Temperature-dependent Circular Dichroism Research Articles

Insights into the mechanism of binding of doxorubicin and a chlorin compound with 22-mer c-Myc G quadruplex

BackgroundThe interaction of small molecules with G quadruplexes is in focus due to its role in molecular recognition and therapeutic drug design. Stabilization of G-quadruplex structures in the promoter regions of oncogenes by small molecule binding has been demonstrated as a potential approach for cancer therapy. MethodsIn this study, electronic spectroscopy (ultraviolet-visible, fluorescence, circular dichroism), differential scanning calorimetry, and molecular modeling were employed to explore the interactions between the chemotherapy drug doxorubicin and a chlorin compound 5,10,15,20-tetraphenyl-[2,3]-[bis(carboxy)-methano]chlorin (H2TPC(DAC)), and the c-Myc 22-mer G quadruplex DNA. ResultsSpectroscopic studies indicated external binding of the compounds with partial stacking at the end quartets. Calorimetric studies and temperature dependent circular dichroism data displayed increased melting temperatures of G quadruplex structure on binding with the compounds. Circular dichroism spectra indicated that the G quadruplex structure is intact upon ligand binding. Both the compounds showed binding affinities of the order of 106 M−1. Fluorescence lifetime studies revealed static quenching as major mechanism for fluorescence quenching. Polymerase chain reaction stop assay hinted that binding of both ligands under study could inhibit the amplification of the DNA sequence. ConclusionResults show that doxorubicin and H2TPC(DAC) bind to the 22-mer c-Myc quadruplex structure with good affinity and induce stability. SignificanceDoxorubicin and H2TPC(DAC) have demonstrated their affinity towards c-Myc G quadruplex DNA, stabilizing it and inhibiting expression and polymerization. The results can be of practical use in designing new analogs for the two compounds, which can become potent anti-cancer agents targeting the c-Myc GQ structure.

Hyper Thermostability and Liquid-Crystal-Like Properties of Designed α-Helical Peptide Nanofibers.

Structural and thermodynamic transitions of artificially designed α-helical nanofibers were investigated using eight peptide variants, including four peptides with amide-modified carboxyl termini (CB peptides) and four unmodified peptides (CF peptides). Temperature-dependent circular dichroism spectroscopy and differential scanning calorimetry showed that CB peptides exhibit thermostability up to 50 °C higher than CF peptides. As a result, one of the denaturation temperatures approached nearly 130 °C, which is exceptionally high for a biomacromolecule. Thermodynamic analysis and microscopy observations also showed that CB peptides undergo a thermal transition similar to the phase transition in liquid crystals. In addition, one of the peptides showed a sharp and highly cooperative transition with a small enthalpy change at around 25 °C, which was ascribed to a giga-bundle burst of the molecular assembly. These macroscopic changes in the thermostability and crystallinity of CB peptides may be attributed to an increased amphiphilicity of the molecule in the direction of the helix axis, originating from the microscopic modification of the carboxyl-terminus.

A good and bad aggregation: Effect of imidazolium- and cholinium-based ionic liquids on the thermal stability of bovine serum albumin

The present work describes the effect of imidazolium- and cholinium-based ionic liquids (ILs) on the thermal stability, binding and aggregation of bovine serum albumin (BSA). The studied ILs include choline chloride [Ch][Cl], choline dihydrogenphosphate [Ch][DHP], choline acetate [Ch][OAc], 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF4], 1-octyl-3-methylimidazolium tetrafluoroborate [OMIM][BF4] 1-butyl-3-methylimidazolium trifluoromethanesulfonate [BMIM][TfO], 1-butyl-3-methylimidazolium acetate [BMIM][OAc], and 1-butyl-3-methylimidazolium methanesulfonate [BMIM][CH3SO3]. The state of BSA in presence of ILs was assessed by circular dichroism (CD), temperature dependent circular dichroism, fluorescence spectroscopy, and dynamic light scattering (DLS). The thermal stability of BSA in presence of ILs increased (above 85 °C) in all studied ILs except [OMIM][BF4]. Differences of binding ILs with BSA depending on alkyl chain length were estimated, the probable binding modes were identified using molecular docking. The influence of ILs on the aggregation of BSA at different temperatures was established. It was found that the ILs with short alkyl chain length (up to butyl) promote aggregation of the native form of BSA increasing its thermal stability.

