Combination Of Circular Dichroism Research Articles

Revealing the Binding Structure of the Protein Corona on Gold Nanorods Using Synchrotron Radiation-Based Techniques: Understanding the Reduced Damage in Cell Membranes

Regarding the importance of the biological effects of nanomaterials, there is still limited knowledge about the binding structure and stability of the protein corona on nanomaterials and the subsequent impacts. Here we designed a hard serum albumin protein corona (BSA) on CTAB-coated gold nanorods (AuNRs) and captured the structure of protein adsorption using synchrotron radiation X-ray absorption spectroscopy, microbeam X-ray fluorescent spectroscopy, and circular dichroism in combination with molecular dynamics simulations. The protein adsorption is attributed to at least 12 Au-S bonds and the stable corona reduced the cytotoxicity of CTAB/AuNRs. These combined strategies using physical, chemical, and biological approaches will improve our understanding of the protective effects of protein coronas against the toxicity of nanomaterials. These findings have shed light on a new strategy for studying interactions between proteins and nanomaterials, and this information will help further guide the rational design of nanomaterials for safe and effective biomedical applications.

Understanding the catalytic activity of single‐chain polymeric nanoparticles in water

ABSTRACTThe structuring role of benzene‐1,3,5‐tricarboxamide (BTA) groups for the catalytic activity of single chain polymeric nanoparticles in water was investigated in the transfer hydrogenation of ketones. To this end, a set of segmented, amphiphilic copolymers was prepared, which comprised oligo(ethylene glycol) side chains to impart water solubility, BTA and/or lauryl side chains to induce hydrophobicity and diphenylphosphinostyrene (SDP) units in the middle part as a ligand to bind a ruthenium catalyst. All copolymers were obtained by reversible addition‐fragmentation chain transfer (RAFT) polymerization and showed low dispersities (Mw/Mn = 1.23–1.38) and controlled molecular weights (Mn = 44–28 kDa). A combination of circular dichroism (CD) spectroscopy and dynamic light scattering (DLS) showed that all copolymers fold into a single chain polymeric nanoparticles (SCPNs) as a result of the helical self‐assembly of the pendant BTA units and/or hydrophilic–hydrophobic phase separation. To create catalytic sites, RuCl2(PPh3)3 was incorporated into the copolymers. The Cotton effects of the copolymers before and after Ru(II) loading were identical, indicating that the helical self‐assembly of the BTA units and the complexation of SDP ligands and Ru(II) occurs in an orthogonal manner. DLS revealed that after Ru(II) loading, SDP‐bearing copolymers retained their single chain character in water, while copolymers lacking SDP units clustered into larger aggregates. The Ru(II) loaded SCPNs were tested in the transfer hydrogenation of cyclohexanone. This study reveals that BTA induced stack formation is not crucial for SCPN formation and catalytic activity; SDP‐bearing copolymers folded by Ru(II) complexation and hydrophobic pendants suffice to provide hydrophobic, isolated reaction pockets around Ru(II) complexes. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014, 52, 12–20

Water-Soluble Co(III) Complexes of Substituted Phenanthrolines with Cell Selective Anticancer Activity

Transition metal complexes with substituted phenanthrolines as ligands represent potential anticancer products without the drawbacks of platinum complexes that are currently marketed. Here, we report the synthesis and cell selective anticancer activity of five new water-soluble Co(III) complexes with methyl substituted phenanthroline ligands. The complexes were characterized by elemental analysis, NMR, FAB-mass spectrometry, FTIR, electronic spectroscopy, and single crystal X-ray diffraction. Possible interaction of these complexes with DNA was assessed by a combination of circular dichroism, UV-vis spectroscopy titration, and ethidium bromide displacement assay, and the results indicated that DNA interaction is weak for these complexes. Cellular uptake and cytotoxicity of complexes at low concentrations were assessed by flow cytometry on PC-3 cells, while their effect on intracellular mitochondrial function was measured by MTS assay on HeLa and PC-3 cell lines. These complexes showed selective cytotoxicity with a significantly higher effect on intracellular mitochondrial function in PC-3 cells than in HeLa cells. At low concentrations, complex 2 had the highest cytotoxic effect on PC-3 cells, inducing around 38% cell death, and the correlation of cytotoxicity of these complexes to their hydrophobicity indicates that an appropriate value of the hydrophobicity is essential for high antitumor activity.

