Solution Structure Determination Research Articles

C(P)XCG Proteins of Haloferax volcanii with Predicted Zinc Finger Domains: The Majority Bind Zinc, but Several Do Not.

In recent years, interest in very small proteins (µ-proteins) has increased significantly, and they were found to fulfill important functions in all prokaryotic and eukaryotic species. The halophilic archaeon Haloferax volcanii encodes about 400 µ-proteins of less than 70 amino acids, 49 of which contain at least two C(P)XCG motifs and are, thus, predicted zinc finger proteins. The determination of the NMR solution structure of HVO_2753 revealed that only one of two predicted zinc fingers actually bound zinc, while a second one was metal-free. Therefore, the aim of the current study was the homologous production of additional C(P)XCG proteins and the quantification of their zinc content. Attempts to produce 31 proteins failed, underscoring the particular difficulties of working with µ-proteins. In total, 14 proteins could be produced and purified, and the zinc content was determined. Only nine proteins complexed zinc, while five proteins were zinc-free. Three of the latter could be analyzed using ESI-MS and were found to contain another metal, most likely cobalt or nickel. Therefore, at least in haloarchaea, the variability of predicted C(P)XCG zinc finger motifs is higher than anticipated, and they can be metal-free, bind zinc, or bind another metal. Notably, AlphaFold2 cannot correctly predict whether or not the four cysteines have the tetrahedral configuration that is a prerequisite for metal binding.

Conformations of Macrocyclic Peptides Sampled by Nuclear Magnetic Resonance: Models for Cell-Permeability.

The biological activities and pharmacological properties of peptides and peptide mimetics are determined by their conformational states. Therefore, a detailed understanding of the conformational landscape is crucial for rational drug design. Nuclear magnetic resonance (NMR) is the only method for structure determination in solution. However, it remains challenging to determine the structures of peptides using NMR because of very weak nuclear Overhauser effects (NOEs), the semiquantitative nature of the rotating frame Overhauser effect (ROE), and the low number of NOEs/ROEs in N-methylated peptides. In this study, we introduce a new approach to investigating the structures of modified macrocyclic peptides. We utilize exact NOEs (eNOEs) in viscous solvent mixtures to replicate various cellular environments. eNOEs provide detailed structural information for highly dynamic modified peptides. Structures of high precision were obtained for cyclosporin A, with a backbone atom rmsd of 0.10 Å. Distinct conformational states in different environments were identified for omphalotin A (OmphA), a fungal nematotoxic and multiple backbone N-methylated macrocyclic peptides. A model for cell-permeation is presented for OmphA, based on its structures in polar, apolar, and mixed polarity solvents. During the transition from a polar to an apolar environment, OmphA undergoes a rearrangement of its H-bonding network, accompanied by a cis to trans isomerization of the ω torsion angle within a type VIa β-turn. We hypothesize that the kinetics of these conformational transitions play a crucial role in determining the membrane-permeation capabilities of OmphA.

The translational repressor Glorund uses interchangeable RNA recognition domains to recognize Drosophila nanos.

The Drosophila melanogaster protein Glorund (Glo) represses nanos (nos) translation and uses its quasi-RNA recognition motifs (qRRMs) to recognize both G-tract and structuredUA-rich motifs within the nos translational control element (TCE). We showed previously that each of the three qRRMs is multifunctional, capable of binding to G-tract and UA-rich motifs, yet if and how the qRRMs combine to recognize the nos TCE remained unclear. Here we determined solution structures of a nos TCEI_III RNA containing the G-tract and UA-rich motifs. The RNA structure demonstrated that a single qRRM is physically incapable of recognizing both RNA elements simultaneously. In vivo experiments further indicated that any two qRRMs are sufficient to repress nos translation. We probed interactions of Glo qRRMs with TCEI_III RNA using NMR paramagnetic relaxation experiments. Our in vitro and in vivo data support a model whereby tandem Glo qRRMs are indeed multifunctional and interchangeable for recognition of TCE G-tract or UA-rich motifs. This study illustrates how multiple RNA recognition modules within an RNA-binding protein may combine to diversify the RNAs that are recognized and regulated.

