Helix In Solution Research Articles

NMR-based solution structure of the Caulobacter crescentus ProXp-ala trans-editing enzyme

ProXp-ala is a key component of the translational machinery in all three Domains of life. This enzyme helps to maintain the fidelity of proline codon translation through aminoacyl-tRNAPro proofreading. In the first step of tRNA aminoacylation, the cognate aminoacyl-tRNA synthetase (aaRS) binds and activates an amino acid in the enzyme’s synthetic active site. If a non-cognate amino acid passes this first selection step and is charged onto the tRNA, a distinct aaRS editing active site may recognize the mischarged tRNA and deacylate it. Alternatively, this editing reaction may be carried out by a separate enzyme that deacylates the mischarged tRNA in trans. ProXp-ala is responsible for editing Ala mischarged onto tRNAPro. Since trans-editing domains such as ProXp-ala bind their substrates after release from the synthetase, they must recognize not only the mischarged amino acid, but also the specific tRNA. Previous studies showed that Caulobacter crescentus (Cc) ProXp-ala distinguishes tRNAPro from tRNAAla, in part, based on the unique tRNAPro acceptor stem base pair C1:G72. Previous crystallographic and NMR data also revealed a role for conformational selection by the ProXp-ala α2 helix in Ala- versus Pro-tRNAPro substrate discrimination. The α2 helix makes lattice contacts in the crystal, which left some uncertainty as to its position in solution. We report resonance assignments for the substrate-free Cc ProXp-ala and the NMR-derived three-dimensional structure of the protein. These data reveal the position of the α2 helix in solution, with implications for substrate binding and recognition.

Backbone 1H, 15N and 13C resonance assignments for dengue NS2B without the NS3 protease cofactor region in detergent micelles.

Dengue virus is an important human pathogen affecting people especially in tropical and subtropical regions. Its genome encodes seven non-structural proteins that are important for viral assembly and replication. Dengue NS2B is a membrane protein containing four transmembrane helices and involved in protein-protein interactions. Its transmembrane helices are critical for location of NS2B on the cell membrane while one cytoplasmic region composed of approximately 40 amino acids serves as a cofactor of viral NS3 protease by forming a tight complex with the N-terminal region of NS3. Here, we report the backbone resonance assignments for a dengue NS2B construct referred to as mini-NS2B containing only the transmembrane regions without NS3 cofactor region in detergent micelles. Mini-NS2B exhibits well-dispersed cross-peaks in the 1H-15N-HSQC spectrum and contains four helices in solution. The available mini-NS2B and its assignment will be useful for determining the structure of NS2B and identifying small molecules binding to the transmembrane regions.

Formation of lanthanide luminescent di-metallic helicates in solution using a bis-tridentate (1,2,3-triazol-4-yl)-picolinamide (tzpa) ligand.

The chiral bis-tridentate (1,2,3-triazol-4-yl)-picolinamide (tzpa) ligand 1 was used in the formation of lanthanide di- and triple stranded di-metallic helicates in acetonitrile solution, where the changes in the ground and the Tb(III) excited state properties were used to monitor the formation of these supramolecular structures in situ under kinetic control.

A Chiral 19F NMR Reporter of Foldamer Conformation in Bilayers.

Understanding and controlling peptide foldamer conformation in phospholipid bilayers is a key step toward their use as molecular information relays in membranes. To this end, a new 19F "reporter" tag has been developed and attached to dynamic peptide foldamers. The (R)-1-(trifluoromethyl)ethylamido ((R)-TFEA) reporter was attached to the C-terminus of α-amino-iso-butyric acid (Aib) foldamers. Crystallography confirmed that the foldamers adopted 310 helical conformations. Variable temperature (VT) NMR spectroscopy in organic solvents showed that the (R)-TFEA reporter had an intrinsic preference for P helicity, but the overall screw-sense was dominated by a chiral "controller" at the N-terminus. The 19F NMR chemical shift of the CF3 resonance was correlated with the ability of different N-terminal groups to induce either an M or a P helix in solution. In bilayers, a similar correlation was found. Solution 19F NMR spectroscopy on small unilamellar vesicle (SUV) suspensions containing the same family of (R)-TFEA-labeled foldamers showed broadened but resolvable 19F resonances, with each chemical shift mirroring their relative positions in organic solvents. These studies showed that foldamer conformational preferences are the same in phospholipid bilayers as in organic solvents and also revealed that phospholipid chirality has little influence on conformation.

