Unveiling the Oxazolidine Character of Pseudoproline Derivatives by Automated Flow Peptide Chemistry.

Pseudoproline derivatives such as Thr(ΨPro)-OH are commonly used in peptide synthesis to reduce the likelihood of peptide aggregation and to prevent aspartimide (Asi) formation during the synthesis process. In this study, we investigate notable by-products such as aspartimide formation and an imine derivative of the Thr(ΨPro) moiety observed in flow peptide chemistry synthesis. To gain insight into the formation of these unexpected by-products, we design a series of experiments. Furthermore, we demonstrate the oxazolidine character of the pseudoproline moiety and provide plausible mechanisms for the two-way ring opening of oxazolidine leading to these by-products. In addition, we present evidence that Asi formation appears to be catalyzed by the presence of the pseudoproline moiety. These observed side reactions are attributed to elevated temperature and pressure; therefore, caution is advised when using ΨPro derivatives under such harsh conditions. In addition, we propose a solution whereby thermodynamically controlled Asi formation can be kinetically prevented.

D27-LIKE1 isomerase has a preference towards trans/cis and cis/cis conversions of carotenoids in Arabidopsis.

Carotenoids contribute to a variety of physiological processes in plants, functioning also as biosynthesis precursors of ABA and strigolactones (SLs). SL biosynthesis starts with the enzymatic conversion of all-trans-ß-carotene to 9-cis-ß-carotene by the DWARF27 (D27) isomerase. In Arabidopsis, D27 has two closely related paralogs, D27-LIKE1 and D27-LIKE2, which were predicted to be ß-carotene-isomerases. In the present study, we characterised D27-LIKE1 and identified some key aspects of its physiological and enzymatic functions in Arabidopsis. d27-like1-1 mutant does not display any strigolactone-deficient traits and exhibits a substantially higher 9-cis-violaxanthin content, which is accompanied by a slightly higher ABA level. In vitro feeding assays with recombinant D27-LIKE1 revealed that the protein exhibits affinity to all ß-carotene isoforms but with an exclusive preference towards trans/cis conversions and the interconversion between 9-cis, 13-cis and 15-cis-ß-carotene forms, and accepts zeaxanthin and violaxanthin as substrates. Finally, we present evidence showing that D27-LIKE1 mRNA is phloem mobile and D27-LIKE1 is an ancient isomerase with a long evolutionary history. In summary, we demonstrate that D27-LIKE1 is a carotenoid isomerase with multi-substrate specificity and has a characteristic preference towards the catalysation of cis/cis interconversion of carotenoids. Therefore, D27-LIKE1 is a potential regulator of carotenoid cis pools and, eventually, SL and ABA biosynthesis pathways.

Acetyl group for proper protection of ß-sugar-amino acids used in SPPS.

The synthesis of D-glucosamine-1-carboxylic acid based ß-sugar amino acids (ß-SAAs) is typically performed in nine consecutive steps via an inefficient OAc → Br → CN conversion protocol with low overall yield. Here, we present the improved and more efficient synthesis of both Fmoc-GlcAPC-OH and Fmoc-GlcAPC(Ac)-OH, ß-SAAs consisting of only 4-5 synthetic steps. Their active ester and amide bond formation with glycine methyl ester (H-Gly-OMe) was completed and monitored by 1H NMR. The stability of the pyranoid OHs protecting the acetyl groups was investigated under three different Fmoc cleavage conditions and was found to be satisfactory even at high piperidine concentration (e.g. 40%). We designed a SPPS protocol using Fmoc-GlcAPC(Ac)-OH to produce model peptides Gly-ß-SAA-Gly as well as Gly-ß-SAA-ß-SAA-Gly with high coupling efficiency. The products were deacetylated using the Zemplén method, which allows the hydrophilicity of a building block and/or chimera to be fine-tuned, even after the polypeptide chain has already been synthesized.

Amide isomerization pathways: Electronic and structural background of protonation- and deprotonation-mediated cis-trans interconversions.