Are Actinyl Cations Good Probes for Structure Determination in Solution by NMR?

We report on NMR spectroscopy, CAS-based method calculations, and X-ray diffraction of AnV and AnVI complexes with a neutral and slightly flexible TEDGA ligand. After checking that pNMR shifts mainly arise from pseudocontact interactions, we analyze pNMR shifts considering the axial and rhombic anisotropy of the actinyl magnetic susceptibilities. The results are compared to those of a previous study performed on [AnVIO2]2+ complexes with dipicolinic acid. It is shown that 5f2 cations (PuVI and NpV) make very good candidates for determining the structure of actinyl complexes in solution by 1H NMR spectroscopy as shown by the invariance of the magnetic properties to the equatorial ligands, as opposed to the NpVI complexes with a 5f1 configuration.

RNA binding induces an allosteric switch in Cyp33 to repress MLL1-mediated transcription

Mixed-lineage leukemia 1 (MLL1) is a transcription activator of the HOX family, which binds to specific epigenetic marks on histone H3 through its third plant homeodomain (PHD3) domain. Through an unknown mechanism, MLL1 activity is repressed by cyclophilin 33 (Cyp33), which binds to MLL1 PHD3. We determined solution structures of Cyp33 RNA recognition motif (RRM) free, bound to RNA, to MLL1 PHD3, and to both MLL1 and the histone H3 lysine N6-trimethylated. We found that a conserved α helix, amino-terminal to the RRM domain, adopts three different positions facilitating a cascade of binding events. These conformational changes are triggered by Cyp33 RNA binding and ultimately lead to MLL1 release from the histone mark. Together, our mechanistic findings rationalize how Cyp33 binding to MLL1 can switch chromatin to a transcriptional repressive state triggered by RNA binding as a negative feedback loop.

Structure Determination of Binuclear Triple-Decker Phthalocyaninato Complexes by NMR via Paramagnetic Shifts Analysis Using Symmetry Peculiarities.

The detailed knowledge about the structure of multinuclear paramagnetic lanthanide complexes for the targeted design of these compounds with special magnetic, sensory, optical and electronic properties is a very important task. At the same time, establishing the structure of such multinuclear paramagnetic lanthanide complexes in solution, using NMR is a difficult task, since several paramagnetic centers act simultaneously on the resulting chemical shift of a particular nucleus. In this paper, we have demonstrated the possibility of molecular structure determination in solution on the example of binuclear triple-decker lanthanide(III) complexes with tetra-15-crown-5-phthalocyanine Ln2[(15C5)4Pc]3 {where Ln = Tb (1) and Dy (2)} by quantitative analysis of the pseudo-contact lanthanide-induced shifts (LIS). The symmetry of complexes was used for the simplification of the calculation of pseudo-contact shifts on the base of the expression for the magnetic susceptibility tensor in the arbitrary oriented magnetic axis system. Good agreement between the calculated and experimental shifts in the 1H NMR spectra indicates the similarity of the structure for the complexes 1 and 2 in solution of CDCl3 and the structure in the crystalline phase, found from the data of the X-ray structural study of the similar complex Lu2[(15C5)4Pc]3. The described approach can be useful for LIS analysis of other polynuclear symmetric lanthanide complexes.

Polymer-Nanodiscs as a Novel Alignment Medium for High-Resolution NMR-Based Structural Studies of Nucleic Acids.

Residual dipolar couplings (RDCs) are increasingly used for high-throughput NMR-based structural studies and to provide long-range angular constraints to validate and refine structures of various molecules determined by X-ray crystallography and NMR spectroscopy. RDCs of a given molecule can be measured in an anisotropic environment that aligns in an external magnetic field. Here, we demonstrate the first application of polymer-based nanodiscs for the measurement of RDCs from nucleic acids. Polymer-based nanodiscs prepared using negatively charged SMA-EA polymer and zwitterionic DMPC lipids were characterized by size-exclusion chromatography, 1H NMR, dynamic light-scattering, and 2H NMR. The magnetically aligned polymer-nanodiscs were used as an alignment medium to measure RDCs from a 13C/15N-labeled fluoride riboswitch aptamer using 2D ARTSY-HSQC NMR experiments. The results showed that the alignment of nanodiscs is stable for nucleic acids and nanodisc-induced RDCs fit well with the previously determined solution structure of the riboswitch. These results demonstrate that SMA-EA-based lipid-nanodiscs can be used as a stable alignment medium for high-resolution structural and dynamical studies of nucleic acids, and they can also be applicable to study various other biomolecules and small molecules in general.

