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In the Alzheimer's disease (AD) brain, the microtubule-associated protein tau aggregates into paired helical filaments in which each protofilament has a C-shaped conformation. In vitro assembly of tau fibrils adopting this fold is highly valuable for both fundamental and applied studies of AD without requiring patient-brain extracted fibrils. To date, reported methods for forming AD-fold tau fibrils have been irreproducible and sensitive to subtle variations in fibrillization conditions. Here, we describe a route to reproducibly assemble tau fibrils adopting the AD fold on the multi-milligram scale. We investigated the fibrillization conditions of two constructs and found that a tau (297-407) construct that contains four AD phospho-mimetic glutamate mutations robustly formed the C-shaped conformation. 2D and 3D correlation solid-state NMR spectra show a single predominant set of chemical shifts, indicating a single molecular conformation. Negative-stain electron microscopy and cryo-EM data confirm that the protofilament formed by 4E-tau (297-407) adopts the C-shaped conformation, which associates into paired, triple, and quadruple helical filaments. In comparison, NMR spectra indicate that a previously reported construct, tau (297-391), forms a mixture of a four-layered dimer structure and the C-shaped structure, whose populations are sensitive to the environmental conditions. The determination of the NMR chemical shifts of the AD-fold tau opens the possibility for future studies of tau fibril conformations and ligand binding by NMR. The quantitative assembly of tau fibrils adopting the AD fold should facilitate the development of diagnostic and therapeutic compounds that target AD tau., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

As sessile organisms, plants need to counteract different biotic and abiotic stresses to survive. RNA interference provides natural immunity against various plant pathogens, especially against viral infections via inhibition of viral genome replication or translation. In plants, DRB3, a multi-domain protein containing two N-terminal dsRNA binding domains (dsRBD), plays a vital role in RNA-directed DNA methylation of the geminiviral genome. Additionally, DRB3 arrests the replication of the viral genome in the viral replication complex of RNA viruses through a mechanism that has yet to be fully deciphered. Therefore, as a first step towards exploring the structural details of DRB3, we present a nearly complete backbone and side chain assignment of the two N-terminal dsRBD domains., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Human K-Ras protein, which is a member of the GTPase Ras family, hydrolyzes GTP to GDP and concomitantly converts from its active to its inactive state. It is a key oncoprotein, because several mutations, particularly those at residue position 12, occur with a high frequency in a wide range of human cancers. The K-Ras protein is therefore an important target for developing therapeutic anti-cancer agents. In this work we report the almost complete sequence-specific resonance assignments of wild-type and the oncogenic G12C and G12D mutants in the GTP-complexed active forms, including the functionally important Switch I and Switch II regions. These assignments serve as the basis for a comprehensive functional dynamics study of wild-type K-Ras and its G12 mutants., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Human small EDRK-rich factor protein SERF2 is a cellular driver of protein amyloid formation, a process that has been linked to neurodegenerative diseases including Alzheimer's and Parkinson's disease. SERF2 is a 59 amino acid protein, highly charged, and well conserved whose structure and physiological function is unclear. SERF family proteins including human SERF2 have shown a tendency to form fuzzy complexes with misfolded proteins such as α-Synuclein which has been linked to Parkinson's disease. SERF family proteins have been recently identified to bind nucleic acids, but the binding mechanism(s) remain enigmatic. Here, using multidimensional solution NMR, we report the 1 H, 15 N, and 13 C chemical shift assignments (~ 86% of backbone resonance assignments) for human SERF2. TALOS-N predicted secondary structure of SERF2 showed three very short helices (3-4 residues long) in the N-terminal region of the protein and a long helix in the C-terminal region spanning residues 37-46 which is consistent with the helical content indicated by circular dichroism spectroscopy. Paramagnetic relaxation enhancement NMR analysis revealed that a short C-terminal region E53-K55 is in the proximity of the N-terminus. Having the backbone assignment of SERF2 allowed us to probe its interaction with α-Synuclein and to identify the residues in SERF2 binding interfaces that likely promote α-Synuclein aggregation., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

S. aureus resistance to antibiotics has increased rapidly. MRSA strains can simultaneously be resistant to many different classes of antibiotics, including the so-called "last-resort" drugs. Resistance complicates treatment, increases mortality and substantially increases the cost of treatment. The need for new drugs against (multi)resistant S. aureus is high. M23B family peptidoglycan hydrolases, enzymes that can kill S. aureus by cleaving glycine-glycine peptide bonds in S. aureus cell wall are attractive targets for drug development because of their binding specificity and lytic activity. M23B enzymes lysostaphin, LytU and LytM have closely similar catalytic domain structures. They however differ in their lytic activities, which can arise from non-conserved residues in the catalytic groove and surrounding loops or differences in dynamics. We report here the near complete 1 H/ 13 C/ 15 N resonance assignment of the catalytic domain of LytM, residues 185-316. The chemical shift data allow comparative structural and functional studies between the enzymes and is essential for understanding how these hydrolases degrade the cell wall., (© 2023. The Author(s).)