The cis-trans isomerization of amide bonds leads to wide range of structural and functional changes in proteins and can easily be the rate-limiting step in folding. The trans isomer is thermodynamically more stable than the cis, nevertheless the cis form can play a role in biopolymers' function. The molecular system of N-methylacetamide · 2H2O is complex enough to reveal energetics of the cis-trans isomerization at coupled cluster single-double and coupled cluster single-double and perturbative triple [CCSD(T)] levels of theory. The cis-trans isomerization cannot be oversimplified by a rotation along ω, since this rotation is coupled with the N-atom pyramidal inversion, requesting the introduction of a second dihedral angle "α." Full f(ω,α) potential energy surfaces of the different amide protonation states, critical points and isomerization reaction paths were determined, and the barriers of the neutral, O-protonated and N-deprotonated amides were found too high to allow cis-trans interconversion at room temperature: ∼85, ∼140, and ∼110 kJ mol-1, respectively. For the N-protonated amide bond, the cis form (ω = 0°) is a maximum rather than a minimum, and each ω state is accessible for less than ∼10 kJ mol-1. Here we outline a cis-trans isomerization pathway with a previously undescribed low energy transition state, which suggests that the proton is transferred from the more favorable O- to the N-protonation site with the aid of nearby water molecules, allowing the trans → cis transition to occur at an energy cost of ≤11.6 kJ mol-1. Our results help to explain why isomerase enzymes operate via protonated amide bonds and how N-protonation of the peptide bond occurs via O-protonation.

NMR-Chemical-Shift-Driven Protocol Reveals the Cofactor-Bound, Complete Structure of Dynamic Intermediates of the Catalytic Cycle of Oncogenic KRAS G12C Protein and the Significance of the Mg2+ Ion.

In this work, catalytically significant states of the oncogenic G12C variant of KRAS, those of Mg2+-free and Mg2+-bound GDP-loaded forms, have been determined using CS-Rosetta software and NMR-data-driven molecular dynamics simulations. There are several Mg2+-bound G12C KRAS/GDP structures deposited in the Protein Data Bank (PDB), so this system was used as a reference, while the structure of the Mg2+-free but GDP-bound state of the RAS cycle has not been determined previously. Due to the high flexibility of the Switch-I and Switch-II regions, which also happen to be the catalytically most significant segments, only chemical shift information could be collected for the most important regions of both systems. CS-Rosetta was used to derive an "NMR ensemble" based on the measured chemical shifts, which, however, did not contain the nonprotein components of the complex. We developed a torsional restraint set for backbone torsions based on the CS-Rosetta ensembles for MD simulations, overriding the force-field-based parametrization in the presence of the reinserted cofactors. This protocol (csdMD) resulted in complete models for both systems that also retained the structural features and heterogeneity defined by the measured chemical shifts and allowed a detailed comparison of the Mg2+-bound and Mg2+-free states of G12C KRAS/GDP.

N6-Methyladenine Progressively Accumulates in Mitochondrial DNA during Aging.

N6-methyladenine (6mA) in the DNA is a conserved epigenetic mark with various cellular, physiological and developmental functions. Although the presence of 6mA was discovered a few years ago in the nuclear genome of distantly related animal taxa and just recently in mammalian mitochondrial DNA (mtDNA), accumulating evidence at present seriously questions the presence of N6-adenine methylation in these genetic systems, attributing it to methodological errors. In this paper, we present a reliable, PCR-based method to determine accurately the relative 6mA levels in the mtDNA of Caenorhabditis elegans, Drosophila melanogaster and dogs, and show that these levels gradually increase with age. Furthermore, daf-2(-)-mutant worms, which are defective for insulin/IGF-1 (insulin-like growth factor) signaling and live twice as long as the wild type, display a half rate at which 6mA progressively accumulates in the mtDNA as compared to normal values. Together, these results suggest a fundamental role for mtDNA N6-adenine methylation in aging and reveal an efficient diagnostic technique to determine age using DNA.

Inhibitor Design Strategy for Myostatin: Dynamics and Interaction Networks Define the Affinity and Release Mechanisms of the Inhibited Complexes.