Frémy's Salt as a Low-Persistence Hyperpolarization Agent: Efficient Dynamic Nuclear Polarization Plus Rapid Radical Scavenging.

Nuclear magnetic resonance (NMR) spectroscopy is a key technique for molecular structure determination in solution. However, due to its low sensitivity, many efforts have been made to improve signal strengths and reduce the required substrate amounts. In this regard, dissolution dynamic nuclear polarization (DDNP) is a versatile approach as signal enhancements of over 10 000-fold are achievable. Samples are signal-enhanced ex situ by transferring electronic polarization from radicals to nuclear spins before dissolving and shuttling the boosted sample to an NMR spectrometer for detection. However, the applicability of DDNP suffers from one major drawback, namely, paramagnetic relaxation enhancements (PREs) that critically reduce relaxation times due to the codissolved radicals. PREs are the primary source of polarization losses canceling the signal improvements obtained by DNP. We solve this problem by using potassium nitrosodisulfonate (Frémy's salt) as polarization agent (PA), which provides high nuclear spin polarization and allows for rapid scavenging under mild reducing conditions. We demonstrate the potential of Frémy's salt, (i) showing that both 1H and 13C polarization of ∼30% can be achieved and (ii) describing a hybrid sample shuttling system (HySSS) that can be used with any DDNP/NMR combination to remove the PA before NMR detection. This gadget mixes the hyperpolarized solution with a radical scavenger and injects it into an NMR tube, providing, within a few seconds, quantitatively radical-free, highly polarized solutions. The cost efficiency and broad availability of Frémy's salt might facilitate the use of DDNP in many fields of research.

1H, 13C, and 15N resonance assignments of human glutathione peroxidase 4.

Glutathione peroxidase 4 (GPx4) behaves as an antioxidant enzyme capable of directly reducing peroxidized phospholipids within cell membranes. Recently, GPx4 has attracted attention as a target molecule for cancer therapy because it induces the immortalization of cancer cells suppressing ferroptosis. In this study, to analyze the function and structure of GPx4 by solution NMR, we performed resonance assignments of GPx4 and assigned almost all backbone 1H, 13C, and 15N resonances and most of the side chain 1H and 13C resonances. Using these assignments, the secondary structure of GPx4 was analyzed by the TALOS + program. GPx4 has six helices and seven strands. Then, the backbone dynamics were examined by the {1H}-15N heteronuclear NOE experiment. GPx4 was found to be rigid except for a short loop region. These results will provide basis for functional analysis and the first solution structure determination of GPx4.

"Invisible" Detergents Enable a Reliable Determination of Solution Structures of Native Photosystems by Small-Angle Neutron Scattering.

Photosystems I (PSI) and II (PSII) are pigment-protein complexes capable of performing the light-induced charge separation necessary to convert solar energy into a biochemically storable form, an essential step in photosynthesis. Small-angle neutron scattering (SANS) is unique in providing structural information on PSI and PSII in solution under nearly physiological conditions without the need for crystallization or temperature decrease. We show that the reliability of the solution structure critically depends on proper contrast matching of the detergent belt surrounding the protein. Especially, specifically deuterated ("invisible") detergents are shown to be properly matched out in SANS experiments by a direct, quantitative comparison with conventional matching strategies. In contrast, protonated detergents necessarily exhibit incomplete matching so that related SANS results systematically overestimate the size of the membrane protein under study. While the solution structures obtained are close to corresponding high-resolution structures, we show that temperature and solution state lead to individual structural differences compared with high-resolution structures. We attribute these differences to the presence of a manifold of conformational substates accessible by protein dynamics under physiological conditions.

Bimetallic Copper/Ruthenium/Osmium Complexes: Observation of Conformational Differences Between the Solution Phase and Solid State by Atomic Pair Distribution Function Analysis.

