial and theoretical models, from natural systems to functional, application-oriented systems. In a systematic review of scaffold architectures, the underlying effects and control options will be demonstrated, and suggestions will be given for designing effective multivalent binding systems, as well as for polyvalent therapeutics.

Synthetic strategies to alpha-trifluoromethyl and alpha-difluoromethyl substituted alpha-amino acids.

The combination of the unique physical and chemical properties of fluorine with proteinogenic amino acids represents a new approach to the design of biologically active compounds including peptides with improved pharmacological parameters. Therefore, the development of routine synthetic methods which enable the effective and selective introduction of fluorine into the desired amino acids from readily available starting materials is of significant synthetic importance. The scope of this critical review is to summarize the most frequently employed strategies for the synthesis of alpha-difluoromethyl and alpha-trifluoromethyl substituted alpha-amino acids (114 references).

Nanoscale imaging reveals laterally expanding antimicrobial pores in lipid bilayers

Antimicrobial peptides are postulated to disrupt microbial phospholipid membranes. The prevailing molecular model is based on the formation of stable or transient pores although the direct observation of the fundamental processes is lacking. By combining rational peptide design with topographical (atomic force microscopy) and chemical (nanoscale secondary ion mass spectrometry) imaging on the same samples, we show that pores formed by antimicrobial peptides in supported lipid bilayers are not necessarily limited to a particular diameter, nor they are transient, but can expand laterally at the nano-to-micrometer scale to the point of complete membrane disintegration. The results offer a mechanistic basis for membrane poration as a generic physicochemical process of cooperative and continuous peptide recruitment in the available phospholipid matrix.

Amide-I and -II Vibrations of the Cyclic β-Sheet Model Peptide Gramicidin S in the Gas Phase

In the condensed phase, the peptide gramicidin S is often considered as a model system for a beta-sheet structure. Here, we investigate gramicidin S free of any influences of the environment by measuring the mid-IR spectra of doubly protonated (deuterated) gramicidin S in the gas phase. In the amide I (i.e., C=O stretch) region, the spectra show a broad split peak between 1580 and 1720 cm(-1). To deduce structural information, the conformational space has been searched using molecular dynamics methods and several structural candidates have been further investigated at the density functional level. The calculations show the importance of the interactions of the charged side-chains with the backbone, which is responsible for the lower frequency part of the amide I peak. When this interaction is inhibited via complexation with two 18-crown-6 molecules, the amide I peak narrows and shows two maxima at 1653 and 1680 cm(-1). A comparison to calculations shows that for this complexed ion, four C=O groups are in an antiparallel beta-sheet arrangement. Surprisingly, an analysis of the calculated spectra shows that these beta-sheet C=O groups give rise to the vibrations near 1680 cm(-1). This is in sharp contrast to expectations based on values for the condensed phase, where resonances of beta-sheet sections are thought to occur near 1630 cm(-1). The difference between those values might be caused by interactions with the environment, as the condensed phase value is mostly deduced for beta-sheet sections that are embedded in larger proteins, that interact strongly with solvent or that are part of partially aggregated species.

How Metal Ions Affect Amyloid Formation: Cu2+‐ and Zn2+‐Sensitive Peptides

The common feature of proteins involved in many neurodegenerative diseases is their ability to adopt at least two different stable conformations. The conformational transition that shifts the equilibrium from the functional, mostly partially α‐helical structure, to the β‐sheet rich amyloid can be triggered by numerous factors, such as mutations in the primary structure or changes in the environment. We present a set of model peptides that, without changes in their primary structure, react in a predictable fashion in the presence of transition metal ions by adopting different conformations and aggregate morphologies. These de novo designed peptides strictly follow the characteristic heptad repeat of the α‐helical coiled‐coil structural motif. Furthermore, domains that favor β‐sheet formation have been incorporated to make the system prone to amyloid formation. As a third feature, histidine residues create sensitivity towards the presence of transition metal ions. CD spectroscopy, ThT fluorescence experiments, and transmission electron microscopy were used to characterize peptide conformation and aggregate morphology in the presence of Cu2+ and Zn2+. Furthermore, the binding geometry within peptide–Cu2+ complexes was characterized by electron paramagnetic resonance spectroscopy.

Chemical Labeling Strategy with (R)- and (S)-Trifluoromethylalanine for Solid State 19F NMR Analysis of Peptaibols in Membranes

Substitution of a single Aib-residue in a peptaibol with (R)- and (S)-trifluoromethylalanine yields two local orientational constraints theta by solid state (19)F NMR. The structure of the membrane-perturbing antibiotic alamethicin in DMPC bilayers was analyzed in terms of two angles tau and rho from six such constraints, showing that the N-terminus (up to a kink at Pro14) is folded as an alpha-helix, tilted away from the membrane normal by 8 degrees, and assembled as an oligomer. The new (19)F NMR label CF(3)-Ala has thus been demonstrated to be highly sensitive, virtually unperturbing, and ideally suited to characterize peptaibols in membranes.