Myostatin, an important negative regulator of muscle mass, is a therapeutic target for muscle atrophic disorders such as muscular dystrophy. Thus, the inhibition of myostatin presents a strategy to treat these disorders. It has long been established that the myostatin prodomain is a strong inhibitor of the mature myostatin, and the minimum peptide of the prodomain-corresponding to the α1-helix of its lasso-region-responsible for the inhibitory efficiency was defined and characterized as well. Here we show that the minimum peptide segment based on the growth differentiation factor 11 (GDF11), which we found to be more helical in its stand-alone solvated stfate than the similar segment of myostatin, is a promising new base scaffold for inhibitor design. The proposed inhibitory peptides in their solvated state and in complex with the mature myostatin were analyzed by in silico molecule modeling supplemented with the electronic circular dichroism spectroscopy measurements. We defined the Gaussian-Mahalanobis mean score to measure the fraction of dihedral angle-pairs close to the desired helical region of the Ramachandran-plot, carried out RING analysis of the peptide-protein interaction networks and characterized the internal motions of the complexes using our rigid-body segmentation protocol. We identified a variant-11m2-that is sufficiently ordered both in solvent and within the inhibitory complex, forms a high number of contacts with the binding-pocket and induces such changes in its internal dynamics that lead to a rigidified, permanently locked conformation that traps this peptide in the binding site. We also showed that the naturally evolved α1-helix has been optimized to simultaneously fulfill two very different roles: to function as a strong binder as well as a good leaving group. It forms an outstanding number of non-covalent interactions with the mature core of myostatin and maintains the most ordered conformation within the complex, while it induces independent movement of the gate-keeper ß-hairpin segment assisting the dissociation and also results in the least-ordered solvated form which provides extra stability for the dissociated state and discourages rebinding.

Off-pathway 3D-structure provides protection against spontaneous Asn/Asp isomerization: shielding proteins Achilles heel.

Spontaneous deamidation prompted backbone isomerization of Asn/Asp residues resulting in - most cases - the insertion of an extra methylene group into the backbone poses a threat to the structural integrity of proteins. Here we present a systematical analysis of how temperature, pH, presence of charged residues, but most importantly backbone conformation and dynamics affect isomerization rates as determined by nuclear magnetic resonance in the case of designed peptide-models. We demonstrate that restricted mobility (such as being part of a secondary structural element) may safeguard against isomerization, but this protective factor is most effective in the case of off-pathway folds which can slow the reaction by several magnitudes compared to their on-pathway counterparts. We show that the geometric descriptors of the initial nucleophilic attack of the isomerization can be used to classify local conformation and contribute to the design of stable protein drugs, antibodies or the assessment of the severity of mutations.At any ­Asn/AspGly­ sites in proteins a spontaneous backbone isomerization occurs within days under physiological conditions leading to various forms of proteopathy. This unwanted transformation especially harmful to long-lived proteins (e.g. hemoglobin and crystallins), can be slowed down, though never stopped, by a rigid three-dimensional protein fold, if it can delay in the conformational maze, on-pathway intermediates from occurring.

The Importance of Mg2+ -Free State in Nucleotide Exchange of Oncogenic K-Ras Mutants.

For efficient targeting of oncogenic K-Ras interaction sites, a mechanistic picture of the Ras-cycle is necessary. Herein, we used NMR relaxation techniques and molecular dynamics simulations to decipher the role of slow dynamics in wild-type and three oncogenic P-loop mutants of K-Ras. Our measurements reveal a dominant two-state conformational exchange on the ms timescale in both GDP- and GTP-bound K-Ras. The identified low-populated higher energy state in GDP-loaded K-Ras has a conformation reminiscent of a nucleotide-bound/Mg2+ -free state characterized by shortened ß2/ß3-strands and a partially released switch-I region preparing K-Ras for the interaction with the incoming nucleotide exchange factor and subsequent reactivation. By providing insight into mutation-specific differences in K-Ras structural dynamics, our systematic analysis improves our understanding of prolonged K-Ras signaling and may aid the development of allosteric inhibitors targeting nucleotide exchange in K-Ras.

Omicron Binding Mode: Contact Analysis and Dynamics of the Omicron Receptor-Binding Domain in Complex with ACE2.

