Temperature-dependent CD Research Articles

Elucidating the stabilization mechanism of a K+-depleted c-MYC DNA G-quadruplex in hydrophobic imidazolium-based ionic liquids using spectroscopy coupled with molecular dynamics simulations

Stabilizing native nucleic acid structures is critical for developing nucleic acid-based therapeutics, which have enormous potential in biomedicine for disease prevention, disease treatment and diagnostics. Imidazolium chloride ionic liquids (ImCl ILs) can increase the thermal stability of G-Quadruplex DNA structures. Generally, G-Quadruplex DNA requires K+ (or sometimes Na+) to stabilize their structures via coordination of the guanine bases. In this work we investigated the ability of ImCl ILs to stabilize G-Quadruplex DNA without K+ present. Temperature-dependent CD spectroscopy showed that the ImCl ILs that can form micelles in aqueous solution ([OMIM]Cl and [DMIM]Cl) increase the thermal stability of G-Quadruplex DNA without K+, while the ImCl ILs that do not form micelles ([EMIM]Cl, [BMIM]Cl, and [HMIM]Cl) do not stabilize the structure. Notably, [OMIM]Cl and [DMIM]Cl only stabilize G-Quadruplex DNA at concentrations above their CMC values; at concentrations equal to their CMC values these ILs actually destabilize the structure. MD simulations were performed to elucidate the mechanism of thermal stabilization. Simulations are consistent with experimental results as [DMIM]Cl micelles keep the G-Quadruplex structure stable without K+ present. These results show how ILs can encapsulate and stabilize DNA, which can help guide the development of new strategies for stabilizing DNA and RNA structures at room temperature.

Solvent-dependent supramolecular assembly behavior of a coumarin-headed amphiphile.

To study the effect of solvent on supramolecular self-assembly behaviors, a chiral courmarin-substituted glutamine amphiphile, L/DG-Cm, was synthesized for investigation. It was found that L/DG-Cm self-assembled into short nanotubes in toluene, while it formed longer nanotubes together with an obvious helix nanobelt structure for L/DG-Cm in DMSO, demonstrating that the nanotubes were formed by nanobelt rolling. The CD and CPL spectra revealed the same chiral property of the L/DG-Cm assemblies formed in toluene and DMSO. Theoretical calculations revealed that LG-Cm was prone to forming similar dimer structures in both DMSO and toluene. However, the distinct hierarchical packing ways in toluene and DMSO led to different nanostructures and chiroptical properties. Based on the temperature-dependent UV-visible and CD spectrometric measurements, LG-Cm was observed to aggregate in different supramolecular self-assembly modes, which was the cooperative (nucleation-elongation) mechanism in toluene and the isodesmic model in DMSO. This work proves that the solvent not only affects the self-assembly morphologies and properties but also determines the self-assembly pathways.

Synthesis and Structural Stability of α-Helical Gold(I)-Metallopeptidesy

AbstractThe synthesis of hexa- and dodecapeptides functionalized with two Au(I)–phosphine complexes is reported. The high stability of the Au(I)–phosphine bond allowed orthogonal peptide-protecting-group chemistry, even when using hard Lewis acids like boron tribromide. This enabled the preparation of an Fmoc-protected lysine derivative carrying the Au(I) complex in a side chain, which was used in standard Fmoc-based solid-phase peptide synthesis protocols. Alanine and leucine repeats in the metallododecapeptide formed α-helical secondary structures in 2,2,2-trifluoroethanol–H2O and 1,1,1,3,3,3-hexafluoroisopropanol–H2O mixtures with high thermal stability, as shown by temperature-dependent CD spectroscopy studies.