Rational design of ultrafast PpcA-Ru(bpy)3 complexes.

Converting light energy into its electrochemical equivalent requires precise control and fine-tuning of relevant kinetic and thermodynamic parameters of electron transfer (ET) steps. A particularly big challenge for artificial photosynthesis is to create the primary charge separation on the ultrafast (sub-nanosecond) time scale, as slower reactions are likely to require precious metal photosensitizers and produce significant amounts of reactive oxygen species. However, the structural requirements for ultrafast ET are poorly understood. The previous work from our lab has demonstrated that ET rates do not strictly depend on the distances. Similarly, we have demonstrated that short ET pathways through covalent bonds do not always result in ultrafast ET. We hypothesize that a rigid attachment of photosensitizers and efficient heat dissipation into the protein framework are needed for ultrafast ET. To test this hypothesis, we developed 13 new mutant forms of PpcA with the attachment cysteine sites showing restricted solvent exposure. We successfully labeled 11 out of 13 with Ru(bpy)3 and observed the expected complex masses in LC-MS. Analytical size-exclusion chromatography revealed the absence of aggregation and predominantly monomeric forms. Temperature-dependent circular dichroism (CD) spectroscopy showed the absence of any significant destabilization of the protein structure due to mutations and photosensitizer attachment, aside from I38CRu. Finally, we measured electron transfer rates that show that 10 of the 11 labeled PpcA-Ru(bpy)3 complexes show ultra-fast electron transfer.

Ligand Coverage and Exciton Delocalization Control Chiral Imprinting in Perovskite Nanoplatelets

Chiral perovskites have recently generated significant interest, yet little is known about how their chiro-optical properties arise. In this study, chiral methylammonium lead halide perovskite nanoplatelets (NPLs) with varied halide and ligand compositions are prepared by using direct synthetic methods. Circular dichroism (CD) and 1H NMR studies find a nonlinear relationship between the chiroptical properties and the ratio of chiral phenylethylammonium (PEA) to achiral octylamine (OA) ligands on the NPL surface. We use density functional theory (DFT) computations and a chiral imprinted particle-in-a-box model to rationalize the experimentally observed CD spectra, and we find that the saturation of the induced chirality depends on the size of the perovskite exciton relative to the size of the ligand moleclues. Temperature-dependent CD and 1H NMR studies, combined with DFT analysis, show that both the CD intensity and sign depend strongly on the structure and orientation of the ligands. This work reveals the complex nature of chiral imprinting in perovskite nanostructures and establishes a simple physical model for ligand-induced chiral imprinting to guide the further development of chiral materials.

Bovine hemoglobin thermal stability in the presence of naringenin: Calorimetric, spectroscopic and molecular modeling studies

Calorimetric, spectroscopic and dynamic light scattering experiments, together with complementary computational investigations afforded a detailed analysis of the thermal stability and conformation of the bovine hemoglobin (BHb)–naringenin (NAR) complex. Differential scanning microcalorimetry (μDSC) revealed complex, multi-step aggregation processes above 330 K. There is a definite enhancement and alteration of the thermal signature of protein aggregation with increasing NAR concentration. Circular dichroism (CD) spectroscopy indicated minor conformational changes in the protein secondary structure upon ligand binding at 298 K. Temperature-dependent CD measurements evidenced steep variations of the α-helix (decrease) and unordered structures (increase), with only a slight increase of β-structure as a result of unfolding and aggregation. UV–Vis absorbance and DLS measurements pointed to large size aggregates formation in the presence of ligand in high concentration.Molecular docking data identified the binding site of NAR, located in the internal cavity of BHb. Molecular dynamics simulations indicated hydrogen bonds, electrostatic and hydrophobic interactions as main contributions in the binding process; the bound ligand produced no sizable structural alterations of the protein molecule. Therefore, the clearly evidenced (μDSC, CD, DLS) influence of the flavonoid on hemoglobin thermal aggregation is exerted as a free (not bound) component of the studied systems.