The backbone 1 H, 13 C and 15 N resonance assignment of Ubiquitin Specific Protease 7 catalytic domain (residues 208-554) was performed in its complex with a small molecule ligand and in its apo form as a reference. The amide 1 H- 15 N signal intensities were boosted by an amide hydrogen exchange protocol, where expressed 2 H, 13 C, 15 N-labeled protein was unfolded and re-folded to ensure exchange of amide deuterons to protons. The resonance assignments were used to determine chemical shift perturbations on ligand binding, which are consistent with the binding site observed by crystallography., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

ModA is a soluble periplasmic molybdate-binding protein found in most gram-negative bacteria. It is part of the ABC transporter complex ModABC that moves molybdenum into the cytoplasm, to be used by enzymes that carry out various redox reactions. Since there is no clear analog for ModA in humans, this protein could be a good target for antibacterial drug design. Backbone 1 H, 13 C and 15 N chemical shifts of apo and molybdate-bound ModA from E. coli were assigned at pHs 6.0 and 4.5. In addition, side chain atoms were assigned for apo ModA at pH 6.0. When comparing apo and molybdate-bound ModA at pH 6.0, large chemical shift perturbations are observed, not only in areas near the bound metal, but also in regions that are distant from the metal-binding site. Given the significant conformational change between apo and holo ModA, we might expect the large chemical shift changes to be more widespread; however, since they are limited to specific regions, the residues with large perturbations may reveal allosteric sites that could ultimately be important for the design of antibiotics that target ModA., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock, considering that vancomycin is the last resort for controlling methicillin-resistant Staphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, the analyses, which are typically not as straightforward as those of small proteins around 10 kDa, were successfully conducted. We assigned the main chain using an amino acid-selective unlabeling method. Because we found that the zinc ion-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by replacing an amino acid at the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR., (© 2024 The Protein Society.)

KKT4 is a multi-domain kinetochore protein specific to kinetoplastids, such as Trypanosoma brucei. It lacks significant sequence similarity to known kinetochore proteins in other eukaryotes. Our recent X-ray structure of the C-terminal region of KKT4 shows that it has a tandem BRCT (BRCA1 C Terminus) domain fold with a sulfate ion bound in a typical binding site for a phosphorylated serine or threonine. Here we present the 1 H, 13 C and 15 N resonance assignments for the BRCT domain of KKT4 (KKT4 463-645 ) from T. brucei. We show that the BRCT domain can bind phosphate ions in solution using residues involved in sulfate ion binding in the X-ray structure. We have used these assignments to characterise the secondary structure and backbone dynamics of the BRCT domain in solution. Mutating the residues involved in phosphate ion binding in T. brucei KKT4 BRCT results in growth defects confirming the importance of the BRCT phosphopeptide-binding activity in vivo. These results may facilitate rational drug design efforts in the future to combat diseases caused by kinetoplastid parasites., (© 2024. The Author(s).)

The BRCA1 carboxyl-terminal (BRCT) domain, an evolutionarily conserved structural motif, is ubiquitous in a multitude of proteins spanning prokaryotic and eukaryotic organisms. In Mycobacterium tuberculosis (Mtb), BRCT domain plays a pivotal role in the catalytic activity of the NAD+-dependent DNA ligase (LigA). LigA is pivotal in DNA replication, catalyzing the formation of phosphodiester bonds in Okazaki fragments and repairing single-strand breaks in damaged DNA, essential for the survival of Mtb. Structural and functional aspects of LigA unveil its character as a highly modular protein, undergoing substantial conformational changes during its catalytic cycle. Although the BRCT domain of Mtb LigA plays an essential role in DNA binding and protein-protein interactions, the precise mechanism of action remains poorly understood. Unravelling the structure of the BRCT domain holds the promise of advancing our understanding of this pivotal domain. Additionally, it will facilitate further exploration of the protein-protein interactions and enhance our understanding of inter domain interactions within LigA, specifically between BRCT and the Adenylation domain. In this study, we demonstrate the overexpression of the BRCT domain of Mtb LigA and conduct its analysis using solution NMR spectroscopy, revealing a well-folded structure and we present the nearly complete chemical shift assignments of both backbone and sidechains. In addition, a secondary structure prediction by TALOS N predicts BRCT consisting of 3 α-helices and 4 β-sheets, closely resembling the typical structural topology of most BRCT domains., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

NCYM is a cis-antisense gene of MYCN oncogene and encodes an oncogenic protein that stabilizes MYCN via inhibition of GSK3b. High NCYM expression levels are associated with poor clinical outcomes in human neuroblastomas, and NCYM overexpression promotes distant metastasis in animal models of neuroblastoma. Using vacuum-ultraviolet circular dichroism and small-angle X-ray scattering, we previously showed that NCYM has high flexibility with partially folded structures; however, further structural characterization is required for the design of anti-cancer agents targeting NCYM. Here we report the 1 H, 15 N and 13 C nuclear magnetic resonance assignments of NCYM. Secondary structure prediction using Secondary Chemical Shifts and TALOS-N analysis demonstrates that the structure of NCYM is essentially disordered, even though residues in the central region of the peptide clearly present a propensity to adopt a dynamic helical structure. This preliminary study provides foundations for further analysis of interaction between NCYM and potential partners., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

In contrast to intracellular gene transfer, the direct delivery of expressed proteins is a significantly challenging yet essential technique for elucidating cellular functions, including protein complex structure, liquid-liquid phase separation, therapeutic applications, and reprogramming. In this study, we developed a hybrid nanotube (HyNT) stamp system that physically inserts the HyNTs into adhesive cells, enabling the injection of target molecules through HyNT ducts. This system demonstrates the capability to deliver multiple proteins, such as lactate oxidase (LOx) and ubiquitin (UQ), to approximately 1.8 × 10 7 adhesive cells with a delivery efficiency of 89.9% and a viability of 97.1%. The delivery of LOx enzyme into HeLa cancer cells induced cell death, while enzyme-delivered healthy cells remained viable. Furthermore, our stamp system can deliver an isotope-labeled UQ into adhesive cells for detection by nuclear magnetic resonance (NMR).