Multi-responsive luminescence sensing behaviour of a pair of temperature-dependent Cd(II) coordination polymers

Adopting the temperature-controlling synthesis strategy, a pair of temperature-dependent Cd(II) coordination polymers (CPs) with the mixed 5-amino-2,4,6-tribromoisophthalic acid (H2ATBIP) and 1,4-bis(triazol-1-ylmethyl)benzene (bbtz), {[Cd(HATBIP)2(bbtz)(H2O)2]·2H2O}n (1) and {[Cd(HATBIP)2(bbtz)2]·2H2O}n (2), were obtained using the typical solution-evaporation method at room temperature and under hydrothermal condition at 100 °C, respectively. The ligand bbtz in 1 exhibits a TT (trans–trans) conformation and links the adjacent Cd(II) atoms into a chain polymer. The hydrogen-bonded centrosymmetric ring R22(8) further holds the neighbouring chains into a two-dimensional (2D) supramolecular network. With the aid of the interlayer hydrogen and halogen bonding, a 3D supramolecular network with the “ABAB” packing mode is formed. In 2, the bbtz ligand bridges the adjacent Cd(II) ions, resulting in a 2D sql topological network with the Schläfli symbol of (44.62), in which the HATBIP− ligand arranges alternatingly on both sides of the layer. The ligand HATBIP− shows monodentate coordination mode in 1 and 2. The indirect band gap exists in 1 and 2. Both 1 and 2 show multi-responsive luminescence sensing behaviour toward the Fe3+ ion, the Cr(VI) (Cr2O72−/CrO42−) anions and some NACs. Interestingly, 1 and 2 demonstrate distinct structure-dependent tuneable sensing sensitivities for these analytes. 1 has higher sensing sensitivity with lower limit of detection (LOD) than that of 2 for the same analyte. The sensing mechanisms have been illustrated.

Two temperature-dependent Cd(II)-based coordination polymers with mixed adenine nucleobase and benzene-1,4-dicarboxylic acid: synthesis, structures and fluorescence properties

Two Hade-based Cd(II) polymers, [Cd(Hade)(tp)]n (1) and {[Cd6(Hade)6(tp)6]·9H2O}n (2) (Hade = adenine, H2tp = benzene-1,4-dicarboxylic acid), were obtained from the same reaction system at different temperatures which have been characterized by single-crystal X-ray diffraction, elemental analysis, FT-IR spectroscopy, thermogravimetric analysis, and luminescence spectroscopy. Significantly resulting from the synergistic coordination of nucleobase and bicarboxylate groups, they are a 3-D pillared-layer structure for 1 and a 2-D grid structure where the dicadmium paddle-wheel motifs are double bridged by benzene-1,4-dicarboxylate anions for 2. The supramolecular architecture of 2 is essentially knitted by hydrogen bonding interactions produced by the exocyclic amino/endocyclic imino groups of the nucleobase and the carboxylate groups of the co-ligand to favorably stabilize the high-dimensional supramolecular architectures. At room temperature, 1 and 2 exhibit intense luminescent emissions originating from a Hade-based intraligand and/or photoinduced charge transfer upon adenine binding mode.

Stable Hg(II)-mediated base pairs with a phenanthroline-derived nucleobase surrogate in antiparallel-stranded DNA

Metal-mediated base pairs involving artificial nucleobases have emerged as a promising means for the site-specific functionalization of nucleic acids with metal ions. In this context, a GNA-appended (GNA: glycol nucleic acid) nucleoside analogue containing the artificial nucleobase 1H-imidazo[4,5-f][1,10]phenanthroline (P) has already been applied successfully in a variety of homo- and heteroleptic metal-mediated base pairs, mainly involving Ag(I) ions. Herein, we report a thorough investigation of the Hg(II)-binding properties of P when incorporated into antiparallel-stranded DNA duplexes. The artificial nucleobase P is able to form Hg(II)-mediated homoleptic base pairs of the type P–Hg(II)–P with a [2 + 2] coordination environment. In addition, the heteroleptic P–Hg(II)–T pair was investigated. The addition of a stoichiometric amount of Hg(II) to a duplex comprising either a P:P pair or a P:T pair stabilizes the DNA duplex by 4.3 °C and 14.5 °C, respectively. The P–Hg(II)–T base pair, hence, represents the most stabilizing non-organometallic Hg(II)-mediated base pair reported to date. The formation of the Hg(II)-mediated base pairs was investigated by means of temperature-dependent UV spectroscopy and CD spectroscopy.Graphic abstract

Interaction of carborane-appended trimer with bovine serum albumin: A spectroscopic investigation

The interaction of symmetrical triazine-cored carborane-appended trimer 5 with bovine serum albumin has been investigated using different spectroscopic methods. The intrinsic fluorescence of BSA was quenched by the carborane trimer 5 through the dynamic quenching mechanism and the estimated binding constant Kb value (3.3–4.1) × 105 M−1 is similar to those obtained from the interaction of commercial drugs such as aspirin, ibuprofen and ceftriaxone with BSA. The conformational changes of BSA were monitored by CD analysis and temperature dependent CD measurements were done to study the thermal stability of BSA in presence of carborane-appended trimer 5.