High-energy X-ray scattering and pair distribution function analysis (HEXS/PDF) is a powerful method to reveal the structure of materials lacking long-range order, but is underutilized for molecular complexes in solution. We demonstrate the application of HEXS/PDF with 0.26 Å resolution to uncover the solution structure of five bimetallic CuI /RuII /OsII complexes. HEXS/PDF of each complex in acetonitrile solution confirms the pairwise distances in the local coordination sphere of each metal center as well as the metal⋅⋅⋅metal distances separated by over 12 Å. The metal⋅⋅⋅metal distance detected in solution is compared with that from the crystal structure and molecular models to confirm that distortions to the metal bridging ligand are unique to the solid state. This work presents the first example of observing sub-Ångström conformational differences by direct comparison of solution phase and solid-state structures and shows the potential for HEXS/PDF in the determination of solution structure of single molecules.

Bimetallic Copper/Ruthenium/Osmium Complexes: Observation of Conformational Differences Between the Solution Phase and Solid State by Atomic Pair Distribution Function Analysis

AbstractHigh‐energy X‐ray scattering and pair distribution function analysis (HEXS/PDF) is a powerful method to reveal the structure of materials lacking long‐range order, but is underutilized for molecular complexes in solution. We demonstrate the application of HEXS/PDF with 0.26 Å resolution to uncover the solution structure of five bimetallic CuI/RuII/OsII complexes. HEXS/PDF of each complex in acetonitrile solution confirms the pairwise distances in the local coordination sphere of each metal center as well as the metal⋅⋅⋅metal distances separated by over 12 Å. The metal⋅⋅⋅metal distance detected in solution is compared with that from the crystal structure and molecular models to confirm that distortions to the metal bridging ligand are unique to the solid state. This work presents the first example of observing sub‐Ångström conformational differences by direct comparison of solution phase and solid‐state structures and shows the potential for HEXS/PDF in the determination of solution structure of single molecules.

Fischer carbene complexes of cobalt(I): Synthesis and structure

Three different aryl substrates thiophene (ThH), ferrocene (FcH) and p-bromodimethylaniline (p-DMABr) were lithiated and reacted with [Co(CO)4SnPh3], according to the Fischer carbene protocol. The products [CoSnPh3(CO)3{C(OEt)R}] (R =Th, 1, p-DMA, 2 and R = Fc, 3) were analysed to unravel the role of the aryl carbene substituent in stabilizing intermediates and final products. The ethoxy carbene complexes were aminolysed by in situ generated HNMe2 to afford [CoSnPh3(CO)3{C(NMe2)R}] (R =Th, 4, p-DMA, 5) and with N,N-dimethylethylenediamine [CoSnPh3(CO)3{C(NHCH2CH2NMe2)R}] (R = p-DMA, 6) in high yields. The trigonal Co(CO)3 in the equatorial plane is very stable and efforts to displace a carbonyl in 6 was unsuccessful, both by heating and irradiation. The role of the aryl carbene substituents in stabilizing the electrophilic carbene carbon was investigated and studied by NMR spectroscopy in solution and single crystal X-ray structure determinations.

Intrinsic anisotropy parameters of a series of lanthanoid complexes deliver new insights into the structure-magnetism relationship

Intrinsic anisotropy parameters of a series of lanthanoid complexes deliver new insights into the structure-magnetism relationship

