Do Ionic Liquids Exhibit the Required Characteristics to Dissolve, Extract, Stabilize, and Purify Proteins? Past-Present-Future Assessment.

Proteins are highly labile molecules, thus requiring the presence of appropriate solvents and excipients in their liquid milieu to keep their stability and biological activity. In this field, ionic liquids (ILs) have gained momentum in the past years, with a relevant number of works reporting their successful use to dissolve, stabilize, extract, and purify proteins. Different approaches in protein-IL systems have been reported, namely, proteins dissolved in (i) neat ILs, (ii) ILs as co-solvents, (iii) ILs as adjuvants, (iv) ILs as surfactants, (v) ILs as phase-forming components of aqueous biphasic systems, and (vi) IL-polymer-protein/peptide conjugates. Herein, we critically analyze the works published to date and provide a comprehensive understanding of the IL-protein interactions affecting the stability, conformational alteration, unfolding, misfolding, and refolding of proteins while providing directions for future studies in view of imminent applications. Overall, it has been found that the stability or purification of proteins by ILs is bispecific and depends on the structure of both the IL and the protein. The most promising IL-protein systems are identified, which is valuable when foreseeing market applications of ILs, e.g., in "protein packaging" and "detergent applications". Future directions and other possibilities of IL-protein systems in light-harvesting and biotechnology/biomedical applications are discussed.

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.

The Complete Assessment of Small Molecule and Peptidomimetic Inhibitors of Sortase A Towards Antivirulence Treatment.

This review covers the most recent advances in the development of inhibitors for the bacterial enzyme sortase A (SrtA). Sortase A (SrtA) is a critical virulence factor, present ubiquitously in Gram-positive bacteria of which many are pathogenic. Sortases are key enzymes regulating bacterial adherence to host cells, by anchoring extracellular matrix-binding proteins to the bacterial outer cell wall. By targeting virulence factors, effective treatment can be achieved, without inducing antibiotic resistance to the treatment. This is a potentially more sustainable, long-term approach to treating bacterial infections, including ones that display multiple resistance to current therapeutics. There are many promising approaches available for SrtA inhibition, some of which have the potential to advance into further clinical development, with peptidomimetic and in vivo active small molecules being among the most promising. There are currently no approved drugs on the market targeting SrtA, despite its promise, adding to the relevance of this review article, as it extends to the pharmaceutical industry additionally to academic researchers.

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.

CA10 regulates neurexin heparan sulfate addition via a direct binding in the secretory pathway.

Neurexins are presynaptic adhesion molecules that shape the molecular composition of synapses. Diversification of neurexins in numerous isoforms is believed to confer synapse-specific properties by engaging with distinct ligands. For example, a subset of neurexin molecules carry a heparan sulfate (HS) glycosaminoglycan that controls ligand binding, but how this post-translational modification is controlled is not known. Here, we observe that CA10, a ligand to neurexin in the secretory pathway, regulates neurexin-HS formation. CA10 is exclusively found on non-HS neurexin and CA10 expressed in neurons is sufficient to suppress HS addition and attenuate ligand binding and synapse formation induced by ligands known to recruit HS. This effect is mediated by a direct interaction in the secretory pathway that blocks the primary step of HS biosynthesis: xylosylation of the serine residue. NMR reveals that CA10 engages residues on either side of the serine that can be HS-modified, suggesting that CA10 sterically blocks xylosyltransferase access in Golgi. These results suggest a mechanism for the regulation of HS on neurexins and exemplify a new mechanism to regulate site-specific glycosylations.

Native Chemical Ligation of Highly Hydrophobic Peptides in Ionic Liquid-Containing Media.

The chemical synthesis of a highly hydrophobic membrane-associated peptide by native chemical ligation (NCL) in an ionic liquid (IL) [C2mim][OAc]/buffer mixture was achieved by employing peptide concentrations up to 11 mM. NCL was studied at different pH and water content and compared to several "gold-standard" ligation protocols. The optimized reaction protocol for the NCL in IL required the addition of 40% water and pH adjustment to 7.0-7.5, resulting in ligation yields of up to 80-95% within 1 to 4 h. This new ligation protocol is generally applicable and outperforms current "gold-standard" NCL methods.

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.

In Silico Analysis of the Subtype Selective Blockage of KCNA Ion Channels through the µ-Conotoxins PIIIA, SIIIA, and GIIIA.

Understanding subtype specific ion channel pore blockage by natural peptide-based toxins is crucial for developing such compounds into promising drug candidates. Herein, docking and molecular dynamics simulations were employed in order to understand the dynamics and binding states of the µ-conotoxins, PIIIA, SIIIA, and GIIIA, at the voltage-gated potassium channels of the KV1 family, and they were correlated with their experimental activities recently reported by Leipold et al. Their different activities can only adequately be understood when dynamic information about the toxin-channel systems is available. For all of the channel-bound toxins investigated herein, a certain conformational flexibility was observed during the molecular dynamic simulations, which corresponds to their bioactivity. Our data suggest a similar binding mode of µ-PIIIA at KV1.6 and KV1.1, in which a plethora of hydrogen bonds are formed by the Arg and Lys residues within the α-helical core region of µ-PIIIA, with the central pore residues of the channel. Furthermore, the contribution of the K+ channel's outer and inner pore loops with respect to the toxin binding. and how the subtype specificity is induced, were proposed.