Do Osmolytes Impact the Structure and Dynamics of Myoglobin?

Osmolytes are small organic compounds that can affect the stability of proteins in living cells. The mechanism of osmolytes’ protective effects on protein structure and dynamics has not been fully explained, but in general, two possibilities have been suggested and examined: a direct interaction of osmolytes with proteins (water replacement hypothesis), and an indirect interaction (vitrification hypothesis). Here, to investigate these two possible mechanisms, we studied myoglobin-osmolyte systems using FTIR, UV-vis, CD, and femtosecond IR pump-probe spectroscopy. Interestingly, noticeable changes are observed in both the lifetime of the CO stretch of CO-bound myoglobin and the spectra of UV-vis, CD, and FTIR upon addition of the osmolytes. In addition, the temperature-dependent CD studies reveal that the protein’s thermal stability depends on molecular structure, hydrogen-bonding ability, and size of osmolytes. We anticipate that the present experimental results provide important clues about the complicated and intricate mechanism of osmolyte effects on protein structure and dynamics in a crowded cellular environment.

Stable Copper(I)-Mediated Base Pairing in DNA.

A GNA (glycol nucleic acid) functionalized nucleoside analogue containing the artificial nucleobase 1H-imidazo[4,5-f][1,10]phenanthroline (P) was used to form a copper(I)-mediated base pair within a DNA duplex. The geometrical constraints imposed by the artificial nucleobase play a pivotal role in this unprecedented stabilization of copper(I) in aqueous medium via metal-mediated base pairing. The formation of the copper(I)-mediated base pair was investigated by temperature-dependent UV spectroscopy and CD spectroscopy. The metal-mediated base pair stabilizes the DNA oligonucleotide duplex by 23 °C. A redox chemistry approach confirmed that this base pair formation was due to the incorporation of copper(I) into the duplex. This first report of a copper(I)-mediated base pair adds metal-based diversity to the field and consequently opens up the range of possible applications of metal-modified nucleic acids.

Probing the quality control mechanism of the Escherichia coli twin-arginine translocase with folding variants of a de novo–designed heme protein

Protein transport across the cytoplasmic membrane of bacterial cells is mediated by either the general secretion (Sec) system or the twin-arginine translocase (Tat). The Tat machinery exports folded and cofactor-containing proteins from the cytoplasm to the periplasm by using the transmembrane proton motive force as a source of energy. The Tat apparatus apparently senses the folded state of its protein substrates, a quality-control mechanism that prevents premature export of nascent unfolded or misfolded polypeptides, but its mechanistic basis has not yet been determined. Here, we investigated the innate ability of the model Escherichia coli Tat system to recognize and translocate de novo–designed protein substrates with experimentally determined differences in the extent of folding. Water-soluble, four-helix bundle maquette proteins were engineered to bind two, one, or no heme b cofactors, resulting in a concomitant reduction in the extent of their folding, assessed with temperature-dependent CD spectroscopy and one-dimensional 1H NMR spectroscopy. Fusion of the archetypal N-terminal Tat signal peptide of the E. coli trimethylamine-N-oxide (TMAO) reductase (TorA) to the N terminus of the protein maquettes was sufficient for the Tat system to recognize them as substrates. The clear correlation between the level of Tat-dependent export and the degree of heme b–induced folding of the maquette protein suggested that the membrane-bound Tat machinery can sense the extent of folding and conformational flexibility of its substrates. We propose that these artificial proteins are ideal substrates for future investigations of the Tat system's quality-control mechanism.