On 26 November 2021, the WHO classified the Omicron variant of the SARS-CoV-2 virus (B.1.1.529 lineage) as a variant of concern (VOC) (COVID-19 Variant Data, Department of Health, 2022). The Omicron variant contains as many as 26 unique mutations of effects not yet determined (Venkatakrishnan, A., Open Science Framework, 2021). Out of its total of 34 Spike protein mutations, 15 are located on the receptor-binding domain (S-RBD) (Stanford Coronavirus Antiviral & Resistance Database, 2022) that directly contacts the angiotensin-converting enzyme 2 (ACE2) host receptor and is also a primary target for antibodies. Here, we studied the binding mode of the S-RBD domain of the Spike protein carrying the Omicron mutations and the globular domain of human ACE2 using molecular dynamics (MD) simulations. We identified new and key Omicron-specific interactions such as R493 (of mutation Q493R), which forms salt bridges both with E35 and D38 of ACE2, Y501 (N501Y), which forms an edge-to-face aromatic interaction with Y41, and Y505 (Y505H), which makes an H-bond with E37 and K353. The glycan chains of ACE2 also bind differently in the WT and Omicron variants in response to different charge distributions on the surface of Spike proteins. However, while the Omicron mutations considerably improve the overall electrostatic fit of the two interfaces, the total number of specific and favorable interactions between the two does not increase. The dynamics of the complexes are highly affected too, making the Omicron S-RBD:ACE2 complex more rigid; the two main interaction sites, Patches I and II, isolated in the WT complex, become connected in the Omicron complex through the alternating interaction of R493 and R498 with E35 and D38.

Dynamically encoded reactivity of Ras enzymes: opening new frontiers for drug discovery.

Decoding molecular flexibility in order to understand and predict biological processes-applying the principles of dynamic-structure-activity relationships (DSAR)-becomes a necessity when attempting to design selective and specific inhibitors of a protein that has overlapping interaction surfaces with its upstream and downstream partners along its signaling cascade. Ras proteins are molecular switches that meet this definition perfectly. The close-lying P-loop and the highly flexible switch I and switch II regions are the site of nucleotide-, assisting-, and effector-protein binding. Oncogenic mutations that also appear in this region do not cause easily characterized overall structural changes, due partly to the inherent conformational heterogeneity and pliability of these segments. In this review, we present an overview of the results obtained using approaches targeting Ras dynamics, such as nuclear magnetic resonance (NMR) measurements and experiment-based modeling calculations (mostly molecular dynamics (MD) simulations). These methodologies were successfully used to decipher the mutant- and isoform-specific nature of certain transient states, far-lying allosteric sites, and the internal interaction networks, as well as the interconnectivity of the catalytic and membrane-binding regions. This opens new therapeutic potential: the discovered interaction hotspots present hitherto not targeted, selective sites for drug design efforts in diverse locations of the protein matrix.

Assessment of Tractable Cysteines for Covalent Targeting by Screening Covalent Fragments.

Targeted covalent inhibition and the use of irreversible chemical probes are important strategies in chemical biology and drug discovery. To date, the availability and reactivity of cysteine residues amenable for covalent targeting have been evaluated by proteomic and computational tools. Herein, we present a toolbox of fragments containing a 3,5-bis(trifluoromethyl)phenyl core that was equipped with chemically diverse electrophilic warheads showing a range of reactivities. We characterized the library members for their reactivity, aqueous stability and specificity for nucleophilic amino acids. By screening this library against a set of enzymes amenable for covalent inhibition, we showed that this approach experimentally characterized the accessibility and reactivity of targeted cysteines. Interesting covalent fragment hits were obtained for all investigated cysteine-containing enzymes.

Synthesis of chimera oligopeptide including furanoid ß-sugar amino acid derivatives with free OHs: mild but successful removal of the 1,2-O-isopropylidene from the building block.

Complementary to hydrophobic five membered ring ß-amino acids (e.g. ACPC), ß-sugar amino acids (ß-SAAs) have found increasing application as hydrophilic building blocks of foldamers and α/ß chimeric peptides. Fmoc-protected ß-SAAs [e.g. Fmoc-RibAFU(ip)-OH] are indeed useful Lego elements, ready to use for SPPS. The removal of 1,2-OH isopropylidene protecting group increasing the hydrophilicity of such SAA is presented here. We first used N3-RibAFU(ip)-OH model compound to optimize mild deprotection conditions. The formation of the 1,2-OH free product N3-RibAFU-OH and its methyl glycoside methyl ester, N3-RibAFU(Me)-OMe were monitored by RP-HPLC and found that either 50% TFA or 8 eqv. Amberlite IR-120 H+ resin in MeOH are optimal reagents for the effective deprotection. These conditions were then successfully applied for the synthesis of chimeric oligopeptide: -GG-X-GG- [X=RibAFU(ip)]. We found the established conditions to be effective and-at the same time-sufficiently mild to remove 1,2-O-isopropylidene protection and thus, it is proposed to be used in the synthesis of oligo- and polypeptides of complex sequence combination.

