Backbone Of Molecule Research Articles

Leaveraging heat shock protein analogues to induce the expression of Interleukin 10. Smart design of emerging anti-inflammatory biomaterials

Event Abstract Back to Event Leaveraging heat shock protein analogues to induce the expression of Interleukin 10. Smart design of emerging anti-inflammatory biomaterials Mariagemiliana Dessi1, Louise Topping1 and Matteo Santin1 1 University of Brighton, School of Pharmacy and Biomolecular Sciences, United Kingdom INTRODUCTION In tissue engineering and regenerative medicine applications, there is a compelling need of novel therapeutics for the treatment of chronic inflammation. Nowadays, the therapeutic approaches mainly help relieve the symptoms of the inflammation. The high rates of non-responders and the evidence of systemic immunosuppression have evolved in the development of selective anti-inflammatory materials. Among the family of heat shock proteins (HSPs), the heat shock protein 70 (HSP70) is the most evolutionary conserved and is widely recognized for its great ability to treat and prevent inflammatory conditions [1]. In the current scenario, the exploitation of the immunoregulatory nature of peptides derived from heat shock proteins offers a potential alternative for the treatment of inflammatory conditions. The peptide MtHSP70 derived from Mycobacterium tuberculosis HSP70 is of clinical relevance due to its immunoregulatory features. In this study, for the first time, the MtHSP70 peptide has been conjugated to a poly-L-lysine (PLL) used as coating biopolymer in tissue engineering applications. We demonstrate that the presence of MtHSP70 is able to encouraging lymphocyte to release anti-inflammatory cytokines (i.e.IL-10) causing the repolarization of macrophages, from a pro-inflammatory (M1) to an anti-inflammatory skew (M2). Aim of the study is to create an advanced and clinically-reflective coating biomaterial with anti-inflammatory cues to locally treat or prevent chronic inflammations. EXPERIMENTAL METHODS Structurally well-defined MtHSP70 mimicking peptide was synthetized through a bottom up strategy based on a multistep sequence of coupling and deprotection reactions on a solid support. The complete molecule was characterized by mass spectrometry (MS) and Fourier Transformed Infrared spectroscopy (FTIR). The peptide was conjugated by carbodiimide chemistry to poly-L-lysine at different concentrations and the functionalized materials has been analysed by FTIR to evaluate the efficacy of peptide conjugation. The cytotoxicity of the biomaterial was assessed by MTS assay and the viability of cells was evaluated by HPI test. ELISA sandwich test was performed to test the ability of lymphocyte to produce the IL-10 cytokine. Co-colture study was performed to simulate the body conditions and immunocytochemistry assessed the expression of CD206 (M2 specific surface receptor). RESULTS AND DISCUSSION Physico-chemical analysis confirmed the successful synthesis of peptide. The accurate mass measurements of MS (Fig.1) confirmed that the actual molecular weight matches the theoretical value (1611 m/z). The absorption bands of FTIR spectra revealed the characteristic elements present on the molecule backbone. As confirmed by FTIR the chemical grafting of the peptide to PLL was successful. Cell viability analysis reveals that after 24hrs in contact with functionalized substrates, both adherent and non-adherent cells are viable. ELISA test showed that lymphocytes incubated upon the functionalized material release significantly high level of IL-10. Type 1 macrophages co-cultured on PLL showed a low amount of CD206 marker and exhibited a rounded outspread cytoplasm (Fig.2). The expression of CD206 is higher on the macrophages co-coltured on the MtHSP70 functionalized coating and their morphology is evolving towards an elongated cytoskeleton (Fig.3) Figure 2. Expression of CD206 and morphology of macrophages co-coltured on PLL. Figure 3. Expression of CD206 and morphology of macrophages co-coltured on functionalized PLL. CONCLUSION In the current study, a new successful route to obtain anti-inflammatory biomaterials is demonstrated. For the first time, the heat shock mimicking peptide MtHSP70 was covalently attached to a biopolymer. The presence of peptide was able to elicit an anti-inflammatory response by stimulating the IL-10 production at levels that could induce a skewing in macrophages phenotype from M1 (inflammatory state) to M2 (anti-inflammatory state). These results are paving the way for the potential use of MtHSP70 analogues as promising molecules to functionalize biomaterials to locally treat chronic inflammatory conditions.