Investigation on the effect of nonpolar amino acids as macromolecular crowders on the stability of globular proteins

Addition of macromolecular crowders to biomolecules can mimic the interior of living cells, however, it is not always very evident whether the effect is general or dependent on the biomolecule-crowder pair specificity. In this study, we investigate the effect of nonpolar hydrophobic amino acids (Glycine, L-alanine, L-valine, L-leucine, L-isoleucine) as crowding agents on the structural and thermal stability of three proteins: human serum albumin (HSA), lysozyme (HEWL) and Ribonuclease-A (RNase-A) of varying α-helical content using temperature dependent circular dichroism (CD) spectroscopic technique. It has been observed that all these five amino acids do not substantially alter the secondary structure of proteins rather they stabilize their respective native forms. Thermal unfolding of each protein is found to be irreversible in nature with the extent of secondary structural loss during refolding being proportional to the α-helicity of the protein. Estimated thermodynamic parameters (van't Hoff enthalpy and heat capacity) show that enthalpic stabilization is protein specific and the added amino acids alter these parameters in different extent without following any specific trend. Our study affirms that conformational stability of proteins and the associated thermodynamic parameters do not necessarily bear a linear correlation with the hydrophobicity of amino acids; rather it is protein specific where the secondary structural content plays an important role.

Supramolecular Nanohelix Fabricated by Pillararene-Based Host–Guest System for Chirality Amplification, Transfer, and Circularly Polarized Luminescence in Water

Supramolecular Nanohelix Fabricated by Pillararene-Based Host–Guest System for Chirality Amplification, Transfer, and Circularly Polarized Luminescence in Water

Biphenyl-Induced Superhelix in l -Phenylalanine-Based Supramolecular Self-Assembly with Dynamic Morphology Transitions

Biphenyl-Induced Superhelix in <scp>l</scp> -Phenylalanine-Based Supramolecular Self-Assembly with Dynamic Morphology Transitions

Evidence of Protein Binding by a Nucleopeptide Based on a Thyminedecorated L-Diaminopropanoic Acid through CD and In Silico Studies.

Nucleopeptides are chimeric compounds of biomedical importance carrying DNA nucleobases anchored to peptide backbones with the ascertained capacity to bind nucleic acids. However, their ability to interact with proteins involved in pathologies of social relevance is a feature that still requires investigation. The worrying situation currently observed worldwide for the COVID-19 pandemic urgently requires the research on novel anti-SARSCoV- 2 molecular weapons, whose discovery can be aided by in silico predictive studies. The aim of this work is to explore by spectroscopic methods novel features of a thymine-bearing nucleopeptide based on L-diaminopropanoic acid, including conformational aspects as well as its ability to bind proteins, starting from bovine serum albumin (BSA) as a model protein. Moreover, in consideration of the importance of targeting viral proteins in the current fight against COVID-19, we evaluated in silico the interaction of the nucleopeptide with some of the most relevant coronavirus protein targets. First, we investigated via circular dichroism (CD) the conformational behaviour of this thymine-bearing nucleopeptide with temperature: we observed CD spectral changes, particularly passing from 15 to 35 °C. Scanning Electron Microscopy (SEM) analysis of the nucleopeptide was also conducted on nucleopeptide solid samples. Additionally, CD binding and preliminary in silico investigations were performed with BSA as a model protein. Moreover, molecular dockings were run using as targets some of the main SARS-CoV-2 proteins. The temperature-dependent CD behaviour reflected the three-dimensional rearrangement of the nucleopeptide at different temperatures, with higher exposure to the solvent of its chromophores at higher temperatures compared to a more stacked structure at a low temperature. SEM analysis of nucleopeptide samples in the solid-state showed a granular morphology, with a low roughness and some thread structures. Moreover, we found through spectroscopic studies that the modified peptide bound the albumin target by inducing significant changes to the protein secondary structure. CD and preliminary in silico studies suggested that the nucleopeptide bound the BSA protein with high affinity according to different binding modes, as testified by binding energy scores lower than -11 kcal/mol. Interestingly, a predictive study performed on 3CLpro and other SARS-CoV-2 protein targets suggested the potential ability of the nucleopeptide to bind with good affinity the main protease of the virus and other relevant targets, including the RNAdependent RNA polymerase, especially when complexed with RNA, the papain-like protease, and the coronavirus helicase at the nucleic acid binding site.

