Structural Studies on Porphyrin–PNA Conjugates in Parallel PNA:PNA Duplexes: Effect of Stacking Interactions on Helicity

Parallel PNA:PNA duplexes were synthesized and conjugated with meso‐tris(pyridyl)phenylporphyrin carboxylic acid at the N‐terminus. The introduction of one porphyrin unit was shown to affect slightly the stability of the PNA:PNA parallel duplex, whereas the presence of two porphyrin units at the same end resulted in a dramatic increase of the melting temperature, accompanied by hysteresis between melting and cooling curves. The circular dichroism (CD) profile of the Soret band and fluorescence quenching strongly support the occurrence of a face‐to‐face interaction between the two porphyrin units. Introduction of a L‐lysine residue at the C‐terminal of one strand of the parallel duplex induced a left‐handed helical structure in the PNA:PNA duplex if the latter contains only one or no porphyrin moiety. The left‐handed helicity was revealed by nucleobase CD profile at 240–280 nm and by the induced‐CD observed in the presence of the DiSC₂(5) cyanine dye at ~500–550 nm. Surprisingly, the presence of two porphyrin units led to the disappearance of the nucleobase CD signal and the absence of CD exciton coupling within the Soret band region. In addition, a dramatic decrease of induced CD of DiSC₂(5) was observed. These results are in agreement with a model where the porphyrin–porphyrin interactions cause partial loss of chirality of the PNA:PNA parallel duplex, forcing it to adopt a ladder‐like conformation. Chirality 27:864–874, 2015. © 2015 Wiley Periodicals, Inc.     

An ochre suppressor of bacteriophage T4 that is associated with a transfer RNA

Ultraviolet circular dichroism spectra have been obtained for native and heat-denatured Drosophila virilis satellite DNAs I, II and III. Gall &; Atherton (1974) have found that these DNAs have simple, unique sequences. We compare here the circular dichroism spectra of these satellite sequences with the circular dichroism spectra of synthetic DNAs of simple sequences which are combined in first-neighbor calculations. We also apply an analytical procedure for determining nearest-neighbor frequencies from the DNA spectra (Allen et al., 1972). The results are an indication of the potential usefulness and present limitations of circular dichroism measurements in confirming or determining the nearestneighbor frequencies of satellite DNAs of simple sequences.     

Circular dichroism spectroscopic study of non‐covalent interactions of poly‐L‐glutamic acid with a porphyrin derivative in aqueous solutions

The interactions of poly‐L ‐glutamic acid and a cationic porphyrin derivative in aqueous solutions were studied by the combination of vibrational circular dichroism (VCD) and electronic circular dichroism (ECD) spectroscopies. It was found that non‐covalent interactions between both agents influence the structure of the polymeric matrix and the guest porphyrins and vice versa, but the physico‐chemical properties of the solutions, especially the pH and the relative permittivity of the solvent, play a key role in the structure of the polypeptide part of the formed complexes. It was shown that the interaction with porphyrins prevents the precipitation of poly‐L ‐glutamic acid in aqueous solution at acidic pH. In special conditions, the porphyrins attached to the polypeptide probably possess face‐to‐face interaction as demonstrated by the enhancement of the characteristic ECD signal and the appearance of sidebands on its short and long wavelength sides. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.     

A Solid Phase Vibrational Circular Dichroism Study of Polypeptide–Surfactant Interaction

We studied the interaction of poly‐l ‐lysine (PLL) and poly‐l ‐arginine (PLAG) with sodium dodecyl sulfate (SDS) surfactant and the interaction of poly‐l‐ glutamic acid (PLGA) and poly‐l ‐aspartic acid (PLAA) with tetradecyltrimethylammonium bromide (TTAB) surfactant using vibrational circular dichroism (VCD) spectroscopy in the region of C‐H stretching vibration and in the Amide I region both in solution and in mulls. A chirality transfer from polypeptides to achiral surfactants was observed in the C‐H stretching region, where measurements in solution were impossible. This observation was enabled by a special sample treatment technique using lyophilization and the preparation of mulls. This technique demonstrated itself as an interesting and beneficial tool for VCD measurements. In addition, we observed that SDS changed the secondary structure of PLL to the β‐sheet and of PLAG to the α‐helix. TTAB disrupted the PLGA and PLAA structure. These results were obtained in the mull but were confirmed by the VCD spectra measured in solution and by electronic circular dichroism. The chirality transfer from the polypeptides to SDS was caused by polypeptides ordered into a specific conformation during the interaction, while in the TTBA system it was induced primarily by the chirality of the amino acid residues. Chirality 27:965–972, 2015. © 2015 Wiley Periodicals, Inc.     