Purification, chitooligosaccharide binding properties and thermal stability of CIA24, a new PP2-like phloem exudate lectin from ivy gourd (Coccinia indica)

PP2-like chitin binding phloem exudate lectins, abundant in the sieve tube of cucurbits, have been implicated to play key roles in wound sealing and antipathogenic responses of the plant. Here we report the affinity purification, macromolecular characterization and carbohydrate binding properties of a new chitooligosaccharide-specific lectin from the phloem exudate of ivy gourds (Coccinia indica). The protein, CIA24, has a subunit mass of 24 kDa. Partial sequence analysis indicated that CIA24 exhibits high homology with CIA17 and other Cucurbitaceae PP2 proteins whereas CD spectroscopic studies suggested that β-sheets constitute the predominant secondary structure. Temperature dependent CD spectroscopic and differential scanning calorimetric studies revealed that CIA24 is a highly thermostable protein, which undergoes complete unfolding at ∼105 °C. Isothermal titration calorimetric studies suggested that binding of chitooligosaccharides to CIA24 is a highly exothermic process. The lectin combining site can accommodate upto a tetrasaccharide with the binding stoichiometry (n) close to unity with respect to each protein subunit, whereas for chitohexaose a sharp decrease in the binding stoichiometry (n) to ∼1:0.5 was observed. This suggests that the protein probably undergoes dimerisation in presence of chitohexaose, wherein two protein molecules bind to the oligosaccharides from the reducing and non-reducing end, respectively.

Biological Semiconductors: Structural Control of Heme Redox Potentials in PpcA, a 3-Heme Cytochrome

In an effort to tune and optimize relative bandgap potentials found in biological nanowires that form from multiheme domains of members of the cytochrome c7 family, we developed and extensively characterized 12 point mutations of PpcA, a 3-heme member of the cytochrome c7 family native to Geobacter sulfurreducens. These mutations were engineered to influence the redox potential (Em) of the middle heme (heme III) in PpcA by using four different strategies: performing charge reversal mutations, decreasing solvent access to the heme plane with bulky residues, altering the native bis-histidine axial ligation of the heme, and by attempting to form hydrogen bonds with the propionates of the heme. The latter strategy is expected not only to increase Em but also to introduce a redox Bohr effect. Out of 12 mutants, 11 were successfully expressed in E.coli and show thermal stability in temperature-dependent CD experiments comparable to the wild-type protein (Tm > 90°C). HPLC-ESI-MS was used to confirm both the purity and the mass of the expressed mutants. Small-angle X-ray scattering confirmed that the mutant proteins formed the expected compact globular structures. In addition to ongoing attempts to obtain protein crystals for X-ray crystallography, we performed extensive all-atom molecular dynamics simulations for all mutants. Formation of propionate hydrogen bonds is supported by simulations for A19R and A23R, while increased solvent access is supported for A19I, A19R, A23R, H20M, and K60E. UV/Vis and NMR redox titrations were performed in order to measure the effect of the mutations on the electrochemical properties of all 3 hemes and to understand the underlying principles and viable approaches in tuning relative heme redox potentials. Successful development of this project may lead to biological semiconductors with much smaller footprints and selectively tunable bandgap properties.

A metal-mediated base pair that discriminates between the canonical pyrimidine nucleobases.

A nucleoside analogue comprising the ligand 1H-imidazo[4,5-f][1,10]phenanthroline (P) was applied to develop a molecular beacon capable of discriminating the canonical nucleobases cytosine and thymine. The beacon is based on the formation of a stable Ag+-mediated base pair between P and cytosine, whereas the presence of Ag+ strongly destabilizes nucleic acids comprising an artificial base pair between P and thymine. Metal-mediated base pair formation was investigated by temperature-dependent UV spectroscopy and CD spectroscopy and complemented by extensive DFT calculations. The molecular beacon significantly extends the application spectrum of nucleic acids with metal-mediated base pairs. It is of potential use in the detection of single-nucleotide polymorphisms.