Nanoparticle‐Induced Folding and Fibril Formation of Coiled‐Coil‐Based Model Peptides

Nanomedicine is a rapidly growing field that has the potential to deliver treatments for many illnesses. However, relatively little is known about the biological risks of nanoparticles. Some studies have shown that nanoparticles can have an impact on the aggregation properties of proteins, including fibril formation. Moreover, these studies also show that the capacity of nanoscale objects to induce or prevent misfolding of the proteins strongly depends on the primary structure of the protein. Herein, light is shed on the role of the peptide primary structure in directing nanoparticle-induced misfolding by means of two model peptides. The design of these peptides is based on the alpha-helical coiled-coil folding motif, but also includes features that enable them to respond to pH changes, thus allowing pH-dependent beta-sheet formation. Previous studies showed that the two peptides differ in the pH range required for beta-sheet folding. Time-dependent circular dichroism spectroscopy and transmission electron microscopy are used to characterize peptide folding and aggregate morphology in the presence of negatively charged gold nanoparticles (AuNPs). Both peptides are found to undergo nanoparticle-induced fibril formation. The determination of binding parameters by isothermal titration calorimetry further reveals that the different propensities of both peptides to form amyloid-like structures in the presence of AuNPs is primarily due to the binding stoichiometry to the AuNPs. Modification of one of the peptide sequences shows that AuNP-induced beta-sheet formation is related to the structural propensity of the primary structure and is not a generic feature of peptide sequences with a sufficiently high binding stoichiometry to the nanoparticles.

Highly dynamic motion of crown ethers along oligolysine peptide chains

Molecular mobility has attracted considerable attention in supramolecular chemistry and biochemistry, but the simple question of whether a small molecule can move directly between different binding sites of a multitopic host without intermediate dissociation has not been addressed so far. To study such processes, we consider hydrogen/deuterium exchange experiments on a model system comprising complexes formed between 18-crown-6 and oligolysine peptides. Because direct binding-site hopping is indistinguishable in solution from a dissociation/reassociation mechanism, here we show that the high vacuum of a mass spectrometer offers a unique environment for probing such processes. The highly dynamic motion of crown ethers along oligolysine peptide chains proceeds mechanistically by a simultaneous transfer of the crown ether from its ammonium ion binding site to a nearby amino group together with a proton. Furthermore, the exchange experiments unambiguously reveal the zwitterionic structure of the 18-crown-6/oligolysine complexes, highlighting the versatility and potential of gas-phase experiments for investigating non-covalent interactions.

Following polypeptide folding and assembly with conformational switches.

Conformational transitions are a crucial factor in the vast majority of protein misfolding diseases. In most of these cases, the change in conformation is accompanied by the formation of insoluble aggregates, which often precludes a detailed characterization at the molecular level. Therefore, much effort has been put into the development of simplified, easy-to-synthesize peptide models that can be used to elucidate the molecular processes that underlie the conformational switch. For a design to be successful, two, sometimes concomitantly fulfilled, requirements are of importance. First, it is essential to create inherent structural ambiguity. This is usually achieved by combining the most prominent characteristics of different folds within a consensus sequence. Second, a stimulus-sensitive functionality that responds to alterations in the environment, such as pH, ionic strength or the presence of metal ions, is often needed to control structural conversion and to shift the equilibrium in either direction.

Position-dependent effects of fluorinated amino acids on the hydrophobic core formation of a heterodimeric coiled coil.

Systematic model investigations of the molecular interactions of fluorinated amino acids within native protein environments substantially improve our understanding of the unique properties of these building blocks. A rationally designed heterodimeric coiled coil peptide (VPE/VPK) and nine variants containing amino acids with variable fluorine content in either position a16 or d19 within the hydrophobic core were synthesized and used to evaluate the impact of fluorinated amino acid substitutions within different hydrophobic protein microenvironments. The structural and thermodynamic stability of the dimers were examined by applying both experimental (CD spectroscopy, FRET, and analytical ultracentrifugation) and theoretical (MD simulations and MM-PBSA free energy calculations) methods. The coiled coil environment imposes position-dependent conformations onto the fluorinated side chains and thus affects their packing and relative orientation towards their native interaction partners. We find evidence that such packing effects exert a significant influence on the contribution of fluorine-induced polarity to coiled coil folding.