Unfolding Mechanisms and Conformational Stability of the Dimeric Endophilin N-BAR Domain

Endophilin, which is a member of the Bin-amphiphysin-Rvs (BAR) domain protein superfamily, contains a homodimeric N-BAR domain of a characteristic crescent shape. The N-BAR domain comprises a six-helix bundle and is known to sense and generate membrane curvature. Here, we characterize aspects of the unfolding mechanism of the endophilin A1 N-BAR domain during thermal denaturation and examine factors that influence the thermal stability of this domain. Far-UV circular dichroism (CD) spectroscopy was applied to monitor changes in the secondary structure above room temperature. The protein’s conformational changes were further characterized through Foerster resonance energy transfer and cross-linking experiments at varying temperatures. Our results indicate that thermal unfolding of the endophilin N-BAR is (minimally) a two-step process, with a dimeric intermediate that displays partial helicity loss. Furthermore, a thermal shift assay and temperature-dependent CD were applied to compare the unfolding processes of several truncated versions of endophilin. The melting temperature of the N-BAR domain decreased when we deleted either the N-terminal H0 helix or the unstructured linker of endophilin. This result suggests that these intrinsically disordered domains may play a role in structurally stabilizing the functional N-BAR domain in vivo. Finally, we show that single-site mutations can also compromise endophilin’s thermal stability.

Wide thermal range, exclusive occurrence of technically significant chiral nematic phase: synthesis and mesomorphism of cholesterol-based non-symmetric dimers

Fifteen new non-symmetric chiral dimers belonging to three different series have been synthesized and evaluated for their mesomorphic properties. They are formed by interlinking cholesterol with salicylaldimine (SAN) cores (with reverse imine groups) via an $$\upomega $$ -oxyalkanoyloxy spacer. Within a series, the length of the terminal n-alkoxy tails has been varied for a fixed even-parity spacer. Three even-parity spacers such as 4-oxybutanoyloxy, 6-oxyhexanoyloxy and 8-oxyoctanoyloxy have been used to join two cores, whereas the terminal tails such as n-butyloxy, n-hexyloxy, n-octyloxy, n-decyloxy and n-dodecyloxy chains have been attached to the SAN core. Microscopic and calorimetric experimental results show that all the dimers behave identically exhibiting the chiral nematic (N*) phase solely, which was authenticated by powder X-ray diffraction studies carried out on some selected samples. In the vast majority of the cases, this phase is thermodynamically stable, and while cooling, it exists over a wide thermal range covering room temperature (RT) due to supercooling. This finding is notable given the fact that the N* phase possesses technologically significant optical properties. At RT, the N* phase displayed one of the iridescent colours characteristically caused by interference and diffraction of the reflected and scattered light. A comparative study reveals that the lengths of both the terminal chain and central spacer influence the clearing temperature of the dimers, and also the temperature range of the N* phase. The selective reflection measurements revealed that the pitch of the N* phase is either temperature sensitive or temperature insensitive. Temperature-dependent circular dichroism (CD) spectra were recorded for the planar texture of the N* phase formed by a dimer, as a representative case. The presence of an intense negative CD band suggests the left-handed screw sense of the N* phase helix.