Direct Measurement of Interhelical DNA Repulsion and Attraction by Quantitative Cross-Linking.

To better understand the forces that mediate nucleic acid compaction in biology, we developed the disulfide cross-linking approach xHEED (X-linking of Helices to measure Electrostatic Effects at Distance) to measure the distance-dependent encounter frequency of two DNA helices in solution. Using xHEED, we determined the distance that the electrostatic potential extends from DNA helices, the dependence of this distance on ionic conditions, and the magnitude of repulsion when two helices approach one another. Across all conditions tested, the potential falls to that of the bulk solution within 15 Å of the major groove surface. For separations of ∼30 Å, we measured a repulsion of 1.8 kcal/mol in low monovalent ion concentration (30 mM Na+), with higher Na+ concentrations ameliorating this repulsion, and 2 M Na+ or 100 mM Mg2+ eliminating it. Strikingly, we found full screening at very low Co3+ concentrations and net attraction at higher concentrations, without the higher-order DNA condensation that typically complicates studies of helical attraction. Our measurements define the relevant distances for electrostatic interactions of nucleic-acid helices in biology and introduce a new method to propel further understanding of how these forces impact biological processes.

Coal-Tar Dye-based Coordination Cages and Helicates.

A strategy to implement four members of the classic coal‐tar dye family, Michler's ketone, methylene blue, rhodamine B, and crystal violet, into [Pd2L4] self‐assemblies is introduced. Chromophores were incorporated into bis‐monodentate ligands using piperazine linkers that allow to retain the auxochromic dialkyl amine functionalities required for intense colors deep in the visible spectrum. Upon palladium coordination, ligands with pyridine donors form lantern‐shaped dinuclear cages while quinoline donors lead to strongly twisted [Pd2L4] helicates in solution. In one case, single crystal X‐ray diffraction revealed rearrangement to a [Pd3L6] ring structure in the solid state. For nine examined derivatives, showing colors from yellow to deep violet, CD spectroscopy discloses different degrees of chiral induction by an enantiomerically pure guest. Ion mobility mass spectrometry allows to distinguish two binding modes. Self‐assemblies based on this new ligand class promise application in chiroptical recognition, photo‐redox catalysis and optical materials.

Directed Evolution Using Stabilized Bacterial Peptide Display.

Chemically stabilized peptides have attracted intense interest by academics and pharmaceutical companies due to their potential to hit currently "undruggable" targets. However, engineering an optimal sequence, stabilizing linker location, and physicochemical properties is a slow and arduous process. By pairing non-natural amino acid incorporation and cell surface click chemistry in bacteria with high-throughput sorting, we developed a method to quantitatively select high affinity ligands and applied the Stabilized Peptide Evolution by E. coli Display technique to develop disrupters of the therapeutically relevant MDM2-p53 interface. Through in situ stabilization on the bacterial surface, we demonstrate rapid isolation of stabilized peptides with improved affinity and novel structures. Several peptides evolved a second loop including one sequence (Kd = 1.8 nM) containing an i, i+4 disulfide bond. NMR structural determination indicated a bent helix in solution and bound to MDM2. The bicyclic peptide had improved protease stability, and we demonstrated that protease resistance could be measured both on the bacterial surface and in solution, enabling the method to test and/or screen for additional drug-like properties critical for biologically active compounds.

Helical Self-Assembly of Optically Active Glycoconjugated Phthalocyanine J-Aggregates.