Electronic and vibrational optical activity of several peptides related to neurohypophyseal hormones: disulfide group conformation

Electronic and vibrational optical activity of the set of neurohypophyseal hormones and their analogs was investigated to clarify the S-S bond solution conformation. The selected compounds include oxytocin (I), lysine vasopressin (II), arginine vasopressin (III), and their analogs (IV-IX), differing widely in their pharmacological properties. We have extended the already known electronic circular dichroism data by new information provided by vibrational circular dichroism (VCD) and Raman optical activity (ROA). The use of VCD brought additional details on three-dimensional structure of the chain reversal in the ring moiety and on its left handedness. Furthermore, Raman scattering and ROA allowed us to deduce the sense of the disulfide bond torsion.     

Conformation of gramicidin A in Triton X‐100 micelles from CD and FTIR data: a clean example of antiparallel double β5.6 helix formation

The linear peptide gramicidin A (gA) forms prototypical ion channels specific for monovalent cations and has been extensively used to study the organization and dynamics of membrane channels. This polymorphic peptide can adopt two different types of structures, the helical dimer β6.3 (‘channel state’) and the double helical structure with two intertwined monomers. The structure of gA in micelles of detergent Triton X‐100 has been studied using CD, Fourier transform infrared, and fluorescence spectroscopy. The results obtained demonstrate that only one thermodynamically stable gA structure, the antiparallel left‐handed double helix β5.6, is formed in this membrane‐mimetic environment. The position of the tryptophan fluorescence maximum at 332 nm is the same as that in phospholipid membranes. The causative factors governing the double helix formation in the micellar medium are discussed on the basis of known physicochemical properties of Triton X‐100. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Structural analysis of lysine‐4 methylated histone H3 proteins using synchrotron radiation circular dichroism spectroscopy

We report structural alterations of histone H3 proteins induced by lysine‐4 (K4) monomethylation, dimethylation, and trimethylation identified by using synchrotron radiation circular dichroism spectroscopy. Compared with unmethylated H3, monomethylation and dimethylation induced increases in α‐helix structures and decreases in β‐strand structures. In contrast, trimethylation decreased α‐helix content but increased β‐strand content. The structural differences among K4‐unmethylated/methylated H3 may allow epigenetic enzymes to discriminate the substrates both chemically and sterically.     

High‐yield membrane protein expression from E. coli using an engineered outer membrane protein F fusion

Obtaining high yields of membrane proteins necessary to perform detailed structural study is difficult due to poor solubility and variability in yields from heterologous expression systems. To address this issue, an Escherichia coli‐based membrane protein overexpression system utilizing an engineered bacterial outer membrane protein F (pOmpF) fusion has been developed. Full‐length human receptor activity‐modifying protein 1 (RAMP1) was expressed using pOmpF, solubilized in FC15 and purified to homogeneity. Using circular dichroism and fluorescence spectroscopy, purified full‐length RAMP1 is composed of approximately 90% α‐helix, and retains its solubility and structure in FC15 over a wide range of temperatures (20–60°C). Thus, our approach provides a useful, complementary approach to achieve high‐yield, full‐length membrane protein overexpression for biophysical studies.     

VCD studies on cyclic peptides assembled from L‐α‐amino acids and a trans‐2‐aminocyclopentane‐ or trans‐2‐aminocyclohexane carboxylic acid

The increasing interest in peptidomimetics of biological relevance prompted us to synthesize a series of cyclic peptides comprising trans‐2‐aminocyclohexane carboxylic acid (Achc) or trans‐2‐aminocyclopentane carboxylic acid (Acpc). NMR experiments in combination with MD calculations were performed to investigate the three‐dimensional structure of the cyclic peptides. These data were compared to the conformational information obtained by electronic circular dichroism (ECD) and vibrational circular dichroism (VCD) spectroscopy. Experimental VCD spectra were compared to theoretical VCD spectra computed quantum chemically at B3LYP/6‐31G(d) density functional theory (DFT) level. The good agreement between the structural features derived from the VCD spectra and the NMR‐based structures underlines the applicability of VCD in studying the conformation of small cyclic peptides. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Interaction of DNA with actinocin amides containing cationoid centers in the amide groups