MON-175 Structural Instability as an Underlying Pathomechanism in Congenital Adrenal Hyperplasia

Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders affecting key enzymes of cortisol biosynthesis. In the majority of cases the underlying cause are detrimental mutations in the steroidogenic cytochrome P450 enzyme 21-hydroxylase (CYP21A2). Early diagnosis via newborn screening programs in most Western countries and lifelong oral cortisol replacement therapy enable survival, however quality of life often is reduced and co-morbidities are substantially increased. Treatment is a major challenge as disease control can only be achieved with supraphysiological glucocorticoid doses. In addition, the currently available drugs cannot ideally mimic the circadian rhythm and stress adaption of cortisol secretion. Currently, disease severity is classified by residual enzyme activity. The goal of our research is to better understand the specific biophysico-chemical pathomechanism of 21-hydroxylase deficiency in order to enable causative therapeutic approaches. To this end, we investigated the structural and stability properties of six clinically relevant mutant variants of CYP21A2 (V282G/L, P31L, D323G, R484Q/W). Difficulty in purification of these CYP21A2 variants and various biophysical studies suggest that the proteins were less stable than wild-type (WT). Structural and thermal stability assessment by circular dichroism (CD) spectroscopy of recombinant, purified CYP21A2 mutant variants revealed high α-helical content for the WT (65% α-helix) and the mutants at the position 282 (V282G: 60.6 %, V282L: 57.6%). Other mutations (P31L, D323G, R484Q/W) disrupt the α-helical organization of CYP21A2 in exchange for a slight increase in ß-sheet content but mainly for random coil. Temperature dependent CD spectroscopy showed that all mutant variants have reduced thermal stability (Tm: 41.3 - 45,6°C) compared to the WT (Tm: 47.1°C). Tryptophane fluorescence showed that mutant variants of the protein were more prone to local unfolding at the hydrophobic core compared to WT using urea as denaturant. Furthermore, in UV/Vis spectroscopy at 280 nm and 418 nm we could demonstrate that all mutant variants had a reduced heme incorporation (A418/A280: 0.20 - 0.63) compared to WT (A418/A280: 0.88). Our results show that correct structural folding and stability pose a major problem in specific mutations involved in CAH. Therefore we propose that structural protein instability, play a key role in the pathophysiology of CAH and thus might constitute a novel tailored therapeutic target for the treatment of affected patients.

Autonomous discovery of optically active chiral inorganic perovskite nanocrystals through an intelligent cloud lab

We constructed an intelligent cloud lab that integrates lab automation with cloud servers and artificial intelligence (AI) to detect chirality in perovskites. Driven by the materials acceleration operating system in cloud (MAOSIC) platform, on-demand experimental design by remote users was enabled in this cloud lab. By employing artificial intelligence of things (AIoT) technology, synthesis, characterization, and parameter optimization can be autonomously achieved. Through the remote collaboration of researchers, optically active inorganic perovskite nanocrystals (IPNCs) were first synthesized with temperature-dependent circular dichroism (CD) and inversion control. The inter-structure (structural patterns) and intra-structure (screw dislocations) dual-pattern-induced mechanisms detected by MAOSIC were comprehensively investigated, and offline theoretical analysis revealed the thermodynamic mechanism inside the materials. This self-driving cloud lab enables efficient and reliable collaborations across the world, reduces the setup costs of in-house facilities, combines offline theoretic analysis, and is practical for accelerating the speed of material discovery.