A precursor-directed biosynthesis approach led to the accumulation of seven new neoantimycin derivatives ( 1 - 7 ) from Streptomyces conglobatus RJ2. Structure elucidation was conducted using NMR and HRESIMS analysis, and the absolute configuration was determined by advanced Marfey's method, Mosher's analysis, and ECD analysis. The obtained compounds revealed selective and significant cytotoxicity, specifically against colorectal cancer cells bearing the K-ras mutation, with IC 50 values ranging from 40 nM to 3.5 μM.

The supramolecular interaction between lanthanide complexes and proteins is at the heart of numerous chemical and biological studies. Some of these complexes have demonstrated remarkable interaction properties with proteins or peptides in solution and in the crystalline state. Here we have used the paramagnetism of lanthanide ions to characterize the affinity of two lanthanide complexes for ubiquitin. As the interaction process is dynamic, the acquired NMR data only reflect the time average of the different steps. We have used molecular dynamics (MD) simulations to get a deeper insight into the detailed interaction scenario at the microsecond scale. This NMR/MD approach enabled us to establish that the tris-dipicolinate complex interacts specifically with arginines and lysines, while the crystallophore explores the protein surface through weak interactions with carboxylates. These observations shed new light on the dynamic interaction properties of these complexes, which will ultimately enable us to propose a crystallization mechanism.

Amyloid fibrils have been implicated in the pathogenesis of several neurodegenerative diseases, the most prevalent example being Alzheimer's disease (AD). Despite the prevalence of AD, relatively little is known about the structure of the associated amyloid fibrils. This has motivated our studies of fibril structures, extended here to the familial Arctic mutant of Aβ 1-42 , E22G-Aβ 1-42 . We found E22G-Aβ M0,1-42 is toxic to Escherichia coli , thus we expressed E22G-Aβ 1-42 fused to the self-cleavable tag N Pro in the form of its EDDIE mutant. Since the high surface activity of E22G-Aβ 1-42 makes it difficult to obtain more than sparse quantities of fibrils, we employed 1 H detected magic angle spinning (MAS) nuclear magnetic resonance (NMR) experiments to characterize the protein. The 1 H detected 13 C- 13 C methods were first validated by application to fully protonated amyloidogenic nanocrystals of GNNQQNY, and then applied to fibrils of the Arctic mutant of Aβ, E22G-Aβ 1-42 . The MAS NMR spectra indicate that the biosynthetic samples of E22G-Aβ 1-42 fibrils comprise a single conformation with 13 C chemical shifts extracted from hCH, hNH, and hCCH spectra that are very similar to those of wild type Aβ 1-42 fibrils. These results suggest that E22G-Aβ 1-42 fibrils have a structure similar to that of wild type Aβ 1-42 .

Guanylate cyclase activating protein-5 (GCAP5) in zebrafish photoreceptors promotes the activation of membrane receptor retinal guanylate cyclase (GC-E). Previously, we showed the R22A mutation in GCAP5 (GCAP5 R22A ) abolishes dimerization of GCAP5 and activates GC-E by more than 3-fold compared to that of wild-type GCAP5 (GCAP5 WT ). Here, we present ITC, NMR, and functional analysis of GCAP5 R22A to understand how R22A causes a decreased dimerization affinity and increased cyclase activation. ITC experiments reveal GCAP5 R22A binds a total of 3 Ca 2+ , including two sites in the nanomolar range followed by a single micromolar site. The two nanomolar sites in GCAP5 WT were not detected by ITC, suggesting that R22A may affect the binding of Ca 2+ to these sites. The NMR-derived structure of GCAP5 R22A is overall similar to that of GCAP5 WT (RMSD = 2.3 Å), except for local differences near R22A (Q19, W20, Y21, and K23) and an altered orientation of the C-terminal helix near the N-terminal myristate. GCAP5 R22A lacks an intermolecular salt bridge between R22 and D71 that may explain the weakened dimerization. We present a structural model of GCAP5 bound to GC-E in which the R22 side-chain contacts exposed hydrophobic residues in GC-E. Cyclase assays suggest that GC-E binds to GCAP5 R22A with ∼25% higher affinity compared to GCAP5 WT , consistent with more favorable hydrophobic contact by R22A that may help explain the increased cyclase activation.

Understanding how the amino acid sequence dictates protein structure and defines its stability is a fundamental problem in molecular biology. It is especially challenging for membrane proteins that reside in the complex environment of a lipid bilayer. Here, we obtain an atomic-level picture of the thermally induced unfolding of a membrane-embedded α-helical protein, human aquaporin 1, using solid-state nuclear magnetic resonance spectroscopy. Our data reveal the hierarchical two-step pathway that begins with unfolding of a structured extracellular loop and proceeds to an intermediate state with a native-like helical packing. In the second step, the transmembrane domain unravels as a single unit, resulting in a heterogeneous misfolded state with high helical content but with nonnative helical packing. Our results show the importance of loops for the kinetic stabilization of the whole membrane protein structure and support the three-stage membrane protein folding model.