Unraveling the interaction of hemoglobin with a biocompatible and cleavable oxy-diester-functionalized gemini surfactant

Surfactant-protein mixtures have attracted considerable research interest in recent years at the interface of chemical biology and medicinal chemistry. Herein, the interaction between a green gemini surfactant (C16-E2O-C16) and a redox protein hemoglobin was examined through a series of in vitro experimental techniques with an attempt to provide a comprehensive knowledge of the surfactant-protein binding interactions. Quantitative appraisal of the fluorescence/CV data showed that the binding of C16-E2O-C16 to Hb leads to the formation of thermodynamically favorable non-covalent adduct with 1:1 stoichiometry. UV–vis spectra demonstrated that the effect of C16-E2O-C16 on Hb is highly concentration dependent. Far-UV and near-UV CD spectra together elucidated the formation of molten globule state of Hb upon C16-E2O-C16 addition. Temperature dependent CD explicated the effect of C16-E2O-C16 on the thermal stability of Hb. Furthermore, the structural investigation of Hb via pyrene/synchronous/three-dimensional fluorescence and FT-IR spectroscopy provided the complementary information related to its microenvironmental and conformational changes. Computational studies delineated that C16-E2O-C16 binds in the vicinity of β-37 Trp at the α1β2 interface of Hb. Overall, this study is expected to clarify the binding mechanism between Hb/other congeners and surfactant at the molecular level that are known to have immense potential in biomedical and industrial areas.

Cooperative supramolecular helical assembly of a pyridinium-tailored methyl glycyrrhetate.

Taking a natural triterpenoid as the building block, we have regulated a pyridinium-functionalized methyl glycyrrhetate (C4-MGP) into P-type helices in water primarily driven by hydrophobic forces. By analysing their temperature-dependent CD and UV-Vis spectra, these hierarchical chiral assemblies were found to be formed in a cooperative supramolecular polymerization manner.

NMR Analysis of Tuning Cross-Strand Phe/Tyr/Trp-Trp Interactions in Designed β-Hairpin Peptides: Terminal Switch from L to D Amino Acid as a Strategy for β-Hairpin Capping.

Interaction among the side chains of aromatic amino acids is a well-known mechanism of protein and peptide structure stabilization, particularly in β sheets. Using short β-hairpin models bearing the sequence Ac-Leu-Xxx-Val-DPro-Gly-Leu-Trp-Val-NH2, we report the surprising observation of significant destabilization in aryl–tryptophan interactions, which results in poorly folded peptide populations accompanied by lowering of stability. We find that such destabilization arises from forced occupancy of the indole ring in the shielded Edge position, in T-shaped aryl geometries. We demonstrate that this destabilizing effect can be efficiently salvaged by replacing the N-terminal LLeu with DLeu, which causes an increase in the folded hairpin population, while retaining Trp in the Edge position. Our observation of unique cross strand NOEs and data from temperature-dependent NMR and CD measurements reveals the formation of a locally stabilized aliphatic–aromatic network, leading to an overall increase in ΔGF° by ∼ −0.6 to −1.2 kcal/mol. Our results suggest that a contextual evaluation of stabilization by tryptophan is necessary in β hairpins. Furthermore, we report for the first time that the use of D isomers of aliphatic amino acids at the terminus is stabilizing, which can serve as a new strategy for increasing β-hairpin stability.

A Highly Stabilizing Silver(I)‐Mediated Base Pair in Parallel‐Stranded DNA

The first parallel-stranded DNA duplex with Hoogsteen base pairing that readily incorporates an Ag(+) ion into an internal mispair to form a metal-mediated base pair has been created. Towards this end, the highly stabilizing 6 FP-Ag(+)-6 FP base pair comprising the artificial nucleobase 6-furylpurine (6 FP) was devised. A combination of temperature-dependent UV spectroscopy, CD spectroscopy, and DFT calculations was used to confirm the formation of this base pair. The nucleobase 6 FP is capable of forming metal-mediated base pairs both by the Watson-Crick edge (i.e. in regular antiparallel-stranded DNA) and by the Hoogsteen edge (i.e. in parallel-stranded DNA), depending on the oligonucleotide sequence and the experimental conditions. The 6 FP-Ag(+)-6 FP base pair within parallel-stranded DNA is the most strongly stabilizing Ag(+)-mediated base pair reported to date for any type of nucleic acid, with an increase in melting temperature of almost 15 °C upon the binding of one Ag(+) ion.