Circular Dichroism Study of Late Embryogenesis Abundant Peptides in Reverse Micelles

Late Embryogenesis Abundant (LEA) proteins are a class of characteristically unstructured proteins found within plants at their later stages of seed development. While LEA proteins lack distinct structural characteristics under standard conditions, certain extreme conditions of temperature or drought may allow the formation of functional structure. Through the use of Circular Dichroism (CD) spectroscopy, the structural characteristics of two different 11-mer repeat units of Group 3 LEA proteins has been studied. Here, solutions of the peptides in AOT reverse micelles have been used to simulate conditions of low peptide hydration, with a variety of w0 values investigated. In comparison to aqueous CD results which confirm the characteristic disordered structure of LEA proteins, CD results in reverse micelles suggest a preference for α-helical character under low hydration conditions. Furthermore, temperature-dependent CD scans show interesting non-monotonic behavior. These results enhance our understanding of how LEA proteins may be structured biologically and allow further speculation regarding their role in seed development.

Dynamic elements and kinetics: Most favorable conformations of peptides in solution with measurements and simulations.

Small peptides in solution adopt a specific morphology as they function. It is of fundamental interest to examine the structural properties of these small biomolecules in solution and observe how they transition from one conformation to another and form functional structures. In this study, we have examined the structural properties of a simple dipeptide and a five-residue peptide with the application of far-UV circular dichroism (CD) spectroscopy as a function of temperature, fluorescence anisotropy, and all-atom molecular dynamics simulation. Analysis of the temperature dependent CD spectra shows that the simplest dipeptide N-acetyl-tryptophan-amide (NATA) adopts helical, beta sheet, and random coil conformations. At room temperature, NATA is found to have 5% alpha-helical, 37% beta sheet, and 58% random coil conformations. To our knowledge, this type of structural content in a simplest dipeptide has not been observed earlier. The pentapeptide (WK5) is found to have four major secondary structural elements with 8% 310 helix, 14% poly-L-proline II, 8% beta sheet, and 14% turns. A 56% unordered structural population is also present for WK5. The presence of a significant population of 310 helix in a simple pentapeptide is rarely observed. Fluorescence anisotropy decay (FAD) measurements yielded reorientation times of 45 ps for NATA and 120 ps for WK5. The fluorescence anisotropy decay measurements reveal the size differences between the two peptides, NATA and WK5, with possible contributions from differences in shape, interactions with the environment, and conformational dynamics. All-atom molecular dynamics simulations were used to model the structures and motions of these two systems in solution. The predicted structures sampled by both peptides qualitatively agree with the experimental findings. Kinetic modeling with optimal dimensionality reduction suggests that the slowest dynamic processes in the dipeptide involve sidechain transitions occurring on a 1 ns timescale. The kinetics in the pentapeptide monitors the formation of a distorted helical structure from an extended conformation on a timescale of 10 ns. Modeling of the fluorescence anisotropy decay is found to be in good agreement with the measured data and correlates with the main contributions of the measured reorientation times to individual conformers, which we define as dynamic elements. In NATA, the FAD can be well represented as a sum of contributions from representative conformers. This is not the case in WK5, where our analysis suggests the existence of coupling between conformational dynamics and global tumbling. The current study involving detailed experimental measurements and atomically detailed modeling reveals the existence of specific secondary structural elements and novel dynamical features even in the simplest peptide systems.

Construction of optical active metallo-supramolecular polymers from enantiopure bis-pybox ligands

Two types of optical active metallo-supramolecular polymers were successfully constructed from the complexation of two enantiopure bis-pyridine-dioxazole (bis-pybox) ligands and 3d transition metal ions such as Zn2+ and Fe2+ in organic solution. The self-assembly process was investigated via UV–vis and fluorescent titration. The chiral characteristic of polymers was detected by circular dichroism. It was revealed that the phenyl-substituted polymers showed higher stability than the benzyl-substituted ones. Temperature-dependent circular dichroism spectra demonstrated the dynamic structure of this type polymer under the variation of temperature. This work highlights the opportunity in designing and constructing of the optical active metallo-supramolecular polymers from the organic privilege ligands in the field of asymmetric catalysis.