Molecular dynamics simulations depend critically on the quality of the force field used to describe the interatomic interactions and the extent to which it has been validated for use in a specific application. Using a curated test set of 52 high-resolution structures, 39 derived from X-ray diffraction and 13 solved using NMR, we consider the extent to which different parameter sets of the GROMOS protein force field can be distinguished based on comparing a range of structural criteria, including the number of backbone hydrogen bonds, the number of native hydrogen bonds, polar and nonpolar solvent-accessible surface area, radius of gyration, the prevalence of secondary structure elements, J -coupling constants, nuclear Overhauser effect (NOE) intensities, positional root-mean-square deviations (RMSD), and the distribution of backbone ϕ and ψ dihedral angles. It is shown that while statistically significant differences between the average values of individual metrics could be detected, these were in general small. Furthermore, improvements in agreement in one metric were often offset by loss of agreement in another. The work establishes a framework and test set against which protein force fields can be validated. It also highlights the danger of inferring the relative quality of a given force field based on a small range of structural properties or small number of proteins.

The substitution of a single hydrogen atom in a protein by fluorine yields a site-specific probe for sensitive detection by 19 F nuclear magnetic resonance (NMR) spectroscopy, where the absence of background signal from the protein facilitates the detection of minor conformational species. We developed genetic encoding systems for the site-selective incorporation of 4-fluorotryptophan, 5-fluorotryptophan, 6-fluorotryptophan, and 7-fluorotryptophan in response to an amber stop codon and used them to investigate conformational heterogeneity in a designed amino acid binding protein and in flaviviral NS2B-NS3 proteases. These proteases have been shown to present variable conformations in X-ray crystal structures, including flips of the indole side chains of tryptophan residues. The 19 F NMR spectra of different fluorotryptophan isomers installed at the conserved site of Trp83 indicate that the indole ring flip is common in flaviviral NS2B-NS3 proteases in the apo state and suppressed by an active-site inhibitor.

Fast collective motions are widely present in biomolecules, but their functional relevance remains unclear. Herein, we reveal that fast collective motions of backbone are critical to the water transfer of aquaporin Z (AqpZ) by using solid-state nuclear magnetic resonance (ssNMR) spectroscopy and molecular dynamics (MD) simulations. A total of 212 residue site-specific dipolar order parameters and 158 15 N spin relaxation rates of the backbone are measured by combining the 13 C- and 1 H-detected multidimensional ssNMR spectra. Analysis of these experimental data by theoretic models suggests that the small-amplitude (~10°) collective motions of the transmembrane α helices on the nanosecond-to-microsecond timescales are dominant for the dynamics of AqpZ. The MD simulations demonstrate that these collective motions are critical to the water transfer efficiency of AqpZ by facilitating the opening of the channel and accelerating the water-residue hydrogen bonds renewing in the selectivity filter region.

The Microtubule-binding repeat region (MTBR) of Tau has been studied extensively due to its pathological implications in neurodegenerative diseases like Alzheimer's disease. The pathological property of MTBR is mainly due to the R3 repeat's high propensity for self-aggregation, highlighting the critical molecular grammar of the repeat. Utilizing the R1R3 construct (WT) and its G326E mutant (EE), we determine the distinct characteristics of various peptide segments that modulate the aggregation propensity of the R3 repeat using NMR spectroscopy. Through time-dependent experiments, we have identified 317 KVTSKCGS 324 in R3 repeat as the aggregation initiating motif (AIM) due to its role at the initial stages of aggregation. The G326E mutation induces changes in conformation and dynamics at the AIM, thereby effectively abrogating the aggregation propensity of the R1R3 construct. We further corroborate our findings through MD simulations and propose that AIM is a robust site of interest for tauopathy drug design.

Understanding small molecule binding to RNA can be complicated by an intricate interplay between binding stoichiometry, multiple binding motifs, different occupancies of different binding motifs, and changes in the structure of the RNA under study. Here, we use native top-down mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy to experimentally resolve these factors and gain a better understanding of the interactions between neomycin B and the 40 nt aptamer domain of a neomycin-sensing riboswitch engineered in yeast. Data from collisionally activated dissociation of the 1:1, 1:2 and 1:3 RNA-neomycin B complexes identified a third binding motif C of the riboswitch in addition to the two motifs A and B found in our previous study, and provided occupancies of the different binding motifs for each complex stoichiometry. Binding of a fourth neomycin B molecule was unspecific according to both MS and NMR data. Intriguingly, all major changes in the aptamer structure can be induced by the binding of the first neomycin B molecule regardless of whether it binds to motif A or B as evidenced by stoichiometry-resolved MS data together with titration data from 1H NMR spectroscopy in the imino proton region. Specific binding of the second and third neomycin B molecules further stabilizes the riboswitch aptamer, thereby allowing for a gradual response to increasing concentrations of neomycin B, which likely leads to a fine-tuning of the cellular regulatory mechanism., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Protein allostery is commonly observed in vitro. But how protein allostery behaves in cells is unknown. In this work, a protein monomer-dimer equilibrium system was built with the allosteric effect on the binding characterized using NMR spectroscopy through mutations away from the dimer interface. A chemical shift linear fitting method was developed that enabled us to accurately determine the dissociation constant. A total of 28 allosteric mutations were prepared and grouped to negative allosteric, nonallosteric, and positive allosteric modulators. ∼ 50% of mutations displayed the allosteric-state changes when moving from a buffered solution into cells. For example, there were no positive allosteric modulators in the buffered solution but eight in cells. The change in protein allostery is correlated with the interactions between the protein and the cellular environment. These interactions presumably drive the surrounding macromolecules in cells to transiently bind to the monomer and dimer mutational sites and change the free energies of the two species differently which generate new allosteric effects. These surrounding macromolecules create a new protein allostery pathway that is only present in cells.