Four galactoconjugated zinc(II) phthalocyanines (Pcs) have been prepared and fully characterized. The carbohydrate-containing phthalonitrile precursors of the Pcs were synthesized through a copper-catalysed azide-alkyne cycloaddition (CuAAC). The Pcs show a remarkable aggregation behaviour in solution, depending on the nature of the solvent, the temperature and the substitution position on the phthalocyanine. Solvent-dependent CD-spectroscopy experiments show that these Pcs aggregate as chiral helices in solution. Crystal structure data of a phthalocyanine bearing two carbohydrate units substantiate the properties shown by CD spectroscopy. Furthermore, the 1,2,3-triazole moieties of the Pcs play a decisive role in the formation of supramolecular aggregates. The glycoconjugated zinc(II) phthalocyanines described here show molar extinction coefficients ϵmax >105 M-1 cm-1 and absorption maxima λmax >680 nm, which make them attractive photosensitizers for Photodynamic Therapy (PDT).

Hydrogen-Deuterium Exchange and Electron Capture Dissociation to Interrogate the Conformation of Gaseous Melittin Ions.

There is a need in the field of biological mass spectrometry for structural tools which can report on regional, rather than solely global, structure of gaseous protein ions. Site-specific hydrogen-deuterium (H/D) exchange has shown promise in fulfilling this need, but requires additional method development to prove its utility. In this study, we use H/D exchange and electron capture dissociation (ECD) to probe the gaseous structure of two peptides which are α-helical in solution and which differ by a single point mutation. Global H/D exchange levels, ECD fragmentation profiles, and region specific H/D exchange profiles are compared between wild type (WT) melittin, which adopts a hinged helix conformation in solution, and a mutant P14A melittin which folds into a single helix in solution. High protection from H/D exchange by both peptides is consistent with retention of secondary structure in the gas phase (or refolding into some other compact structure). The P14A mutant melittin exhibits lower ECD fragmentation efficiency than WT melittin, suggesting that it contains more secondary structure in the gas phase, which may indicate that these peptides retain some memory of their solution-phase structures. Examination of the isotopic distributions of fragment ions derived from H/D exchange with subsequent ECD reveals that the C-terminus of these peptides adopts multiple conformations. The results reported here offer insight into the stability of alpha helices in the gas phase, and also highlight the value of combining gas-phase H/D exchange with electron capture dissociation to interrogate gaseous peptide conformation.

A Long-Lived Halogen-Bonding Anion Triple Helicate Accommodates Rapid Guest Exchange.

Anion-templated helical structures are emerging as a dynamic and tractable class of supramolecules that exhibit anion-switchable self-assembly. We present the first kinetic studies of an anion helicate by utilizing halogen-bonding m-arylene-ethynylene oligomers. These ligands formed high-fidelity triple helicates in solution with surprisingly long lifetimes on the order of seconds even at elevated temperatures. We propose an associative ligand-exchange mechanism that proceeded slowly on the same timescale. In contrast, intrachannel anion exchange occurred rapidly within milliseconds or faster as determined by stopped-flow visible spectroscopy. Additionally, the helicate accommodated bromide in solution and the solid state, while the thermodynamic stability of the triplex favored larger halide ions (bromide≈iodide≫chloride). Taken together, we elucidate a new class of kinetically stable helicates. These anion-switchable triplexes maintain their architectures while accommodating fast intrachannel guest exchange.

A Long‐Lived Halogen‐Bonding Anion Triple Helicate Accommodates Rapid Guest Exchange

Anion-templated helical structures are emerging as a dynamic and tractable class of supramolecules that exhibit anion-switchable self-assembly. We present the first kinetic studies of an anion helicate by utilizing halogen-bonding m-arylene-ethynylene oligomers. These ligands formed high-fidelity triple helicates in solution with surprisingly long lifetimes on the order of seconds even at elevated temperatures. We propose an associative ligand-exchange mechanism that proceeded slowly on the same timescale. In contrast, intrachannel anion exchange occurred rapidly within milliseconds or faster as determined by stopped-flow visible spectroscopy. Additionally, the helicate accommodated bromide in solution and the solid state, while the thermodynamic stability of the triplex favored larger halide ions (bromide≈iodide≫chloride). Taken together, we elucidate a new class of kinetically stable helicates. These anion-switchable triplexes maintain their architectures while accommodating fast intrachannel guest exchange.