Dipolar-energy-activated magnetic domain pattern transformation driven by thermal fluctuations

Two-dimensional ferromagnetic layers can serve as a playground for the study of basic physical properties of various pattern forming systems by virtue of their tuneable magnetic properties. Here we use threshold photoemission magnetic circular dichroism in combination with photoemission electron microscopy to investigate ultra-thin ferromagnetic Fe/Ni/Cu(001) films in the stripe domain phase near the spin reorientation transition as a function of film thickness, temperature and effective anisotropy. Here we report a metastable domain state with domain width larger than the thermodynamically stable one as a result of a rapid reduction of the anisotropy. The transformation into the equilibrium state takes place via the propagation of a transition front, which originates from defined steps in the film thickness.

Structures and absolute configuration of three α-pyrones from an endophytic fungus Aspergillus niger

Three α-pyrone derivatives, campyrones A–C (1–3), were isolated from Aspergillus niger, an endophytic fungus occurring in Zanthoxylum lemairei leaves. Their structures were elucidated by detailed spectroscopic and spectrometric analyses and confirmed by X-ray diffraction. The absolute configurations were assigned by a combination of circular dichroism (CD) spectra and X-ray analyses. Biosynthetically, pyrophen (4) and the new derivatives may be derived in a mixed biosynthesis from l-phenylalanine (for 4), l-isoleucine (for 1), l-leucine (for 2), and l-valine (for 3) as starter units, respectively. In bioassays, the new compounds exhibited weak toxicity on brine shrimp larvae (Artemia salina).

Single domain antibody–alkaline phosphatase fusion proteins for antigen detection — Analysis of affinity and thermal stability of single domain antibody

Single domain antibody (sdAb)–alkaline phosphatase (AP) fusion proteins have been demonstrated to be useful immunodiagnostic reagents for bio-threat agent detection. The bivalent nature of sdAb–AP fusion proteins significantly increases effective affinity and thus the sensitivity of detection, but the thermal stability of the fusion protein had not been explored. This property is critical for the development of immunoassays for use in austere environments. In this study four sdAbs with specificity for MS2 phage coat protein (CP) were expressed as fusions with AP in order to evaluate the thermal stability and affinity of the resulting constructs. The melting temperature (Tm) of the sdAb and sdAb–AP fusion proteins was measured by a combination of Circular Dichroism (CD), differential scanning calorimetry (DSC) and Fluorescence-based Thermal Shift assay. Binding kinetics were assessed using surface plasmon resonance (SPR). Our results indicated that the AP fusion protein did not increase the Tm or enhance thermal stability of the sdAb, but did provide the expected increase in binding affinity as compared to the original sdAb.

Trivalent chromium and aluminum affect the thermostability and conformation of collagen very differently

Ultrasensitive differential scanning calorimetry (US-DSC) was used to directly measure the thermal transition temperature and energy change of acid soluble collagen in the presence of Cr3+ and Al3+ sulfates. The behavior of Cr3+ was analogous to kosmotropes in the cation Hofmeister series and increased the stability of collagen in dilute solutions. Meanwhile, the denaturational enthalpy change (ΔH) of collagen was substantially reduced with change to increasing Cr3+ concentration. This is likely due to the uni-point binding of Cr3+ with carboxyl groups of collagen side chains that could decrease the hydrogen-bonding in collagen and result in the increase of protein hydrophobicity. In the case of Al3+, the interactions between the ions and collagen showed very different properties: at low and medium ion concentrations, the stability of the collagen was decreased; however, a further increase of Al3+ concentration led to a salting-out effect of collagen, indicating the Al3+ is a typical chaotropic ion. This striking difference of the two ions in the stabilization of collagen can be explained in terms of the different interactions between the cations and the carboxyl groups of collagen side chains. Additionally, we studied metal ion induced conformational change by the combination of circular dichroism (CD) and atomic force microscopy (AFM). CD measurements revealed that neither metal ion interactions of collagen with Cr3+ nor Al3+ ions destroyed the triple-helical backbone structure of collagen in the solution. AFM results further confirmed that the dehydration of collagen by Cr3+ is more significant than Al3+, thus inducing the aggregation of collagen fibrils.