Chromatinized DNA is targeted by proteins and small molecules to regulate chromatin function. For example, anthracycline cancer drugs evict nucleosomes in a mechanism that is still poorly understood. We here developed a flexible method for specific isotope labeling of nucleosomal DNA enabling NMR studies of such nucleosome interactions. We describe the synthesis of segmental one-strand 13 C-thymidine labeled 601-DNA, the assignment of the methyl signals, and demonstrate its use to observe site-specific binding to the nucleosome by aclarubicin, an anthracycline cancer drug that intercalates into the DNA minor grooves. Our results highlight intrinsic conformational heterogeneity in the 601 DNA sequence and show that aclarubicin binds an exposed AT-rich region near the DNA end. Overall, our data point to a model where the drug invades the nucleosome from the terminal ends inward, eventually resulting in histone eviction and nucleosome disruption., (© 2024 The Authors. ChemBioChem published by Wiley-VCH GmbH.)

G-protein-coupled receptor (GPCR) activation relies on conformational sampling, a nuanced but functionally key behavior well suited to elucidation by nuclear magnetic resonance (NMR) spectroscopy. In this issue of Structure, Thakur et al. 1 demonstrate that judicious choice of experimental conditions for 19 F NMR studies of a GPCR enables rationalization of functional and pharmacological behavior, leading to testable hypotheses correlating structure, dynamics, and function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Elucidating how damage impacts DNA dynamics is essential for understanding the mechanisms of damage recognition and repair. Many DNA lesions alter their propensities to form low-populated and short-lived conformational states. However, NMR methods to measure these dynamics require isotopic enrichment, which is difficult for damaged nucleotides. Here, we demonstrate the utility of the 1 H chemical exchange saturation transfer (CEST) NMR experiment in measuring the dynamics of oxidatively damaged 8-oxoguanine (8OG) in the mutagenic 8OG syn ·A anti mismatch. Using 8OG-H7 as an NMR probe of the damaged base, we directly measured 8OG syn-anti flips to form a lowly populated (pop. ∼ 5%) and short-lived (lifetime ∼50 ms) nonmutagenic 8OG anti ·A anti . These exchange parameters were in quantitative agreement with values from 13 C off-resonance R 1ρ and CEST on the labeled partner adenine. The Watson-Crick-like 8OG syn ·A anti mismatch also rescued the kinetics of Hoogsteen motions at distant A-T base pairs, which the G·A mismatch had slowed down. The results lend further support for 8OG anti ·A anti as a minor conformational state of 8OG·A, reveal that 8OG damage can impact Hoogsteen dynamics at a distance, and demonstrate the utility of 1 H CEST for measuring damage-dependent dynamics in unlabeled DNA.

We leveraged variable-temperature 19 F-NMR spectroscopy to compare the conformational equilibria of the human A 2A adenosine receptor (A 2A AR), a class A G protein-coupled receptor (GPCR), across a range of temperatures ranging from lower temperatures typically employed in 19 F-NMR experiments to physiological temperature. A 2A AR complexes with partial agonists and full agonists showed large increases in the population of a fully active conformation with increasing temperature. NMR data measured at physiological temperature were more in line with functional data. This was pronounced for complexes with partial agonists, where the population of active A 2A AR was nearly undetectable at lower temperature but became evident at physiological temperature. Temperature-dependent behavior of complexes with either full or partial agonists exhibited a pronounced sensitivity to the specific membrane mimetic employed. Cellular signaling experiments correlated with the temperature-dependent conformational equilibria of A 2A AR in lipid nanodiscs but not in some detergents, underscoring the importance of the membrane environment in studies of GPCR function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Two oxygenated ergostane-type steroids including one new compound, 3 β -hydroxy-5 α ,6 β -methoxyergosta-7,22-dien-15-one ( 1 ) along with a known analogue ergosta-6,22-dien-3 β ,5 α ,8 α -triol ( 2 ) were isolated from the crude extracts of the marine sponge-derived fungus Aspergillus sp. Their structures were elucidated on the basis of combined NMR and MS spectroscopic methods. Compound 1 was a marine ergostane-type steroid with two methoxy groups at C-5 and C-6, respectively. These oxygenated ergostane-type steroids were evaluated for their antibacterial activities against human or aquatic pathogens. Among them, compound 1 exhibited antibacterial activity against Staphylococcus aureus.

New dammarane-type triterpenoid saponin, named 22( R )-notoginsenoside Ab1 ( 1 ), together with thirteen known dammarane-type triterpenoid saponins ( 2 - 14 ) was isolated from the EtOH extract of black ginseng and their structures were elucidated on the basis of one- and two-dimensional NMR (including 1 H-NMR, 13 C-NMR, HSQC, HMBC, ROESY) and calculated ECD. Among them, compounds 1-2 and 6 - 8 were isolated for the first time from ginseng and black ginseng. Besides, the absolute structure of 22( R )- and 22( S )- notoginsenoside Ab1 were distinguished by ECD for the first time.

Functional bacterial amyloids play a crucial role in the formation of biofilms, which mediate chronic infections and contribute to antimicrobial resistance. This study focuses on the FapC amyloid fibrillar protein from Pseudomonas, a major contributor to biofilm formation. We investigate the initial steps of FapC amyloid formation and the impact of the chaperone-like protein FapA on this process. Using solution nuclear magnetic resonance (NMR), we recently showed that both FapC and FapA are intrinsically disordered proteins (IDPs). Here, the secondary structure propensities (SSPs) are compared to alphafold (DeepMind, protein structure prediction tool/algorithm: https://alphafold.ebi.ac.uk/) models. We further demonstrate that the FapA chaperone interacts with FapC and significantly slows down the formation of FapC fibrils. Our NMR titrations reveal ~ 18% of the resonances show FapA-induced chemical shift perturbations (CSPs), which has not been previously observed, the largest being for A82, N201, C237, C240, A241, and G245. These sites may suggest a specific interaction site and/or hotspots of fibrillation inhibition/control interface at the repeat-1 (R1)/loop-2 (L2) and L2/R3 transition areas and at the C-terminus of FapC. Remarkably, ~ 90% of FapA NMR signals exhibit substantial CSPs upon titration with FapC, the largest being for S63, A69, A80, and I92. A temperature-dependent effect of FapA was observed on FapC by thioflavin T (ThT) and NMR experiments. This study provides a detailed understanding of the interaction between the FapA and FapC, shedding light on the regulation and slowing down of amyloid formation, and has important implications for the development of therapeutic strategies targeting biofilms and associated infections., (© 2024 Federation of European Biochemical Societies.)