Glatiramer acetate persists at the injection site and draining lymph nodes via electrostatically-induced aggregation

Glatiramer acetate (GA) is widely prescribed for the treatment of relapsing-remitting multiple sclerosis, however, the mechanism of action is still not fully understood. We investigated the structural properties of GA and examined alterations to the drug upon injection into the subcutaneous space. First, a variety of biophysical characterization techniques were employed to characterize GA in solution. GA was found to exist as alpha helices in solution with a hydrodynamic radius of ~3 nm in size. To simulate GA behavior at the site of injection, GA was injected into a solution of 1.5 MDa hyaluronic acid (HA). Visible aggregates were observed immediately upon injection and subsequent testing indicated aggregation was driven by electrostatic interactions between the positively-charged GA and negatively-charged HA. In vivo testing confirmed GA formed spherical particles in the nano- to micrometer size range, suggesting this mechanism contributes to persistence at the injection site and in draining lymph nodes. The aggregates were found to associate with glycosaminoglycans, suggesting an electrostatic mechanism of induced aggregation like the simulated injection. These novel observations may help explain the complex immunomodulatory mechanisms of GA and adverse injection site reactions seen in patients.

Steric and Electronic Factors Affecting the Conformation of Bimetallic CuI Complexes: Effect of the Aliphatic Spacer of Tetracoordinating Schiff-Base Ligands.

A family of six homoleptic [CuI (Ln )]2 (ClO4 )2 and six heteroleptic [CuI (Ln )(PPh3 )2 ]2 (ClO4 )2 bimetallic complexes, in which Ln are bis-Schiff base ligands with alkyl spacers of variable length (n=2-7 -CH2 -), were prepared to evaluate the role of the spacer on the formation of helicates or mesocates. In the homoleptic series, spectroscopic and theoretical studies indicate that preferences for a conformation are based on energetic parameters, mainly, the establishment of noncovalent interactions. The odd-even nature of the spacers preconditions the superposition of the aromatic rings to allow the juxtaposition necessary for noncovalent interactions, whereas the increase of the length reduces the strength of such interactions. Consequently, complexes with even-spacers of short length were identified as helicates in solution, [CuI (Ln )]22+ (n=2, 4). Complexes [CuI (Ln )]22+ (n=3-7) dissociate in solution to produce the monometallic complexes in equilibrium, [CuI (Ln )]+ . The stability of the bimetallic species is discussed in terms of their conformations. The set of heteroleptic complexes was prepared to evaluate the reach of the "odd-even rule" in the solid, which is based on the "zig-zag" arrangements of the spacers. Based on crystallographic information, "S-" and "C"-type conformations of Ln are related to even and odd spacers, respectively. This trend is considered in addition to other factors to explain preferences for either a mesocate or helicate conformation in the homoleptic series.

Symmetry Breaking and Amplification in a Self-Assembled Helix from Achiral trans-3-Nitrocinnamic Acid

Understanding the process of chiral symmetry breaking, amplification, and transfer to the macroscopic chirality in the self-assembled system has been a subject of essential challenge and immense interest. Here, we show that a simple trans-3-nitrocinnamic acid (3-NCA) self-assembles into helix in solution with symmetry breaking. Upon casting on a solid substrate, the tiny chiral bias in solution was amplified into macroscopic chirality, which could be detected via circular dichroism measurements. Furthermore, the scanning electron microscopy observation revealed the unequal numbers of left- and right-handed helixes, which account for the net supramolecular chirality. In comparison with the other isomers or related molecules, it was found that only 3-NCA showed symmetry breaking. It was revealed that the hydrogen bond and dipole–dipole interaction are responsible for the spontaneous symmetry breaking. The work provides a further insight into the emergence and amplification of supramolecular chirality in sel...