Two new neolignan glycosides from Pittosporum glabratum Lindl.

Two new neolignan glycosides, named pittogoside A (1) and pittogoside B (2) were isolated from the roots of Pittosporum glabratum Lindl. Their structures, including the absolute stereochemistry, were determined on the basis of spectroscopic analysis and chemical evidence, with combination of circular dichroism.

Structural Investigations of the Transmembrane Segment of the PDGF Receptor Beta ant the Oncoprotein E5 by Circular Dichroism and NMR

The Platelet-derived growth factor receptor β (PDGFR) is a member of the receptor-tyrosine-kinase family involved in development. The receptor is activated when the natural ligand PDGF binds simultaneously to the ectodomains of two receptors, resulting in spatial proximity and subsequent cross-activation of the cytosolic kinase domains1). The receptor can also be activated independently of the natural ligand by binding of the small E5 oncoprotein from bovine papillomavirus2). E5 is a small membrane protein of only 44 amino acids and it is thought to manipulate the function of the receptor by specific helix-helix-contacts to the transmembrane of the receptor which then result in sustained receptor activation and cell transformation.To elucidate the helix-helix-interactions in receptor complex, we investigate the structure and membrane alignment of both proteins, first for each protein and later in the heterotetrameric four-helix-bundle. For the structural analysis we combined circular dichroism (CD) spectroscopy and liquid-state NMR to determine the secondary structure. Oriented CD and solid-state NMR were used to resolve the orientation of both proteins in their native environment. Therefore the E5 protein and the transmembrane domain of the receptor (PDGFRTMD) were 15N-isotope labelled by bacterial protein expression and reconstituted in detergent micelles and in oriented lipid bilayers. By CD and liquid-state NMR we found that both proteins are predominantly α-helical in detergent micelles and in lipid bilayers3,4). Furthermore, high resolution NMR measurements showed that PDGFRTMD forms a left-handed coiled-coil structure. A complementary OCD and solid-state NMR analysis of E5 and PDGFRTMD reconstituted in different lipid bilayers showed that the orientation of both proteins depends on the bilayer thickness, where both proteins were more tilted in thin membranes and less tilted in thick membranes.

Inhibition of Translation by Small RNA-Stabilized mRNA Structures in Human Cells

RNA-mediated gene regulation and expression are critically dependent on both nucleic acid architecture and recognition. We present a novel mechanism for the regulation of gene expression through direct RNA-RNA interactions between small RNA and mRNA in human cells. Using mRNA reporters containing G-rich sequences in the 5'-untranslated region (5'-UTR), in the coding region, or both, we showed that G-rich small RNAs bind to the reporter mRNAs and form an intermolecular RNA G-quadruplex that can inhibit gene translation in living cells. Using a combination of circular dichroism (CD) and RNase footprinting in vitro, we found that the intermolecular G-quadruplexes show a parallel G-quadruplex structure. We next investigated whether the intermolecular G-quadruplex is present in living cells. Employing the fluorophore-labeled probes, we found that two G-rich RNA molecules form an intermolecular G-quadruplex structure in living cells. These results extend the concept of small RNA-mediated expression and suggest an important role for such RNA structures in the inhibition of mRNA translation.

Biophysical Analysis of Binding of WW Domains of the YAP2 Transcriptional Regulator to PPXY Motifs within WBP1 and WBP2 Adaptors

The YAP2 transcriptional regulator mediates a plethora of cellular functions, including the newly discovered Hippo tumor suppressor pathway, by virtue of its ability to recognize WBP1 and WBP2 signaling adaptors among a wide variety of other ligands. Herein, using isothermal titration calorimery and circular dichroism in combination with molecular modeling and molecular dynamics, we provide evidence that the WW1 and WW2 domains of YAP2 recognize various PPXY motifs within WBP1 and WBP2 in a highly promiscuous and subtle manner. Thus, although both WW domains strictly require the integrity of the consensus PPXY sequence, nonconsensus residues within and flanking this motif are not critical for high-affinity binding, implying that they most likely play a role in stabilizing the polyproline type II helical conformation of the PPXY ligands. Of particular interest is the observation that both WW domains bind to a PPXYXG motif with highest affinity, implicating a preference for a nonbulky and flexible glycine one residue to the C-terminal side of the consensus tyrosine. Importantly, a large set of residues within both WW domains and the PPXY motifs appear to undergo rapid fluctuations on a nanosecond time scale, suggesting that WW-ligand interactions are highly dynamic and that such conformational entropy may be an integral part of the reversible and temporal nature of cellular signaling cascades. Collectively, our study sheds light on the molecular determinants of a key WW-ligand interaction pertinent to cellular functions in health and disease.