Enzymes play an increasingly important role in improving the benignity and efficiency of chemical production, yet the diversity of their applications lags heavily behind chemical catalysts as a result of the relatively narrow range of reaction mechanisms of enzymes. The creation of enzymes containing non-biological functionalities facilitates reaction mechanisms outside nature's canon and paves the way towards fully programmable biocatalysis 1-3 . Here we present a completely genetically encoded boronic-acid-containing designer enzyme with organocatalytic reactivity not achievable with natural or engineered biocatalysts 4,5 . This boron enzyme catalyses the kinetic resolution of hydroxyketones by oxime formation, in which crucial interactions with the protein scaffold assist in the catalysis. A directed evolution campaign led to a variant with natural-enzyme-like enantioselectivities for several different substrates. The unique activation mode of the boron enzyme was confirmed using X-ray crystallography, high-resolution mass spectrometry (HRMS) and 11 B NMR spectroscopy. Our study demonstrates that genetic-code expansion can be used to create evolvable enantioselective enzymes that rely on xenobiotic catalytic moieties such as boronic acids and access reaction mechanisms not reachable through catalytic promiscuity of natural or engineered enzymes., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

A newly discovered trihydroxynaphthalenone derivative, epoxynaphthalenone ( 1 ) involving the condensation of ortho-hydroxyl groups into an epoxy structure, and a novel pyrone metabolite characterized as pyroneaceacid ( 2 ), were extracted from Talaromyces purpurpgenus , an endophytic fungus residing in Rhododendron molle . The structures of these compounds were elucidated through a comprehensive analysis of their NMR and HRESIMS data. The determination of absolute configurations was accomplished using electronic circular dichroism (ECD) calculations and CD spectra. Notably, these recently identified metabolites exhibited a moderate inhibitory activity against xanthine oxidase (XOD).

A new quinochalcone C -glycoside featuring a unique quinonoid moiety, named celochalcoside ( 1 ), was isolated from the n -butanol extract of the aerial parts of Celosia trigyna L. The structure was determined by extensive spectroscopic analysis as well as mass spectrometric data. Compound 1 showed moderate cytotoxic activities against breast cancer cell lines (MCF-7), colon cancer cell lines (HT-29) and hepatocellular carcinoma cell lines (HepG2) with IC 50 values of 23.16, 37.05 and 18.35 μg/ml, respectively.

The catalytic cycle of Enzyme I (EI), a phosphotransferase enzyme responsible for converting phosphoenolpyruvate (PEP) into pyruvate, is characterized by a series of local and global conformational rearrangements. This multistep process includes a monomer-to-dimer transition, followed by an open-to-closed rearrangement of the dimeric complex upon PEP binding. In the present study, we investigate the thermodynamics of EI dimerization using a range of high-pressure solution NMR techniques complemented by SAXS experiments. 1 H- 15 N TROSY and 1 H- 13 C methyl TROSY NMR spectra combined with 15 N relaxation measurements revealed that a native-like engineered variant of full-length EI fully dissociates into stable monomeric state above 1.5 kbar. Conformational ensembles of EI monomeric state were generated via a recently developed protocol combining coarse-grained molecular simulations with experimental backbone residual dipolar coupling measurements. Analysis of the structural ensembles provided detailed insights into the molecular mechanisms driving formation of the catalytically competent dimeric state, and reveals that each step of EI catalytical cycle is associated with a significant reduction in either inter- or intra-domain conformational entropy. Altogether, this study completes a large body work conducted by our group on EI and establishes a comprehensive structural and dynamical description of the catalytic cycle of this prototypical multidomain, oligomeric enzyme., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

A chemical investigation of the hydrophilic fraction of a cultured Nodularia sp. (NIES-3585) afforded six new cyclic lipopeptides, noducyclamides A1-A4 ( 1 - 4 ) containing 10 amino acid residues and dodecapeptides noducyclamides B1 and B2 ( 5 and 6 ). The planar structures of these lipopeptides were elucidated based on the combination of HRMS and 1D and 2D NMR spectroscopic data analyses. These peptides are structurally analogous to laxaphycins and contain the nonproteinogenic amino acids 3-hydroxyvaline and 3-hydroxyleucine and a β-amino decanoic acid residue. The absolute configurations of the noducyclamides ( 1 - 6 ) were determined by acid hydrolysis, followed by advanced Marfey's analysis. Noducyclamide B1 ( 5 ) showed cytotoxic activities against MCF7 breast cancer cell lines with an IC 50 value of 3.0 μg/mL (2.2 μM).