The effect of the linker size in C2-symmetrical chiral ligands on the self-assembly formation of luminescent triple-stranded di-metallic Eu(iii) helicates in solution.

Chiral lanthanide-based supramolecular structures have gained significant importance in view of their application in imaging, sensing and other functional purposes. We have designed chiral C2-symmetrical ligands (L) based on the use of two 2,6-pyridine-dicarboxylic-amide moieties (pda), that differ from one another by the nature of the diamine spacer groups (from 1,3-phenylenedimethanamine (1(S,S), 2(R,R)) and benzene-1,3-diamine (3(S,S), 4(R,R)) to much bulkier 4,4'-(cyclohexane-1,1-diyl)bis(2,6-dimethylaniline) (5(S,S), 6(R,R))) between these two pda units. The self-assembly between L and Eu(iii) ions were investigated in CH3CN solution at low concentration whereby the changes in the absorbance, fluorescence and Eu(iii)-centred emission spectra allowed us to model the binding equilibria occurring in the solution to the presence of [Eu:L2], [Eu2:L2], [Eu2:L3] assemblies and reveal their high binding constant values. The self-assembly in solution were also studied at higher concentration by following the changes in the 1H NMR spectra of the ligands upon Eu(iii) addition, as well as by using MALDI-MS of the isolated solid state complexes. The chiroptical properties of the ligands were used in order to study the structural changes upon self-assembly between the ligands and Eu(iii) ions using circular dichroism (CD) and circularly polarised luminescence (CPL) spectroscopies. The photophysical properties of [Eu2:L3] complexes were evaluated in solution and showed a decrease of luminescence quantum yield when going from the ligand with smaller (1(S,S)) to bulkier (5(S,S)) linker from ∼5.8% to ∼2.6%. While mass-spectrometry revealed the possible formation of trinucler assemblies such as [Eu3:L3] and [Eu3:L2].

Lactam-Stapled Cell-Penetrating Peptides: Cell Uptake and Membrane Binding Properties.

Stapling of side chains to stabilize an α-helical structure has been generally associated with an increased uptake of CPPs. Here, we compare four amphiphilic stapled peptides with their linear counterparts in terms of their membrane binding and conformational features in order to correlate these with uptake efficiency and toxicological effects. The impact of lactam stapling was found to vary strongly with regard to the different aspects of peptide-membrane interactions. Nearly all stapled peptides caused less membrane perturbation (vesicle leakage, hemolysis, bacterial lysis) than their linear counterparts. In one case (MAP-1) where stapling enhanced α-helicity in aqueous and lipid environments, leakage was eliminated while cell uptake in HEK293 and HeLa cells remained high, which improved the overall characteristics. The other systems (DRIM, WWSP, KFGF) did not improve, however. The data suggest that cell uptake of amphipathic CPPs correlates with their adopted α-helix content in membranes rather than their helicity in solution.

Non-linearity of the collagen triple helix in solution and implications for collagen function.

Collagen adopts a characteristic supercoiled triple helical conformation which requires a repeating (Xaa-Yaa-Gly)n sequence. Despite the abundance of collagen, a combined experimental and atomistic modelling approach has not so far quantitated the degree of flexibility seen experimentally in the solution structures of collagen triple helices. To address this question, we report an experimental study on the flexibility of varying lengths of collagen triple helical peptides, composed of six, eight, ten and twelve repeats of the most stable Pro-Hyp-Gly (POG) units. In addition, one unblocked peptide, (POG)10unblocked, was compared with the blocked (POG)10 as a control for the significance of end effects. Complementary analytical ultracentrifugation and synchrotron small angle X-ray scattering data showed that the conformations of the longer triple helical peptides were not well explained by a linear structure derived from crystallography. To interpret these data, molecular dynamics simulations were used to generate 50 000 physically realistic collagen structures for each of the helices. These structures were fitted against their respective scattering data to reveal the best fitting structures from this large ensemble of possible helix structures. This curve fitting confirmed a small degree of non-linearity to exist in these best fit triple helices, with the degree of bending approximated as 4–17° from linearity. Our results open the way for further studies of other collagen triple helices with different sequences and stabilities in order to clarify the role of molecular rigidity and flexibility in collagen extracellular and immune function and disease.