Spectroscopic investigations to reveal the nature of interactions between the haem protein myoglobin and the dye rhodamine 6G

In the present investigation, steady-state and time-resolved fluorescence with the combination of circular dichroism (CD) spectroscopic techniques were applied to study the interactions of the well-known dye rhodamine 6 G (R6G) with the haem protein human myoglobin (Mb). From the analysis of the results it appears that the static type of fluorescence quenching mechanism is primarily involved, due to ground-state interactions. Although considerable overlapping of fluorescence emission of the dye R6G with the absorption of Mb in the Q-band region exists, the possibility of occurrences of the excitational singlet-singlet non-radiative energy transfer process from R6G to Mb appears to be unlikely, according to time-resolved fluorescence measurements. From the determinations of the thermodynamic parameters, it was apparent that the combined effect of van der Waals' interactions and hydrogen bonding plays a vital role in Mb-R6G interactions. Induced circular dichroism (ICD) studies demonstrate the possibility of interactions between R6G and Mb. The binding constants, number of binding sites and thermodynamic parameters have been computed. From CD measurements it is apparent that the binding of the dye R6G with the haem protein Mb induces negligible conformational changes in the protein and Mb retains its secondary structure and helicity when it interacts with R6G. The present detailed studies on the interactions with Mb should be helpful in further advancement of medical diagnostics and biotechnology.

From Fibers to Micelles Using Point-Mutated Amphiphilic Peptides

Biocompatible, self-assembled nanostructures are attracting ever more attention, in particular in aqueous media for biomedical applications. Here, we present the successful, solid-phase peptide synthesis (SPPS) and characterization of short amino acid sequences with amphiphilic character with the aim of gaining insight into their self-assembled, supramolecular structures. The peptide design includes three parts: (a) a charged lysine part, (b) an acetylated lysine part, and (c) a constant hydrophobic rodlike helix, based on gramicidin A (gA). By stepwise replacement of free lysine (K) with acetylated lysine (X) we generated a library of a total of 10 peptides, Ac-X(8)-gA and K(m)X(8-m)-gA (m ranging from 0 to 8). By using point mutations, we adjusted the degree of acetylation (DA) and thus the overall amphiphilicity of the peptides, which led to a change in the secondary structure in the aqueous environment from a β-sheet to an α-helix. This transition generated a significant change in the morphology of the self-assembled structures from fibers to micelles. Two different regions were observed for the conformation of the hydrophilic part of the peptide: one region, a β-sheet-like secondary structure, inducing fiber formation (high DA), the other an α-helical-like secondary structure, generating micelle formation (moderate and low DA). The micellar structures depended on the degree of acetylation, which influenced their critical micelle concentration (cmc). These morphology regions were determined by a combination of circular dichroism, dynamic light scattering, surface tension, and transmission electron microscopy, which allowed us to correlate the generated supramolecular architectures with the fine changes obtained by means of the point mutation strategy.

Magnetostatic coupling of 90° domain walls in Fe19Ni81/Cu/Co trilayers

The magnetic interlayer coupling of Fe19Ni81/Cu/Co trilayered microstructures has been studied by means of x-ray magnetic circular dichroism in combination with photoelectron emission microscopy (XMCD-PEEM). We find that a parallel coupling between magnetic domains coexists with a non-parallel coupling between magnetic domain walls (DWs) of each ferromagnetic layer. We attribute the non-parallel coupling of the two magnetic layers to local magnetic stray fields arising at DWs in the magnetically harder Co layer. In the magnetically softer FeNi layer, non-ordinary DWs, such as 270° and 90° DWs with overshoot of the magnetization either inwards or outwards relative to the turning direction of the Co magnetization, are identified. Micromagnetic simulations reveal that in the absence of magnetic anisotropy, both types of overshooting DWs are energetically equivalent. However, if a uniaxial in-plane anisotropy is present, the relative orientation of the DWs with respect to the anisotropy axis determines which of these DWs is energetically favorable.