Brownian motion, spin diffusion and protein structure determination in solution

This paper presents my recollections on the development of protein structure determination by NMR in solution from 1968 to 1992. The key to success was to identify NMR-accessible parameters that unambiguously determine the spatial arrangement of polypeptide chains. Inspired by work with cyclopeptides, model considerations showed that enforcing short non-bonding interatomic distances imposes «ring closure conditions» on polypeptide chains. Given that distances are scalar parameters, this indicated an avenue for studies of proteins in solution, i.e., under the regime of stochastic rotational and translational motions at frequencies in the nanosecond range (Brownian motion), where sharp pictures could not be obtained by photography-related methods. Later-on, we used distance geometry calculations with sets of inter-atomic distances derived from protein crystal structures to confirm that measurements of short proton—proton distances could provide atomic-resolution structures of globular proteins. During the years 1976–1984 the following four lines of research then led to protein structure determination by NMR in solution. First, the development of NMR experiments enabling the use of the nuclear Overhauser effect (NOE) for measurements of interatomic distances between pairs of hydrogen atoms in proteins. Second, obtaining sequence-specific resonance assignment solved the “phase problem” for protein structure determination by NMR. Third, generating and programming novel distance geometry algorithms enabled the calculation of atomic-resolution protein structures from limited sets of distance constraints measured by NMR. Fourth, the introduction of two-dimensional NMR provided greatly improved spectral resolution of the complex spectra of proteins as well as efficient delineation of scalar and dipole–dipole 1H–1H connectivities, thus making protein structure determination in solution viable and attractive.

Solution Structure Determination of Arabidopsis Thaliana RALF8 Illustrates the use of Cutting-Edge Software Developed at the National Magnetic Resonance Facility at Madison

Solution Structure Determination of Arabidopsis Thaliana RALF8 Illustrates the use of Cutting-Edge Software Developed at the National Magnetic Resonance Facility at Madison

Three-Dimensional Structure Determination of Peptides Using Solution Nuclear Magnetic Resonance Spectroscopy.

Nuclear magnetic resonance (NMR) spectroscopy has over the last few decades proven to be an extremely useful technique for, and indeed an integral part of, investigating the structural features of peptides and small proteins directly in solution, without the need for crystallization. This advantage over X-ray methods is important when dealing with peptides and small proteins that do not readily form crystals. In this chapter we outline what specific NMR experiments are useful, considerations about how to acquire and interpret these experiments, and how information derived from the NMR data can be used to determine solution structures of small peptides.

Abstract 2752: Recognition of the hybrid-1 human telomeric G-quadruplex by a platinum(II)-based compound

Abstract The human telomeric DNA, which consists of tandem repeat sequences of d(TTAGGG)n, plays an important role in cancers and cell aging. Previous studies in human cancer cells have demonstrated DNA G-quadruplex (G4) structure formation in telomeres. G-quadruplexes are four-stranded structures formed in guanine-rich sequences that are held together by guanine-guanine Hoogsteen hydrogen bonding, and G-quadruplex stabilization by small molecules induces tumor cell senescence and apoptosis by repressing telomerase activity and the DNA damage response pathway. Additionally, telomeric-overhang DNA can form biologically relevant higher-order DNA structures containing consecutive G-quadruplexes which provides additional binding sites for small molecules. Thus, telomeric DNA G-quadruplexes have attracted increasing interest as potential drug targets for cancer therapy. Here, we found that a novel platinum (II)-based tripod (Pt-tripod) specifically recognizes the hybrid-1 human telomeric G4, which is formed in the physiologically relevant K+ solution. Pt-tripod strongly represses telomerase activity and exhibits DNA-targeted photodynamic therapy anticancer activity. Using NMR, we show that Pt-tripod binds the hybrid-1 human telomeric G4 over other G-quadruplex structures and dsDNA. We determined solution structures of the 1:1 and the dimeric 4:2 Pt-tripod-hybrid-1 telomeric G4 complexes by NMR. Our 1:1 complex structure shows preferential binding of the Pt-tripod to the 5′-end of hybrid-1 telomeric G4. At higher ligand ratio, Pt-tripod binds to the 3′-end of the hybid-1 G4 and induces an unprecedented dimeric 4:2 structure interlocked by a non-canonical A:A pair at the 3′-end. The specific binding of the Pt-tripod with the hybrid-1 G4 is achieved by unique interaction modes including the π-π stacking, hydrogen bonding, and electrostatic interactions with the loop and flanking segments. The non-planar tertiary amine conformation and three properly sized cationic platinum arms are critical for the specific and strong binding. Our structures provide significant insights into understanding the dynamic binding of small molecules with G-quadruplexes, and a structural basis for future rational anticancer drug design of non-planar small molecules targeting the human telomeric G4. Our studies also provide a potential handle to study the specific protein interactions and biological functions of hybrid-1 human telomeric G4. Citation Format: Wenting Liu, Clement Lin, Zong-wan Mao, Danzhou Yang. Recognition of the hybrid-1 human telomeric G-quadruplex by a platinum(II)-based compound [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2752.