Complexes of DNA with actinocin derivatives containing ω-dialkylaminoalkyl groups in the1 and/or 9 positions of the chromophore were studied by spectrophotometric titration, circular dichroism, and viscometry. Induced circular dichroism (ICD) spectra of the DNA-ligand complexes were compared for the cases of the complexes of known structure established by other methods. It was shown that the presence of an isoelliptic point in the long-wavelength absorption band of the ICD spectra of the ligand under monomeric binding conditions could indicate intercalation of the actinocin chromophore into DNA. The separation of the cationoid center and the chromosphore upon elongation of the methylene chain increases the aggregability of the ligand pn the surface of the DNA double helix, which prevents the intercalation of the chromophore.     

Conformation of membrane‐bound proteins revealed by vacuum‐ultraviolet circular‐dichroism and linear‐dichroism spectroscopy

Knowledge of the conformations of a water‐soluble protein bound to a membrane is important for understanding the membrane‐interaction mechanisms and the membrane‐mediated functions of the protein. In this study we applied vacuum‐ultraviolet circular‐dichroism (VUVCD) and linear‐dichroism (LD) spectroscopy to analyze the conformations of α‐lactalbumin (LA), thioredoxin (Trx), and β‐lactoglobulin (LG) bound to phosphatidylglycerol liposomes. The VUVCD analysis coupled with a neural‐network analysis showed that these three proteins have characteristic helix‐rich conformations involving several helical segments, of which two amphiphilic or hydrophobic segments take part in interactions with the liposome. The LD analysis predicted the average orientations of these helix segments on the liposome: two amphiphilic helices parallel to the liposome surface for LA, two hydrophobic helices perpendicular to the liposome surface for Trx, and a hydrophobic helix perpendicular to and an amphiphilic helix parallel to the liposome surface for LG. This sequence‐level information about the secondary structures and orientations was used to formulate interaction models of the three proteins at the membrane surface. This study demonstrates the validity of a combination of VUVCD and LD spectroscopy in conformational analyses of membrane‐binding proteins, which are difficult targets for X‐ray crystallography and nuclear magnetic resonance spectroscopy. Proteins 2016; 84:349–359. © 2016 Wiley Periodicals, Inc.     

Insights into the helix‐sense inversion of poly(β‐phenethyl‐L‐aspartate) by two‐dimensional Raman correlation spectroscopy

In this study, the transition process of the helix‐sense inversion of poly(β‐phenethyl‐L‐aspartate) was investigated by Raman scattering and 2‐dimensional correlation spectroscopy. Temperature‐dependent Raman spectra were obtained during the helix‐sense inversion. The results of 2‐dimensional correlation analysis in the spectral regions of 1600‐1800 and 3200‐3400 cm^?1 showed that the intensity changes of the side‐chain ester C═O stretching bands occurred prior to those of amide A and amide I bands in the unwinding process of αR‐helix on heating. The sequential order of the intensity changes for amide A, amide I, and the side‐chain ester C═O stretching bands during the inversion process was determined. It was found that the conformation change of the side chain occurred prior to that of the main chain for the αR‐helix on heating. Thus, we concluded that the transformation of the backbone chain from right‐handed to left‐handed is triggered by the conformational change of the side chains.     

Right‐ and left‐handed three‐helix proteins. I. Experimental and simulation analysis of differences in folding and structure