Investigation of the Three-Dimensional Structure of the Caveolin-1 membrane Interacting Domain

The importance of understanding membrane protein structure and function is growing after scientists have recognized that approximately one third of the genome encodes for membrane proteins. Caveolin is an integral membrane protein that is found in high abundance in caveolae. Caveolae are invaginations of the plasma membrane that play an important role in many cellular functions. Interestingly, caveolin plays a dual-role in caveolae. On one hand it is responsible for forming the “flask-like” shape of caveolae, and on the other hand it is intimately involved in cellular processes such as signal transduction and protein trafficking. Alterations in the levels of caveolin protein and mutations of the caveolin protein have been implicated in a variety of disease states including cancer, heart disease, and muscular dystrophy. Caveolin possesses a unique hairpin-like topology (N- and C- termini are on the same side of the membrane) in the membrane which is thought to facilitate its functions. In particular, mutations of two proline residues (P110A and P132L) in the caveolin-1 transmembrane domain appear to be critical for proper biological function, and serve as markers for various disease states. To study the effects of these important mutations, we have prepared constructs of the caveolin protein that encompass the transmembrane domain. Both Caveolin(99-137) and caveolin(122-142) were over-expressed and isotopically labeled in E.coli, purified to homogeneity, and incorporated into dodecylphosphocholine micelles. The three-dimensional structure of the constructs will be revealed using a combination of circular dichroism and NMR spectroscopy. Moreover, comparison of the wild-type structure with that of the constructs containing the proline mutations will give valuable insight into the unusual nature of the caveolin-1 transmembrane domain.

Elasticity of Intrinsically Disordered Nebulin Modules

The elasticity of native full length nebulin, demonstrated recently via atomic force microscopy with site-specific antibody pairs as force handles (Langmuir, 2009, 25, 7496), suggests that in the thin filaments, nebulin is stretched to cope with the actin length and imposes significant force and influences the functions of the underlying actins. This pre-stressed mechanical state of thin filaments may have important implications for the role of nebulin as a length ruler and as a regulator of actomyosin interaction. The structural basis of nebulin elasticity remains open. We report here the structural characterization of modules from the super-repeat and single repeat regions by a combination of circular dichroism (CD), NMR, SAXS, AFM, structural predictions and steered molecular dynamics simulations. In aqueous solutions of common buffers, these modules are intrinsically disordered, but are poised to form alpha-helices, especially in the presence of trifluoroethanol. SAXS analysis of a four-module construct indicates an elongated structure with a radius of gyration of 3.6 nm and, as modeled with DAMMIN, shows a contour length of ∼15 nm. Interestingly, this extended structure is also evident in a small population of the structural models as predicted by ROSETTA++. AFM images of the modules on an inert surface are predominantly compact with an average height of ∼ 2.5 nm, consistent with the bulk of the ROSETTA predictions. These structural ensembles of compact and extended structures are significantly shorter than what it would take for nebulin modules to wrap around the perimeter of actin filaments (∼6 nm per module). We propose that nebulin modules’ disorder-order transition of alpha helices, contributes to its elasticity and how nebulin juxtapositions itself onto the actin to form a pre-stressed thin filaments in the muscle sarcomere.

Autonomous folding in the membrane proximal HIV peptide gp41659–671: pH tuneability at micelle interfaces

The flexibility of the Membrane Proximal Region (MPR) of the HIV-1 gp41 envelope glycoprotein is believed to be relevant to its biological function. Its conformational bias is potentially influenced by the various environmental conditions experienced during viral fusion. Using a combination of Circular Dichroism and Molecular Dynamics simulations, we show that a very short MPR fragment gp41(659-671) spanning the 2F5 monoclonal antibody epitope, exhibits autonomous helical folding in the presence of membrane mimicking SDS micelles and the extent of which can be tuned by pH variation: Specifically, the peptide shows no defined fold type at basic pH but is helical at physiological and lower pH environments. By contrast, no such control of helical folding by pH is observed in aqueous solutions in the absence of SDS. Instead, the experimental data imply that unfolded structures persist and that pH has negligible influence on conformational bias. We also explore the pronounced sensitivity to standard empirical potentials and conclude that AMBER-ff03 provides a reasonably accurate description of the solution state structure and is therefore a good choice for future exploration of membrane-induced phenomena.