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.

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.

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.

Enzymatic Activity of Xyloglucan Xylosyltransferase 5.

Xyloglucan, the most abundant hemicellulosic component of the primary cell wall of flowering plants, is composed of a ß-(1,4)-glucan backbone decorated with d-xylosyl residues. Three xyloglucan xylosyltransferases (XXTs) participate in xyloglucan biosynthesis in Arabidopsis (Arabidopsis thaliana). Two of these, XXT1 and XXT2, have been shown to be active in vitro, whereas the catalytic activity of XXT5 has yet to be demonstrated. By optimizing XXT2 expression in a prokaryotic system and in vitro activity assay conditions, we demonstrate that nonglycosylated XXT2 lacking its cytosolic amino-terminal and transmembrane domain displays high catalytic activity. Using this optimized procedure for the expression of XXT5, we report, to our knowledge for the first time, that recombinant XXT5 shows enzymatic activity in vitro, although at a significantly slower rate than XXT1 and XXT2. Kinetic analysis showed that XXT5 has a 7-fold higher Km and 9-fold lower kcat compared with XXT1 and XXT2. Activity assays using XXT5 in combination with XXT1 or XXT2 indicate that XXT5 is not specific for their products. In addition, mutagenesis experiments showed that the in vivo function and in vitro catalytic activity of XXT5 require the aspartate-serine-aspartate motif. These results demonstrate that XXT5 is a catalytically active xylosyltransferase involved in xylosylation of the xyloglucan backbone.

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.

A homology model of Xyloglucan Xylosyltransferase 2 reveals critical amino acids involved in substrate binding.

In dicotyledonous plants, xyloglucan (XyG) is the most abundant hemicellulose of the primary cell wall. The enzymes involved in XyG biosynthesis have been identified through reverse-genetics and activity was characterized by heterologous expression. Currently, there is no information on the atomic structures or amino acids involved in activity or substrate binding of any of the Golgi-localized XyG biosynthetic enzymes. A homology model of the xyloglucan xylosyltransferase 2 (XXT2) catalytic domain was built on the basis of the crystal structure of A64Rp. Molecular dynamics simulations revealed that the homology model retains the glycosyltransferase (GT)-A fold of the template structure used to build the homology model indicating that XXT2 likely has a GT-A fold. According to the XXT2 homology model, six amino acids (Phe204, Lys207, Asp228, Ser229, Asp230, His378) were selected and their contribution in catalytic activity was investigated. Site-directed mutagenesis studies show that Asp228, Asp230 and His378 are critical for XXT2 activity and are predicted to be involved in coordination of manganese ion. Lys207 was also found to be critical for protein activity and the homology model indicates a critical role in substrate binding. Additionally, Phe204 mutants have less of an impact on XXT2 activity with the largest effect when replaced with a polar residue. This is the first study that investigates the amino acids involved in substrate binding of the XyG-synthesizing xylosyltransferases and contributes to the understanding of the mechanisms of polysaccharide-synthesizing GTs and XyG biosynthesis.

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.

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.

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.

Substrate-derived Sortase A inhibitors: targeting an essential virulence factor of Gram-positive pathogenic bacteria.

The bacterial transpeptidase Sortase A (SrtA) is a surface enzyme of Gram-positive pathogenic bacteria. It has been shown to be an essential virulence factor for the establishment of various bacterial infections, including septic arthritis. However, the development of potent Sortase A inhibitors remains an unmet challenge. Sortase A relies on a five amino acid sorting signal (LPXTG), by which it recognizes its natural target. We report the synthesis of a series of peptidomimetic inhibitors of Sortase A based on the sorting signal, supported by computational binding analysis. By employing a FRET-compatible substrate, our inhibitors were assayed in vitro. Among our panel, we identified several promising inhibitors with IC50 values below 200 µM, with our strongest inhibitor - LPRDSar - having an IC50 of 18.9 µM. Furthermore, it was discovered that three of our compounds show an effect on growth and biofilm inhibition of pathogenic Staphylococcus aureus, with the inclusion of a phenyl ring seemingly key to this effect. The most promising compound in our panel, BzLPRDSar, could inhibit biofilm formation at concentrations as low as 32 µg mL-1, manifesting it as a potential future drug lead. This could lead to treatments for MRSA infections in clinics and diseases such as septic arthritis, which has been directly linked with SrtA.

Protein cohabitation: long-term immunoglobulin G storage at room temperature.

Long-term functional storage of therapeutic proteins at room temperature has been an eternal challenge. Inspired by the cellular cooperativity of proteins, we have taken a step forward to address this challenge by cohabitating Immunoglobulin G (IgG1) with a food protein gelatin in the solid-state at room temperature. Interestingly, IgG1 remained functionally active for a record 14 months revealed from the western-blot assay. Further quantification by HP-LC analysis showed 100% structural integrity of IgG1 with no degradation in the gelatin matrix during this period. The developed formulation has a direct application in oral medical nutrition therapy to cure gastrointestinal microbial infections. Also the strategy provides a robust energy economic alternative to the protein engineering methods for long-term functional storage of therapeutic proteins at room temperature.