Effect of Transient Helicity of cMyb TAD on its Binding Affinity to the Kix Domain of CBP/p300

Intrinsically disordered proteins (IDPs) are involved in various protein‐protein interactions and they often experience a coupled folding and binding reaction when they bind to their protein partners. Many IDPs, like the cMyb trans activation domain (cMyb TAD), exhibit transient helical structures in solution that resemble their bound structures. We hypothesize that the levels of transient helicity in the free IDP will affect the binding affinity to their protein partners in a predictable way. Previous studies have shown that increasing the transient helicity of intrinsically disordered p53 tumor suppressor increases the binding affinity to MDM2 and had negative effects on cellular function. We aim to study the effect of changing cMyb TAD transient helicity on its binding affinity to the kix domain of CBP/p300.cMyb is a hematopoietic regulator which is known to play a role in tumorigenesis. Unlike p53 TAD, cMyb TAD has a high residual helicity in solution in the absence of binding partners. Prolines that flank the N and C‐terminus of this helical region were mutated to alanines to determine whether there was a change in helicity that would affect the binding of Kix. Upon mutation, the increase in helicity had almost no effect on the binding affinity of the cMyb‐Kix complex. Further, mutations that reduce the helicity of cMyb‐TAD were designed to test the effect of reducing transient helicity on the binding affinity to Kix. To achieve this goal, conservative mutations of the solvent exposed amino acids in cMyb were made. We are currently measuring the transient helicity and binding affinity of these mutants with the long‐range goal of identifying a general relationship between changes in transient helicity and binding affinity of IDPs.Support or Funding InformationThis research was supported by funding to GWD by National Institutes of Health (2R01CA14124406‐A1 and 1R01GM115556‐01A1). This work was also supported by funding to JC by Wellcome trust (WT 095195MA) and by a Biotechnology and Biological Sciences Research Council (BBSRC) studentship to MDC.

A Halogen-Bond-Induced Triple Helicate Encapsulates Iodide.

The self-assembly of higher-order anion helicates in solution remains an elusive goal. Herein, we present the first triple helicate to encapsulate iodide in organic and aqueous media as well as the solid state. The triple helicate self-assembles from three tricationic arylethynyl strands and resembles a tubular anion channel lined with nine halogen bond donors. Eight strong iodine⋅⋅⋅iodide halogen bonds and numerous buried π-surfaces endow the triplex with remarkable stability, even at elevated temperatures. We suggest that the natural rise of a single-strand helix renders its linear halogen-bond donors non-convergent. Thus, the stringent linearity of halogen bonding is a powerful tool for the synthesis of multi-strand anion helicates.

A Halogen‐Bond‐Induced Triple Helicate Encapsulates Iodide

AbstractThe self‐assembly of higher‐order anion helicates in solution remains an elusive goal. Herein, we present the first triple helicate to encapsulate iodide in organic and aqueous media as well as the solid state. The triple helicate self‐assembles from three tricationic arylethynyl strands and resembles a tubular anion channel lined with nine halogen bond donors. Eight strong iodine⋅⋅⋅iodide halogen bonds and numerous buried π‐surfaces endow the triplex with remarkable stability, even at elevated temperatures. We suggest that the natural rise of a single‐strand helix renders its linear halogen‐bond donors non‐convergent. Thus, the stringent linearity of halogen bonding is a powerful tool for the synthesis of multi‐strand anion helicates.