Solution Structure and Acid-Base Properties of Reduced α-Conotoxin MI.

The reduced derivative of α-conotoxin MI, a 14 amino acid peptide is characterized by NMR-pH titrations and molecular dynamics simulations to determine the protonation constants of the nine basic moieties, including four cysteine thiolates, and the charge-dependent structural properties. The peptide conformation at various protonation states was determined. The results show that the disulfide motifs in the native globular α-conotoxin MI occur between those cysteine moieties that exhibit the most similar thiolate basicities. Since the basicity of thiolates correlates to its redox potential, this phenomenon can be explained by the higher reactivity of the two thiolates with higher basicities. The folding of the oxidized peptide is further facilitated by the loop-like structure of the reduced form, which brings the thiolate groups into sufficient proximity. The 9 group-specific protonation constants and the related, charge-dependent, species-specific peptide structures are presented.

Structural Water Stabilizes Protein Motifs in Liquid Protein Phase: The Folding Mechanism of Short ß-Sheets Coupled to Phase Transition.

Macromolecular associates, such as membraneless organelles or lipid-protein assemblies, provide a hydrophobic environment, i.e., a liquid protein phase (LP), where folding preferences can be drastically altered. LP as well as the associated phase change from water (W) is an intriguing phenomenon related to numerous biological processes and also possesses potential in nanotechnological applications. However, the energetic effects of a hydrophobic yet water-containing environment on protein folding are poorly understood. Here, we focus on small ß-sheets, the key motifs of proteins, undergoing structural changes in liquid-liquid phase separation (LLPS) and also model the mechanism of energy-coupled unfolding, e.g., in proteases, during W → LP transition. Due to the importance of the accurate description for hydrogen bonding patterns, the employed models were studied by using quantum mechanical calculations. The results demonstrate that unfolding is energetically less favored in LP by ~0.3-0.5 kcal·mol-1 per residue in which the difference further increased by the presence of explicit structural water molecules, where the folded state was preferred by ~1.2-2.3 kcal·mol-1 per residue relative to that in W. Energetics at the LP/W interfaces was also addressed by theoretical isodesmic reactions. While the models predict folded state preference in LP, the unfolding from LP to W renders the process highly favorable since the unfolded end state has >1 kcal·mol-1 per residue excess stabilization.

Compactness of Protein Folds Alters Disulfide-Bond Reducibility by Three Orders of Magnitude: A Comprehensive Kinetic Case Study on the Reduction of Differently Sized Tryptophan Cage Model Proteins.

A new approach to monitor disulfide-bond reduction in the vicinity of aromatic cluster(s) has been derived by using the near-UV range (λ=266-293 nm) of electronic circular dichroism (ECD) spectra. By combining the results from NMR and ECD spectroscopy, the 3D fold characteristics and associated reduction rate constants (k) of E19_SS, which is a highly thermostable, disulfide-bond reinforced 39-amino acid long exenatide mimetic, and its N-terminally truncated derivatives have been determined under different experimental conditions. Single disulfide bond reduction of the E19_SS model (with an 18-fold excess of tris(2-carboxyethyl)phosphine, pH 7, 37 °C) takes hours, which is 20-30 times longer than that expected, and thus, would not reach completion by applying commonly used reduction protocols. It is found that structural, steric, and electrostatic factors influence the reduction rate, resulting in orders of magnitude differences in reduction half-lives (900>t1/2 >1 min) even for structurally similar, well-folded derivatives of a small model protein.

Configuration-Controlled Crystal and/or Gel Formation of Protected d-Glucosamines Supported by Promiscuous Interaction Surfaces and a Conformationally Heterogeneous Solution State.

