Improving cryo-EM grids for amyloid fibrils using interface-active solutions and spectator proteins.

Preparation of cryoelectron microscopy (cryo-EM) grids for imaging of amyloid fibrils is notoriously challenging. The human islet amyloid polypeptide (hIAPP) serves as a notable example, as the majority of reported structures have relied on the use of nonphysiological pH buffers, N-terminal tags, and seeding. This highlights the need for more efficient, reproducible methodologies that can elucidate amyloid fibril structures formed under diverse conditions. In this work, we demonstrate that the distribution of fibrils on cryo-EM grids is predominantly determined by the solution composition, which is critical for the stability of thin vitreous ice films. We discover that, among physiological pH buffers, HEPES uniquely enhances the distribution of fibrils on cryo-EM grids and improves the stability of ice layers. This improvement is attributed to direct interactions between HEPES molecules and hIAPP, effectively minimizing the tendency of hIAPP to form dense clusters in solutions and preventing ice nucleation. Furthermore, we provide additional support for the idea that denatured protein monolayers forming at the interface are also capable of eliciting a surfactant-like effect, leading to improved particle coverage. This phenomenon is illustrated by the addition of nonamyloidogenic rat IAPP (rIAPP) to a solution of preaggregated hIAPP just before the freezing process. The resultant grids, supplemented with this "spectator protein", exhibit notably enhanced coverage and improved ice quality. Unlike conventional surfactants, rIAPP is additionally capable of disentangling the dense clusters formed by hIAPP. By applying the proposed strategies, we have resolved the structure of the dominant hIAPP polymorph, formed in vitro at pH 7.4, to a final resolution of 4 Å. The advances in grid preparation presented in this work hold significant promise for enabling structural determination of amyloid proteins which are particularly resistant to conventional grid preparation techniques.

Mimicking Nonribosomal Peptides from the Marine Actinomycete Streptomyces sp. H-KF8 Leads to Antimicrobial Peptides.

Microorganisms within the marine environment have been shown to be very effective sources of naturally produced antimicrobial peptides (AMPs). Several nonribosomal peptides were identified based on genome mining predictions of Streptomyces sp. H-KF8, a marine Actinomycetota isolated from a remote Northern Chilean Patagonian fjord. Based on these predictions, a series of eight peptides, including cyclic peptides, were designed and chemically synthesized. Six of these peptides showed antimicrobial activity. Mode of action studies suggest that two of these peptides potentially act on the cell membrane via a novel mechanism allowing the passage of small ions, resulting in the dissipation of the membrane potential. This study shows that though structurally similar peptides, determined by NMR spectroscopy, the incorporation of small sequence mutations results in a dramatic influence on their bioactivity including mode of action. The qualified hit sequence can serve as a basis for more potent AMPs in future studies.

Ionic Liquid-Mediated Approach for the Synthesis of Site-Specific Thioether Conjugates.

Site-specific conjugation approaches are of great importance in drug discovery, notably for the synthesis of biochemical probes or molecular conjugates for targeted delivery. Herein, we report a mild ionic liquid (IL)-mediated thiolation technique that relies on the use of 1,3-ethyl-methyl imidazolium acetate, [C2 mim][OAc] as a solvent and precursor to generate activated IL, as well as a solvent for the conjugation reaction. First, a focused library of active ILs was prepared for functionalizing/conjugating cysteine-containing small molecules and unprotected peptides. Interestingly, a bifunctional active IL could also be successfully employed as a linker for the conjugation of peptides lacking Cys. This study sets the ground for further investigation of the use of active ILs for modifying, labeling or conjugating larger and more complex therapeutic modalities such as proteins and antibodies.

Ultrasensitive and Selective Copper(II) Detection: Introducing a Bioinspired and Robust Sensor.

A nanopore-based CuII -sensing system is reported that allows for an ultrasensitive and selective detection of CuII with the possibility for a broad range of applications, for example in medical diagnostics. A fluorescent ATCUN-like peptide 5/6-FAM-Dap-ß-Ala-His is employed to selectively bind CuII ions in the presence of NiII and ZnII and was crafted into ion track-etched nanopores. Upon CuII binding the fluorescence of the peptide sensor is quenched, permitting the detection of CuII in solution. The ion transport characteristics of peptide-modified nanopore are shown to be extremely sensitive and selective towards CuII allowing to sense femtomolar CuII concentrations in human urine mimics. Washing with EDTA fully restores the CuII -binding properties of the sensor, enabling multiple repetitive measurements. The robustness of the system clearly has the potential to be further developed into an easy-to-use, lab-on-chip CuII -sensing device, which will be of great importance for bedside diagnosis and monitor of CuII levels in patients with copper-dysfunctional homeostasis.