Despite the large number of publications on three‐helix protein folding, there is no study devoted to the influence of handedness on the rate of three‐helix protein folding. From the experimental studies, we make a conclusion that the left‐handed three‐helix proteins fold faster than the right‐handed ones. What may explain this difference? An important question arising in this paper is whether the modeling of protein folding can catch the difference between the protein folding rates of proteins with similar structures but with different folding mechanisms. To answer this question, the folding of eight three‐helix proteins (four right‐handed and four left‐handed), which are similar in size, was modeled using the Monte Carlo and dynamic programming methods. The studies allowed us to determine the orders of folding of the secondary‐structure elements in these domains and amino acid residues which are important for the folding. The obtained data are in good correlation with each other and with the experimental data. Structural analysis of these proteins demonstrated that the left‐handed domains have a lesser number of contacts per residue and a smaller radius of cross section than the right‐handed domains. This may be one of the explanations of the observed fact. The same tendency is observed for the large dataset consisting of 332 three‐helix proteins (238 right‐ and 94 left‐handed). From our analysis, we found that the left‐handed three‐helix proteins have some less‐dense packing that should result in faster folding for some proteins as compared to the case of right‐handed proteins.Proteins 2013; © 2013 Wiley Periodicals, Inc.     

Complex of fd gene 5 protein and double-stranded RNA

We report the formation of complexes of the single-stranded DNA binding protein encoded by gene 5 of fd virus, with natural double-stranded RNAs. In the first direct visualization of a complex of the fd gene 5 protein with a double-stranded nucleic acid, we show by electron microscopy that the double-stranded RNA complex has a structure which is distinct from that of complexes with single-stranded DNA and is consistent with uniform coating of the exterior of the double-stranded RNA helix by the protein. Circular dichroism spectral data demonstrate that the RNA double helix in the complex is undisrupted, and that perturbation of the 228-nm circular dichroism assigned to protein tyrosines can occur in the absence of intercalation of nucleotide bases with protein aromatic residues. Our findings emphasize the potential importance of interaction with the sugar-phosphate polynucleotide backbone in binding of the fd gene 5 protein to nucleic acids.     

Circular dichroism spectroscopy and DFT calculations in determining absolute configuration and E/Z isomers of conjugated oximes

The primary purpose of this work was to demonstrate the suitability of circular dichroism (CD) spectroscopy in stereochemical studies of α,β ‐unsaturated oximes, with particular emphasis on determination of E and Z geometry of the oxime double bond. As models for this study, O‐phenyl and O‐triphenylmethyl (trityl) oximes of 4‐hydroxy‐2‐methylcyclopent‐2‐en‐1‐one were selected. These model compounds differ in both absolute configuration at C4 carbon atom and E ‐Z configuration of the oxime double bond. The basic dichroic technique applied was electronic circular dichroism (ECD) assisted by quantum‐chemical calculations and vibrational circular dichroism (VCD) for selected cases. Such an approach enabled effective implementation of both goals. Thus, we were able to associate the signs of Cotton effects in the range of 190–240 nm with the absolute configuration at C4 and within 240–300 nm with the E ‐ or Z ‐geometry of the oxime double bond. Within this work, optical activity of the protecting trityl group was also studied towards formation of the propeller‐shaped conformations by using the same combined CD/DFT methodology. As shown, the helical structure of the trityl group has a considerable influence on the ECD spectra. However, the MPM and PMP conformers of the trityl group are in fact almost equally populated in the conformational equilibrium, making it impossible to distinguish them. On the other hand, rotamers of the hydroxyl group at C4 show a decisive impact on the VCD spectra in both phenoxy and trityl oximes.     

Circular Dichroism Spectroscopy Study of Crystalline‐to‐Amorphous Transformation in Chiral Platinum(II) Complexes

Two couples of enantiomeric platinum(II) complexes: Pt(L_1a)Cl ( 1a ), Pt(L_1b)Cl ( 1b ) and Pt(L_1a)(C ≡ C ? Ph) ( 2a ), Pt(L_1b)(C ≡ C ? Ph) ( 2b ) (L_1a = (+)‐1,3‐di‐(2‐(4,5‐pinene)pyridyl)benzene, L_1b = (?)‐1,3‐di‐(2‐(4,5‐pinene)pyridyl)benzene) were synthesized and characterized. Their absolute configurations were determined by single crystal X‐ray diffraction and further verified by circular dichroism (CD) spectra (including electronic circular dichroism [ECD] and vibrational circular dichroism [VCD]). These complexes show interesting mechanoluminescence and/or vapoluminescence due to crystalline‐to‐amorphous transformation. The crystalline solids, grinding‐induced amorphous powders, and vapor‐induced amorphous powders of complexes 2a and 2b were comparatively investigated by solid‐state ECD and VCD spectra. The transformation from crystalline solids to amorphous powders was accompanied by significant variances of the spectral feature in both ECD and VCD spectra. Chirality 25:384–392, 2013. © 2013 Wiley Periodicals, Inc.     