Hydration Is a Major Determinant of the G-Quadruplex Stability and Conformation of the Human Telomere 3′ Sequence of d(AG3(TTAG3)3)

The factors that determine the conformation and stability of G-quadruplex forming sequences remain poorly understood. Here we demonstrate the influence of cosolvents on the conformation and stability of the human telomeric sequence d(A(GGGTTA)3GGG)) in both K(+) and Na(+) containing solutions using a combination of circular dichroism, NMR, and thermodynamics. Molecular crowding arguments have previously been used to suggest that the parallel quadruplex form may be biologically relevant. However, the small cosolvents previously used, PEG 200 and 400, are actually dehydrating agents. We have used acetonitrile as a non-hydrogen-bonding dehydrating agent; similar conformational transitions were observed in K(+) solution. Moreover, NMR analysis shows that the resulting structure contains non-anti guanine glycosyl torsion angles suggesting that the conformation present in acetonitrile is not identical to the all-parallel crystal structure, despite the supposed parallel type CD spectrum.

In vitro and in silico binding study of the peptide derived from HIV-1 CA-CTD and LysRS as a potential HIV-1 blocking site

The assembly process in HIV-1 has become a new target for infected HIV-1 patient treatment. During this process, the viral genomic RNA and precursor protein are assembled at the permeable membrane and tRNALys3 is packed into a new virion as the primer for the reverse transcription process. The packaging of tRNALys3 arises from the interaction of HIV-1 Gag and hLysRS. To better understand the formation of this ternary complex, the interaction study of LysRS–peptide complex using a combination of circular dichroism, molecular dockings and molecular dynamic simulations are reported here. The circular dichroism experiments confirm that the sh-H4 peptide, containing 10 amino acid residues from helix4 of C-terminal domain of HIV-1 capsid protein (CA-CTD), can be induced to form a helical structure upon binding to hLysRS. Molecular docking analysis of LysRS (hLysRS and eLysRS) with the sh-H4 peptide revealed the two possible arrangements of the peptide upon the binding event. Molecular dynamics based free energy calculations of the peptide binding process are used to determine the interactions as well as the important amino acid residues involving in binding. The peptide is found to lie against helix 7 of LysRS in a perpendicular fashion. Additionally, the peptide preferably interacts with hLysRS over eLysRS including strong hydrogen bond interactions between R247-Q219 and R241-E212. Interestingly, these amino acid residues are found in both LysRS and CA-CTD. These important residues appear to be a vital feature of the LysRS-CA-CTD complex and may ultimately lead to the inhibitor design to block the Gag–LysRS interaction.

Structure, Dynamics and Topology of the N-terminus and First Transmembrane Segment of APJ

APJ is a G-protein coupled receptor expressed in the cardiovascular system, central nervous system and several other tissues. Activation of APJ by the peptide ligand apelin has defined roles in cardiovascular regulation, in glucose metabolism and in tumour growth. Transmembrane proteins such as APJ are difficult to study due to expression, solubility and refolding difficulties. For this reason we have produced a fragment of APJ containing the functionally essential N-terminal region and first transmembrane helix of the receptor (APJ55). Through a combination of circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy we have characterized APJ55. CD spectroscopy indicates that APJ55 only properly refolds in specific detergents, with the anionic detergents sodium dodecylsulphate and 1-palmitoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(1-glycerol)] (LPPG) being the best. NMR spectroscopy has provided an initial structure of APJ55. As a complement to this structure, relaxation studies and paramagnetic spin label titration demonstrated the dynamics and topology of APJ55 in the LPPG micelle. Finally the structure of APJ55 has been placed into the context of full length APJ using a homology model. APJ55 provides a new system to probe apelin-APJ interactions and is a basis for study of additional regions of APJ.