Conformational Bias in Unfolded Proteins Studied by Sequence Reversal

Sequence patterns of charge, hydrophobicity, hydrogen bonding, and other amino acid physicochemical properties contribute to mechanisms of protein folding, but how sequence composition and patterns influence the conformational dynamics of the denatured state ensemble is not fully understood. To investigate structure-sequence relationships in the denatured state, we reversed the sequence of staphylococcal nuclease and characterized its structure, thermodynamic character, and hydrodynamic radius using circular dichroism spectroscopy, dynamic light scattering, analytical ultracentrifugation, and size-exclusion chromatography as a function of temperature. The macromolecular size of “Retro-nuclease” was found to be highly expanded in solution with characteristics similar to biological intrinsically disordered proteins. In contradistinction to a disordered state, Retro-nuclease exhibits a broad sigmoid transition of its hydrodynamic dimensions as temperature is increased, indicating a thermodynamically controlled compaction. Counterintuitively, the magnitude of these temperature-induced hydrodynamic changes exceed that observed from thermal denaturation of folded unaltered staphylococcal nuclease. Undetectable by calorimetry and intrinsic tryptophan fluorescence, the lack of heat capacity or fluorescence changes throughout the thermal transition indicate canonical hydrophobic collapse did not drive the Retro-nuclease structural transitions. Instead, temperature-dependent circular dichroism spectroscopy and computer simulation correlate the temperature sensitivity to the intrinsic sampling of backbone conformations for polyproline II and α-helix. Using analytical techniques that relate backbone conformational bias to hydrodynamic size, the observed changes in Retro-nuclease structure with temperature indicate backbone conformational propensities and coil transition enthalpies that are in good agreement with calorimetric and spectroscopic studies of short peptides.

Retinal-Salinixanthin Interactions in a Thermophilic Rhodopsin.

In microbial rhodopsins (also called retinal proteins), the retinal chromophore is used for harvesting light. A carotenoid molecule has been reported to complement the retinal as light harvesting antenna in bacterial retinal proteins, although examples are scarce. In this paper, we present the formation of a novel antenna complex between thermophilic rhodopsin (TR) and the carotenoid salinixanthin (Sal). The complex formation and its structure were studied using UV-visible absorption as well as circular dichroism (CD) spectroscopies. Our studies indicate that the complex is formed in both the trimeric and monomeric forms of TR. CD spectroscopy suggests that excitonic coupling takes place between retinal and Sal. The binding of Sal with artificial TR pigments derived from synthetic retinal analogues further supports the contribution of the retinal chromophore to the CD spectrum. These studies further support the possibility of interaction between the 4-keto ring of the Sal and the retinal in TR-Sal complexes. Temperature-dependent CD spectra indicate that the positive band (ca. 482 nm) of the bisignate CD spectra of the studied complexes originates from the contribution of excitonic coupling and induced chirality of Sal in the protein binding site. The presence of a relatively smaller glycine residue in the vicinity of the retinal chromophore in TR is proposed to be crucial for binding with Sal. The results are expected to shed light on the mechanism of retinal-carotenoid interactions in other biological systems.

Peculiar Triarylamine-Based Co-assembled Supramolecular Polymers That Exhibit Two Transition Temperatures in the Formation of a Coiled Helical Bundle.

This paper describes the peculiar co-assembly supramolecular polymerization behavior of triphenylamine trisamide derivatives with d-alanine (T-ala) or glycine (T-gly) moieties. Concentration and temperature-dependent circular dichroism (CD) spectroscopy revealed that the heating curves of co-assemblies obtained at various molar ratios of T-ala to T-gly exhibited two distinct transition temperatures. The first transition was due to the transformation from coiled helical bundles to single helical fibers without handedness. The second was due to a change from typical elongation to nucleation. These phenomena were confirmed by solvent-dependent decoiling of coiled helical structures and concentration-dependent morphological analysis. The two transitioning temperatures were dependent on the concentration of T-ala in the co-assemblies, suggesting that T-ala concentration plays an important role in the formation of coiled helical bundles. Our study demonstrated the first observation of two distinct transition temperatures in supramolecular polymers.