Investigation of the interaction between quercetin and human serum albumin by multiple spectra,electrochemical impedance spectra and molecular modeling

Quercetin (Qu), a flavonoid compound, exists widely in the human diet and exhibits a variety of pharmacological activities. This work is aimed at studying the effect of Qu on the bioactive protein, human serum albumin (HSA) under simulated biophysical conditions. Multiple spectroscopic methods (including fluorescence and circular dichroism), electrochemical impedance spectra (EIS) and molecular modeling were employed to investigate the interaction between Qu and HSA. The fluorescence quenching and EIS experimental results showed that the fluorescence quenching of HSA was caused by formation of a Qu–HSA complex in the ground state, which belonged to the static quenching mechanism. Based on the calculated thermodynamic parameters, it concluded that the interaction was a spontaneous process and hydrogen bonds combined with van der Waal's forces played a major role in stabilizing the Qu–HSA complex. Molecular modeling results demonstrated that several amino acids participated in the binding process and the formed Qu–HSA complex was stabilized by H‐bonding network at site I in sub‐domain IIA, which was further confirmed by the site marker competitive experiments. The evidence from circular dichroism (CD) indicated that the secondary structure and microenvironment of HSA were changed. Alterations in the conformation of HSA were observed with a reduction in the amount of α helix from 59.9% (free HSA) to 56% (Qu–HSA complex), indicating a slight unfolding of the protein polypeptides. Copyright © 2014 John Wiley & Sons, Ltd.     

Right‐ and left‐handed three‐helix proteins. II. Similarity and differences in mechanical unfolding of proteins

Here, we study mechanical properties of eight 3‐helix proteins (four right‐handed and four left‐handed ones), which are similar in size under stretching at a constant speed and at a constant force on the atomic level using molecular dynamics simulations. The analysis of 256 trajectories from molecular dynamics simulations with explicit water showed that the right‐handed three‐helix domains are more mechanically resistant than the left‐handed domains. Such results are observed at different extension velocities studied (192 trajectories obtained at the following conditions: v = 0.1, 0.05, and 0.01 Å ps^?1, T = 300 K) and under constant stretching force (64 trajectories, F = 800 pN, T = 300 K). We can explain this by the fact, at least in part, that the right‐handed domains have a larger number of contacts per residue and the radius of cross section than the left‐handed domains. Proteins 2014; 82:90–102. © 2013 Wiley Periodicals, Inc.     

The part of a long beta hairpin from the scrapie form of the human prion protein is reconstructed in the synthetic CC36 protein

Mechanisms of beta sheet formation by the human prion protein are not clear yet. In this work, we clarified the role of the region containing C‐half of the second helix and N‐half of the third helix of that protein in the process of alpha helix to beta sheet transition. Solid phase automatic synthesis of the original peptide (CC36: Cys179–Cys214) failed because of the beta hairpin formation in the region 206‐MERVVEQMC‐214 with a high beta strand potential. Using Met206Arg and Val210Arg substitutions, we increased the probability of alpha helix formation by that sequence. After that modification, the complete CC36 peptide with disulfide bond has been synthesized. Modified peptide has been studied by circular dichroism (CD) and fluorescence spectrography. According to the CD spectra analysis, the CC36 peptide contains 37% of residues in beta sheet and just 15% in helix. Thermal analysis under the control of CD shows that the secondary structure content of the peptide is stable from 5°C to 80°C. Dissociation of oligomers of the CC36 peptide finishes at 37°C according to the fluorescence analysis. The CC36 peptide is able to bind Mn²⁺ cations, which causes small temperature‐associated structural shifts at concentrations of 2 – 10·10^?6 M. Predicted beta hairpin of the CC36 peptide (two beta strands are: 184‐IKQHTVT‐190 and 197‐TETDVKM‐205) should be the part of a longer beta hairpin from the scrapie form of the prion protein (PrPSc). Analogs of the CC36 peptide may be considered as antigens for the future development of a vaccine against PrPSc. Proteins 2016; 84:1462–1479. © 2016 Wiley Periodicals, Inc.