Kagimminols A ( 1 ) and B ( 2 ), new cembrene-type diterpenoids, were isolated from an Okeania sp. marine cyanobacterium. By combining DP4 analysis with an efficient NMR chemical shift calculation protocol, we clarified the relative configurations of 1 and 2 without consuming precious natural products. We determined the absolute configurations by a comparison of theoretical electronic circular dichroism (ECD) spectra with experimental spectra, and the absolute configuration of 1 was verified experimentally. Finally, we found that 1 and 2 showed selective growth-inhibitory activity against the causative agent of human African trypanosomiasis. This study exemplifies that computational chemistry is an efficient tool for clarifying the configurations of natural products possessing tautomers in equilibrium.

The chemical diversity of annelids, particularly those belonging to the class Sipuncula, remains largely unexplored. However, as part of a Marine Biodiscovery program in Ireland, the peanut worm Phascolosoma granulatum emerged as a promising source of unique metabolites. The purification of the MeOH/CH 2 Cl 2 extract of this species led to the isolation of six new linear guanidine amides, named phascolosomines A-F ( 1 - 6 ). NMR analysis allowed for the elucidation of their structures, all of which feature a terminal guanidine, central amide linkage, and a terminal isobutyl group. Notably, these guanidine amides were present in unusually high concentrations, comprising ∼3% of the dry mass of the organism. The primary concentration of the phascolosomines in the viscera is similar to that previously identified in linear amides from sipunculid worms and marine fireworms. The compounds from sipunculid worms have been hypothesized to be toxins, while those from fireworms are reported to be defensive irritants. However, screening of the newly isolated compounds for inhibitory bioactivity showed no significant inhibition in any of the assays conducted.

Two new compounds, kinanthraquinone C ( 1 ) and kinanthraquinone D ( 2 ), were isolated along with two known compounds, kinanthraquinone ( 3 ) and kinanthraquinone B ( 4 ), produced by the heterologous expression of the kiq biosynthetic gene cluster and its pathway-specific regulator, kiqA , in Streptomyces lividans TK23. The chemical structures of compounds 1 and 2 were determined using mass spectrometry and nuclear magnetic resonance analyses. To examine a biosynthetic pathway of compounds 1 and 2 , incubation experiments were conducted using S. lividans TK23 to supply the compounds 3 and 4 . These experiments indicated that compounds 3 and 4 were converted to compounds 2 and 1 , respectively, by the endogenous enzymes of S. lividans TK23. Compounds 2 , 3 , and 4 had antimalarial activities at half-maximal inhibitory concentration values of 0.91, 1.2, and 15 μM, respectively, without cytotoxicity up to 30 μM.

A detailed chemical study of the extract from the soft coral Stereonephthya bellissima resulted in the isolation and identification of seven new sesquiterpenoids, bellissinanes A-G ( 1 - 7 ), along with four new diterpenes ( 8 - 11 ). Bellissinane A ( 1 ) is the third reported nardosinane-type sesquiterpene bearing a 6/5/6 tricyclic system. Bellissinanes C and D ( 3 , 4 ) contain a phenylethylamine fragment, which is relatively unusual in marine organisms. Bellissinanes E-G ( 5 - 7 ) belong to the rare class of nornardosinane sesquiterpenoids. Structurally uncommon octahydro-1 H -indenyl-type and prenyleudesmane-type skeletons were characterized for herpetopanone B ( 8 ) and bellissimain A ( 9 ), respectively. Bellissinane E ( 5 ) exhibited in vivo angiogenesis-promoting activity.

The aggregation of proteins into amyloid-like fibrils is seen in many neurodegenerative diseases. Recent years have seen much progress in our understanding of these misfolded protein inclusions, thanks to advances in techniques such as solid-state nuclear magnetic resonance (ssNMR) spectroscopy and cryogenic electron microscopy (cryo-EM). However, multiple repeat-expansion-related disorders have presented special challenges to structural elucidation. This review discusses the special role of ssNMR analysis in the study of protein aggregates associated with CAG repeat expansion disorders. In these diseases, the misfolding and aggregation affect mutant proteins with expanded polyglutamine segments. The most common disorder, Huntington's disease (HD), is connected to the mutation of the huntingtin protein. Since the discovery of the genetic causes for HD in the 1990s, steady progress in our understanding of the role of protein aggregation has depended on the integrative and interdisciplinary use of multiple types of structural techniques. The heterogeneous and dynamic features of polyQ protein fibrils, and in particular those formed by huntingtin N-terminal fragments, have made these aggregates into challenging targets for structural analysis. ssNMR has offered unique insights into many aspects of these amyloid-like aggregates. These include the atomic-level structure of the polyglutamine core, but also measurements of dynamics and solvent accessibility of the non-core flanking domains of these fibrils' fuzzy coats. The obtained structural insights shed new light on pathogenic mechanisms behind this and other protein misfolding diseases., (© 2024 The Author(s).)