Structural and Dynamical Basis of G Protein Inhibition by YM-254890 and FR900359: An Inhibitor in Action.

Specific inhibition of G proteins holds a great pharmacological promise to, e.g., target oncogenic Gq/11 proteins and can be achieved by the two natural products FR900359 (FR) and YM-254890 (YM). Unfortunately, recent rational-design-based approaches to address G proteins other than Gq/11/14 subtypes were not successful mainly due to the conformational complexity of these new modalities-like compounds. Here, we report the water-derived NMR structure of YM, which strongly differs from the conformation of Gq-bound YM as found in the crystal structure. Reanalysis of the crystal structure suggests that the water-derived NMR structure of YM also represents a valid solution of the electron density. Extensive molecular dynamic simulations unveiled much higher binding affinities of the water-derived NMR structure compared to the original YM conformation of pdb 3ah8 . Employing a in-silico-designed, fast activating G protein conformation molecular dynamics data ultimately show how the inhibitor impairs the domain motion of the G protein necessary to hinder nucleotide exchange.

NiII Complex Formation and Protonation States at the Active Site of a Nickel Superoxide Dismutase-Derived Metallopeptide: Implications for the Mechanism of Superoxide Degradation.

A small, catalytically active metallopeptide (Nim6 SOD, m6 SOD=ACDLAC), which was derived from the nickel superoxide dismutase (NiSOD) active site was employed to study the mechanism of superoxide degradation, especially focusing on the protonation states of the NiII donor atoms, the proton source, and the role of the N-terminal proton(s). Therefore, the NiII -metallopeptide was studied at various pHs and temperatures using UV/Vis and NMR spectroscopy. These studies indicate a strong reduction of the pKa of the NiII -ligating donor atoms, resulting in a fully deprotonated NiII active-site environment. Furthermore, no titratable proton could be observed within a pH ranging from 6.5 to 10.5. This rules out a recently discussed adiabatic proton tunneling-like hydrogen-atom transfer process for the metallopeptides, not found in the native enzyme. Furthermore, variable-temperature 1 H NMR measurements uncovered an extended hydrogen-bond network within the NiII active site of the metallopeptide similar to the enzyme. With respect to the deprotonated NiII active site, the residual N-terminal proton, which is a prerequisite for catalytic activity, cannot act as proton source. Most likely, it stabilizes the NiII -coordinated substrate in an end-on fashion, thus allowing for an inner-sphere electron transfer. Lastly, and unlike the enzyme, the catalytic rate constant of superoxide degradation by the metallopeptides was determined to be strongly pH dependent, suggesting bulk water to be directly involved in proton donation, which in turn strongly suggests the N-terminal histidine to be the respective proton donor in the enzyme.

Conformational µ-Conotoxin PIIIA Isomers Revisited: Impact of Cysteine Pairing on Disulfide-Bond Assignment and Structure Elucidation.

Peptides and proteins carrying high numbers of cysteines can adopt various 3D structures depending on their disulfide connectivities. The unambiguous verification of such conformational isomers with more than two disulfide bonds is extremely challenging, and experimental strategies for their unequivocal structural analysis are largely lacking. We synthesized all 15 possible isomers of the 22mer conopeptide µ-PIIIA and applied 2D NMR spectroscopy and MS/MS for the elucidation of its structure. This study provides intriguing insights in how the disulfide connectivity alters the global fold of a toxin. We also show that analysis procedures involving comprehensive combinations of conventional methods are required for the unambiguous assignment of disulfides in cysteine-rich peptides and proteins and that standard compounds are crucially needed for the structural analysis of such complex molecules.

Reactions of Sulfur-Containing Organic Compounds and Peptides in 1-Ethyl-3-methyl-imidazolium Acetate.