A series of temperature-dependent CdII-complexes containing an important family of N-rich heterocycles from in situ conversion of pyridine-type Schiff base

An efficient method for the synthesis of N-rich heterocycles has been systematically explored through solvothermal in situ generation from ligand (L) mediated by Cd2+. Their in situ formation mechanisms and coordination chemistry are investigated.

Foldameric α/β-Peptide Analogs of the β-Sheet-Forming Antiangiogenic Anginex: Structure and Bioactivity

The principles of β-sheet folding and design for α-peptidic sequences are well established, while those for sheet mimetics containing homologated amino acid building blocks are still under investigation. To reveal the structure-function relations of β-amino-acid-containing foldamers, we followed a top-down approach to study a series of α/β-peptidic analogs of anginex, a β-sheet-forming antiangiogenic peptide. Eight anginex analogs were developed by systematic α → β(3) substitutions and analyzed by using NMR and CD spectroscopy. The foldamers retained the β-sheet tendency, though with a decreased folding propensity. β-Sheet formation could be induced by a micellar environment, similarly to that of the parent peptide. The destructuring effect was higher when the α → β(3) exchange was located in the β-sheet core. Analysis of the β-sheet stability versus substitution pattern and the local conformational bias of the bulky β(3)V and β(3)I residues revealed that a mismatch between the H-bonding preferences of the α- and β-residues played a minor role in the structure-breaking effect. Temperature-dependent CD and NMR measurements showed that the hydrophobic stabilization was scaled-down for the α/β-peptides. Analysis of the biological activity of the foldamer peptides showed that four anginex derivatives dose-dependently inhibited the proliferation of a mouse endothelial cell line. The α → β(3) substitution strategy applied in this work can be a useful approach to the construction of bioactive β-sheet mimetics with a reduced aggregation tendency and improved pharmacokinetic properties.

Thioamides: Versatile Bonds To Induce Directional and Cooperative Hydrogen Bonding in Supramolecular Polymers

The amide bond is a versatile functional group and its directional hydrogen-bonding capabilities are widely applied in, for example, supramolecular chemistry. The potential of the thioamide bond, in contrast, is virtually unexplored as a structuring moiety in hydrogen-bonding-based self-assembling systems. We report herein the synthesis and characterisation of a new self-assembling motif comprising thioamides to induce directional hydrogen bonding. N,N',N''-Trialkylbenzene-1,3,5-tris(carbothioamide)s (thioBTAs) with either achiral or chiral side-chains have been readily obtained by treating their amide-based precursors with P2S5. The thioBTAs showed thermotropic liquid crystalline behaviour and a columnar mesophase was assigned. IR spectroscopy revealed that strong, three-fold, intermolecular hydrogen-bonding interactions stabilise the columnar structures. In apolar alkane solutions, thioBTAs self-assemble into one-dimensional, helical supramolecular polymers stabilised by three-fold hydrogen bonding. Concentration- and temperature-dependent self-assembly studies performed by using a combination of UV and CD spectroscopy demonstrated a cooperative supramolecular polymerisation mechanism and a strong amplification of supramolecular chirality. The high dipole moment of the thioamide bond in combination with the anisotropic shape of the resulting cylindrical aggregate gives rise to sufficiently strong depolarised light scattering to enable depolarised dynamic light scattering (DDLS) experiments in dilute alkane solution. The rotational and translational diffusion coefficients, D(trans) and D(rot), were obtained from the DDLS measurements, and the average length, L, and diameter, d, of the thioBTA aggregates were derived (L = 490 nm and d = 3.6 nm). These measured values are in good agreement with the value L(w) = 755 nm obtained from fitting the temperature-dependent CD data by using a recently developed equilibrium model. This experimental verification validates our common practice for determining the length of BTA-based supramolecular polymers from model fits to experimental CD data. The ability of thioamides to induce cooperative supramolecular polymerisation makes them effective and broadly applicable in supramolecular chemistry.