Water-induced isomerism of salicylaldehyde and 2-acetylpyridine mono- and bis-(thiocarbohydrazones) improves the antioxidant activity: spectroscopic and DFT study

Thiocarbohydrazones (TCHs) and structurally related molecules are versatile organic compounds which exert antioxidant, anticancer, and other beneficial health effects. The combination of UV/Vis, NMR spectroscopy, and quantum chemical calculations was used to rationalize the experimentally observed increase in the radical scavenging activity upon the addition of water in DMSO solution of TCHs. Mono- and bis(salicylaldehyde) TCHs (compounds 1 and 2) undergo water-induced E-to-Z isomerization which is followed by disruption of intramolecular hydrogen bond, ground state destabilization, and 11 kcal/mol decrease in the bond dissociation enthalpy (BDE). Electron spin delocalization is more pronounced in Z-isomers of 1 and 2. On the other hand, 2-acetylpyridine TCHs (compounds 3 and 4) undergo thione-to-thiol tautomerism which also decreases the BDE and facilitates the hydrogen atom transfer to 2,2-diphenyl-1-picrylhydrazyl radical (DPPH∙). The appearance of thiolic –SH group as another reactive site toward free radicals improves the antioxidant activity of 3 and 4. The spin density of 3- and 4-thiol radicals is delocalized over the entire thiocarbohydrazide moiety compared to more localized spin of thione radicals. Additional stabilization of thiol radicals corroborates with the increased antioxidant activity. This study provides the new insights on the solution structure of TCHs, and also highlights the importance of solution structure determination when studying the structure-antioxidant relationships of isomerizable compounds.

Site-Specific Tagging of Proteins with Paramagnetic Ions for Determination of Protein Structures in Solution and in Cells.

High-resolution NMR spectroscopy is sensitive to local structural variations and subtle dynamics of biomolecules and is an important technique for studying the structures, dynamics, and interactions of these molecules. Small-molecule probes, including paramagnetic tags, have been developed for this purpose. Paramagnetic effects manifested in magnetic resonance spectra have long been recognized as valuable tools for chemical analysis of small molecules, and these effects were later applied in the fields of chemical biology and structural biology. However, such applications require the installation of a paramagnetic center in the biomolecules of interest. Paramagnetic metal ions and stable free radicals are the most widely used paramagnetic probes for biological magnetic resonance spectroscopy, and therefore mild, high-yielding approaches for chemically attaching paramagnetic tags to biomolecules are in high demand. In this Account, we begin by discussing paramagnetic species, especially transition metal ions and lanthanide ions, that are suitable for NMR and EPR studies, particularly for in-cell applications. Thereafter, we describe approaches for site-specific tagging of proteins with paramagnetic ions and discuss considerations involved in designing high-quality paramagnetic tags, including the strength of the binding between the metal-chelating moiety and the paramagnetic ion, the chemical stability, and the flexibility of the tether between the paramagnetic tag and the target protein. The flexibility of a tag correlates strongly with the averaging of paramagnetic effects observed in NMR spectra, and we describe methods for increasing tag rigidity and applications of such tags in biological systems. We also describe specific applications of established site-specific tagging approaches and newly developed paramagnetic tags for the elucidation of protein structures and dynamics at atomic resolution both in solution and in cells. First, we describe the determination of the 3D structure of a short-lived, low-abundance enzyme intermediate complex in real time by using pseudocontact shifts as structural restraints. Second, we demonstrate the utility of stable paramagnetic tags for determining 3D structures of proteins in live cells, and pseudocontact shifts are shown to be valuable structural restraints for in-cell protein analysis. Third, we show that a NMR optimized paramagnetic tag allows one to determine distance restraints on proteins by double electron-electron resonance (DEER) measurements with high spatial resolution both in vitro and in cells. Finally, we summarize recent advances in site-specific tagging of proteins to achieve atomic-resolution information about structural changes of proteins, and the advantages and challenges of magnetic resonance spectroscopy in biological systems.