We are entering an exciting time in structural biology where artificial intelligence can be used to predict protein structures with greater accuracy than ever before. Extending this level of accuracy to the predictions of disulfide-rich peptide structures is likely to be more challenging, at least in the short term, given the tight packing of cysteine residues and the numerous ways that the disulfide bonds can potentially be linked. It has been previously shown in many cases that several disulfide bond connectivities can be accommodated by a single set of NMR-derived structural data without significant violations. Disulfide-rich peptides are prevalent throughout nature, and arguably the most well-known are those present in venoms from organisms such as cone snails. Here, we have determined the first three-dimensional structure and disulfide connectivity of a U-superfamily cone snail venom peptide, TxVIIB. TxVIIB has a VI/VII cysteine framework that is generally associated with an inhibitor cystine knot (ICK) fold; however, AlphaFold predicted that the peptide adopts a mini-granulin fold with a granulin disulfide connectivity. Our experimental studies using NMR spectroscopy and orthogonal protection of cysteine residues indicate that TxVIIB indeed adopts a mini-granulin fold but with the ICK disulfide connectivity. Our findings provide structural insight into the underlying features that govern formation of the mini-granulin fold rather than the ICK fold and will provide fundamental information for prediction algorithms, as the subtle complexity of disulfide isomers may be not adequately addressed by the current prediction algorithms., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Side-chain motions play an important role in understanding protein structure, dynamics, protein-protein, and protein-ligand interactions. However, our understanding of protein side-chain dynamics is currently limited by the lack of analytical tools. Here, we present a novel analytical framework employing experimental nuclear magnetic resonance (NMR) relaxation measurements at atomic resolution combined with molecular dynamics (MD) simulation to characterize with a high level of detail the methyl side-chain dynamics in insoluble protein assemblies, using amyloid fibrils formed by the prion HET-s. We use MD simulation to interpret experimental results, where rotameric hops, including methyl group rotation and χ 1 /χ 2 rotations, cannot be completely described with a single correlation time but rather sample a broad distribution of correlation times, resulting from continuously changing local structure in the fibril. Backbone motion similarly samples a broad range of correlation times, from ∼100 ps to μs, although resulting from mostly different dynamic processes; nonetheless, we find that the backbone is not fully decoupled from the side-chain motion, where changes in side-chain dynamics influence backbone motion and vice versa. While the complexity of side-chain motion in protein assemblies makes it very challenging to obtain perfect agreement between experiment and simulation, our analytical framework improves the interpretation of experimental dynamics measurements for complex protein assemblies.

Nuclear magnetic resonance (NMR) relaxation experiments shine light onto the dynamics of molecular systems in the picosecond to millisecond timescales. As these methods cannot provide an atomically resolved view of the motion of atoms, functional groups, or domains giving rise to such signals, relaxation techniques have been combined with molecular dynamics (MD) simulations to obtain mechanistic descriptions and gain insights into the functional role of side chain or domain motion. In this work, we present a comparison of five computational methods that permit the joint analysis of MD simulations and NMR relaxation experiments. We discuss their relative strengths and areas of applicability and demonstrate how they may be utilized to interpret the dynamics in MD simulations with the small protein ubiquitin as a test system. We focus on the aliphatic side chains given the rigidity of the backbone of this protein. We find encouraging agreement between experiment, Markov state models built in the χ1/χ2 rotamer space of isoleucine residues, explicit rotamer jump models, and a decomposition of the motion using ROMANCE. These methods allow us to ascribe the dynamics to specific rotamer jumps. Simulations with eight different combinations of force field and water model highlight how the different metrics may be employed to pinpoint force field deficiencies. Furthermore, the presented comparison offers a perspective on the utility of NMR relaxation to serve as validation data for the prediction of kinetics by state-of-the-art biomolecular force fields., (© 2024 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).)

For the A 2A adenosine receptor (A 2A AR), a class A G-protein-coupled receptor (GPCR), reconstituted in n-dodecyl-β-D-maltoside (DDM)/‌‌‌‌‌cholesteryl hemisuccinate (CHS) mixed micelles, previous 19 F-NMR studies revealed the presence of multiple simultaneously populated conformational states. Here, we study the influence of a different detergent, lauryl maltose neopentyl glycol (LMNG) in mixed micelles with CHS, and of lipid bilayer nanodiscs on these conformational equilibria. The populations of locally different substates are pronouncedly different in DDM/‌‌‌‌‌CHS and LMNG/‌‌‌‌‌CHS micelles, whereas the A 2A AR conformational manifold in LMNG/‌‌‌‌‌CHS micelles is closely similar to that in the lipid bilayer nanodiscs. Considering that nanodiscs represent a closer match of the natural lipid bilayer membrane, these observations support that LMNG/‌‌‌‌‌CHS micelles are a good choice for reconstitution trials of class A GPCRs for NMR studies in solution., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Reducing sugars can spontaneously react with free amines in protein side chains leading to posttranslational modifications (PTMs) called glycation. In contrast to glycosylation, glycation is a non-enzymatic modification with consequences on the overall charge, solubility, aggregation susceptibility and functionality of a protein. Glycation is a critical quality attribute of therapeutic monoclonal antibodies. In addition to glucose, also disaccharides like maltose can form glycation products. We present here a detailed NMR analysis of the Amadori product formed between proteins and maltose. For better comparison, data collection was done under denaturing conditions using 7 M urea-d 4 in D 2 O. The here presented correlation patterns serve as a signature and can be used to identify maltose-based glycation in any protein that can be denatured. In addition to the model protein BSA, which can be readily glycated, we present data of the biotherapeutic abatacept containing maltose in its formulation buffer. With this contribution, we demonstrate that NMR spectroscopy is an independent method for detecting maltose-based glycation, that is suited for cross-validation with other methods., (© 2023. The Author(s).)

In this study, we present the synthesis and incorporation of a metabolic isoleucine precursor compound for selective methylene labeling. The utility of this novel α-ketoacid isotopologue is shown by incorporation into the protein Brd4-BD1, which regulates gene expression by binding to acetylated histones. High quality single quantum 13 C- 1  H-HSQC were obtained, as well as triple quantum HTQC spectra, which are superior in terms of significantly increased 13 C-T 2 times. Additionally, large chemical shift perturbations upon ligand binding were observed. Our study thus proves the great sensitivity of this precursor as a reporter for side-chain dynamic studies and for investigations of CH-π interactions in protein-ligand complexes., (© 2023. The Author(s).)