The configuration-dependent self-association mode of the two anomers of O-Ac,N-Fmoc-d-glucosamine, a foldamer building block, leading to gel and/or single crystal formation is described. The ß-anomer of the sugar amino acid (2) forms a gel from various solvents (confirmed by SEM, rheology measurements, NMR, and ECD spectroscopy), whereas the α-anomer (1) does not form a gel with any solvent tested. Transition from the solution state to a gel is coupled to a concurrent shift of the Fmoc-groups: from a freely rotating (almost symmetrical) to a specific, asymmetric orientation. Whereas the crystal structure of the α-anomer is built as an evenly packed 3D system, the ß-anomer forms a looser superstructure of well-packed 2D layers. Modeling indicates that in the lowest energy, but scarcely sampled conformer of the ß-anomer, the Fmoc-group bends above the sugar moiety, stabilized by intramolecular CH↔π interactions between the aromatic rings. It is concluded that possessing an extended and promiscuous interaction surface and a conformationally heterogeneous solution state are among the basic requirements of gel formation for a candidate molecule.

The Route from the Folded to the Amyloid State: Exploring the Potential Energy Surface of a Drug-Like Miniprotein.

Invited for the cover of this issue is the group of András Perczel at Eötvös Loránd University, Budapest, Hungary and colleagues from Osaka University, Japan. The image depicts the amyloid buildup of an Exenatide derivate miniprotein (E5) monitored on a simplified hyperspace. Read the full text of the article at 10.1002/chem.201903826.

The Route from the Folded to the Amyloid State: Exploring the Potential Energy Surface of a Drug-Like Miniprotein.

The amyloid formation of the folded segment of a variant of Exenatide (a marketed drug for type-2 diabetes mellitus) was studied by electronic circular dichroism (ECD) and NMR spectroscopy. We found that the optimum temperature for E5 protein amyloidosis coincides with body temperature and requires well below physiological salt concentration. Decomposition of the ECD spectra and its barycentric representation on the folded-unfolded-amyloid potential energy surface allowed us to monitor the full range of molecular transformation of amyloidogenesis. We identified points of no return (e.g.; T=37 °C, pH 4.1, cE5 =250 µm, cNaCl =50 mm, t>4-6 h) that will inevitably gravitate into the amyloid state. The strong B-type far ultraviolet (FUV)-ECD spectra and an unexpectedly strong near ultraviolet (NUV)-ECD signal (Θ≈275-285   nm ) indicate that the amyloid phase of E5 is built from monomers of quasi-elongated backbone structure (φ≈-145°, ψ≈+145°) with strong interstrand Tyr↔Trp interaction. Misfolded intermediates and the buildup of "toxic" early-stage oligomers leading to self-association were identified and monitored as a function of time. Results indicate that the amyloid transition is triggered by subtle misfolding of the α-helix, exposing aromatic and hydrophobic side chains that may provide the first centers for an intermolecular reorganization. These initial clusters provide the spatial closeness and sufficient time for a transition to the ß-structured amyloid nucleus, thus the process follows a nucleated growth mechanism.

Assignment of Vibrational Circular Dichroism Cross-Referenced Electronic Circular Dichroism Spectra of Flexible Foldamer Building Blocks: Towards Assigning Pure Chiroptical Properties of Foldamers.

Assignment of the most established electronic circular dichroism (ECD) spectra of polypeptides and foldamers is either "evidence based" or relies on the 3D structures of longer oligomers of limited internal dynamics, which are derived from NMR spectroscopy (or X-ray) data. Critics warn that the use of NMR spectroscopy and ECD side by side has severe limitations for flexible molecules because explicit knowledge of conformational ensembles is a challenge. Herein, an old-new method of comparing ab initio computed and measured vibrational circular dichroism (VCD) data is presented to validate both the structures (conf(i)) and their relative weights (c(i)) that make up the conformational ensemble. Based on the array of {conf(i), c(i)}, the pure ECD spectra, g(i)conf(i) , can be ab initio calculated. The reconstructed spectrum Σc(i)g(i)conf(i) can thus help to assign any experimental ECD counterparts. Herein, such a protocol is successfully applied to flexible foldamer building blocks of sugar ß-amino acid diamides. The epimeric pair of the model system was selected because these molecules were conformationally tunable by simple chemical modification, and thus, the robustness of the current approach could be probed. The initial hydrogen bond (NH⋅⋅⋅O) eliminated by N-methylation reorients the amide plain, which influences the chiroptical properties of the foldamer building block; this structural change is successfully monitored by changes to the VCD and ECD transitions, which are now assigned to pure conformers. The current method seems to be general and effective without requiring extensive CPU and spectroscopic resources.