The neat ionic liquid (IL) [C2mim][OAc] is not just capable of dissolving thiol- and disulfide-containing compounds, but is able to chemically react with them without addition of any catalytic reagent. Through the analysis of four small organic molecules and a cysteine-containing peptide we could postulate a general reaction mechanism. Here, the imidazolium-carbenes preferentially react with the disulfide bond, but not thiol group. Moreover, the imidazole moiety was found to abstract the sulfur atom from the cysteine residue, providing an alternative way to transform Cys residues, which were artificially inserted into a peptide sequence in order to perform native chemical ligation (NCL) of two peptide fragments. Finally, the chemical reaction of [C2mim][OAc] with a cysteine-containing biomolecules can be tuned or even suppressed through the addition of at least 30% of water to the reaction mixture.

Subtype-specific block of voltage-gated K+ channels by µ-conopeptides.

The neurotoxic cone snail peptide µ-GIIIA specifically blocks skeletal muscle voltage-gated sodium (NaV1.4) channels. The related conopeptides µ-PIIIA and µ-SIIIA, however, exhibit a wider activity spectrum by also inhibiting the neuronal NaV channels NaV1.2 and NaV1.7. Here we demonstrate that those µ-conopeptides with a broader target range also antagonize select subtypes of voltage-gated potassium channels of the KV1 family: µ-PIIIA and µ-SIIIA inhibited KV1.1 and KV1.6 channels in the nanomolar range, while being inactive on subtypes KV1.2-1.5 and KV2.1. Construction and electrophysiological evaluation of chimeras between KV1.5 and KV1.6 revealed that these toxins block KV channels involving their pore regions; the subtype specificity is determined in part by the sequence close to the selectivity filter but predominantly by the so-called turret domain, i.e. the extracellular loop connecting the pore with transmembrane segment S5. Conopeptides µ-SIIIA and µ-PIIIA, thus, are not specific for NaV channels, and the known structure of some KV channel subtypes may provide access to structural insight into the molecular interaction between µ-conopeptides and their target channels.

Application of room-temperature aprotic and protic ionic liquids for oxidative folding of cysteine-rich peptides.

The oxidation of the conotoxin µ-SIIIA in different ionic liquids was investigated, and the results were compared with those obtained in [C2 mim][OAc]. Conversion of the reduced precursor into the oxidized product was observed in the protic ILs methyl- and ethylammonium formate (MAF and EAf, respectively), whereas choline dihydrogenphosphate and Ammoeng 110 failed to yield folded peptide. However, the quality and yield of the peptide obtained in MAF and EAF were lower than in the case of the product from [C2 mim][OAc]. Reaction conditions (temperature, water content) also had an impact on peptide conversion. A closer look at the activities of µ-SIIIA versions derived from an up-scaled synthesis in [C2 mim][OAc] revealed a significant loss of the effect on ion channel NaV 1.4 relative to the buffer-oxidized peptide, whereas digestion of either µ-SIIIA product by trypsin was unaffected. This was attributed to adherence of ions from the IL to the peptide, because the disulfide connectivity is basically the same for the differentially oxidized µ-SIIIA versions.

On the nature of interactions between ionic liquids and small amino-acid-based biomolecules.

During the last decade, ionic liquids (ILs) have revealed promising properties and applications in many research fields, including biotechnology and biological sciences. The focus of this contribution is to give a critical review of the phenomena observed and current knowledge of the interactions occurring on a molecular basis. As opposed to the huge advances made in understanding the properties of proteins in ILs, complementary investigations dealing with interactions between ILs and peptides or oligopeptides are underrepresented and are mostly only of phenomenological nature. However, the field has received more attention in the last few years. This Review features a meta-analysis of the available data and findings and should, therefore, provide a basis for a scientifically profound understanding of the nature and mechanisms of interactions between ILs and structured or nonstructured peptides. Fundamental aspects of the interactions between different peptides/oligopeptides and ILs are complemented by sections on the experimental (spectroscopy, structural biology) and theoretical (computational chemistry) possibilities to explain the phenomena reported so far in the literature. In effect, this should lead to the development of novel applications and support the understanding of IL-solute interactions in general.

Ionic liquid applications in peptide chemistry: synthesis, purification and analytical characterization processes.

This review aims to provide a comprehensive overview of the recent advances made in the field of ionic liquids in peptide chemistry and peptide analytics.