Rational design of keratin-based injectable hydrogels for biomedical applications

Event Abstract Back to Event Rational design of keratin-based injectable hydrogels for biomedical applications Najeong Park1, Yunki Lee1, Suji Choi2, Kyung Min Park3, Yu-Shik Hwang2 and Ki Dong Park1 1 Ajou university, Molecular Science and Technology, Korea 2 Kyung Hee University, Maxillofacial Biomedical Engineering and Institute of Oral Biology, Korea 3 Incheon National University, Bioengineering, Korea Introduction: Injectable hydrogel systmes have received a great attention in the biomedical research fields due to the minimally invasiveness as well as the structural similarity to the natural extracellular matrix and multi-tunable properties. Up to date, various kinds of biomaterials have been utilized to create injectable hydrogel matrices for therapeutic implants and therapeutic vesicles[1]. Keratin, derived from hair, has emerged as a fascinating biomaterial due to excellent biocompatibility, biodegradability, low immunogenecity, and favorable cellular interactions. While the natural polymer has been widely used for biomedical applications, poor solubility of keratin in aqueous solutions has been recognized as a critical limitation for use in a broad range of applications including development of injectable biomaterial systems[2]. Herein, we developed keratin-based in situ crosslinkable hydrogels through the enzymatic reaction. We fisrt desinged a water soluble karatin by conjugating poly(ethylene glycol) (PEG) molecules tethered with tyrmine (TA), which can be crosslinked to form the hydrogel network. We next evaluated its physico-chemical and biological properteis, showing a great potential as bioactive injectable materials Materials and Methods: Human hair keratin was extracted by slight modified Shindai method[3]. Keratin-PEG-TA (KPT), a water soluble keratin, was synthesized by conjugating amine-PEG-TA (PEG M.W. = 4,000) to keratin backbone via EDC/NHS chemistry with different PEG feed amount. The chemical structure of the KPT was confirmed using 1H NMR and TGA analysis. The physico-chemical properties of KPT hydrogels were examined, such as gelation time, rheological properties depending on HRP or H2O2 concentrations. In vitro cytocompatibility of hydrogels was also evaluated using human dermal fibroblasts cultured with KPT hydrogel extracts. Results and Discussion: The chemical structures of KPT were confirmed by 1H-NMR and TGA analysis, demonstrating PEG and TA were sucessfully conjugated into keratin. We conjugated PEG as a linker to increase water solubility of keratin and confirmed that 5 wt% of KPT conjugate was quickly (~10 sec) and homogeneously dissolved in distilled water though the same concentration of intact keratin was not completely dissolved even with the vigorous vortexing for 30 min. Figure 1a illustrates the schemetic representation of polymer synthesis and hydrogel formation through HRP-mediated crosslinking reaction, forming a C-C bond or C-O bond between phenol moieties. We also evaluated the physico-chemical properties, such as gelation time (ranged from 2 to 100 sec) and mechanical strength (ranged from 100 to 2400 Pa) of the KPT hydrogels (Figure 1b) depending on concentrations of HRP and H2O2, showing the multi-tunable properties. Futhermore, we carried out in vitro cell study to confirm cytocompatibility of the KPT hydrogels, exhibiting excellent cytocompatibility. Conclusions: We have developed a keratin-based in situ cross-linkable hydrogels. It was demonstrated that water solubility of KPT was significantly increased by introducing PEG molecules between the polymer backbone and TA molecules, and the physico-chemical properties could be controlled easily by varying the concentrations of HRP and H2O2 with excellent cytocompatibility. Taken together, we believe that our Keratin-based hydrogels have a great potential for use as injectable materials for a broad range of biomedical applications, including either therapeutic implnats or therapeutic vesicles. This work was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) grant funded by the Ministry of Science, ICT & Future Planning (2015R1A2A1A14027221)

Non-specific immune potentiating activity of fucoidan from a tropical brown algae (Phaeophyceae), Sargassum cristaefolium in tilapia (Oreochromis niloticus)

Fucoidan mainly produced by brown algae is a sulfated polysaccharide with fucose as a main molecule backbone. The objective of this research was to evaluate the non-specific immune potentiating activity of fucoidan from a tropical brown alga, Sargassum cristaefolium indigenous to the Gunungkidul coast, Yogyakarta, Indonesia. Fucoidan was extracted by acidic extraction, separated from alginate by CaCl2 precipitation and precipitated with ethanol. To know the component of fucoidan, acid hydrolysis was conducted and analyzed by using silica gel thin layer chromatography. In these analyses, a commercial fucoidan from Fucus vesiculosus was used as a standard fucoidan. Fucoidan isolated from S. cristaefolium was composed by monosaccharide components of fucose and galactose. The result revealed that injection of fucoidan increased phagocytic activity, total plasma protein, leukocrit and leukocyte count significantly (p < 0.05). The immunomodulating activity of fucoidan from S. cristaefolium was comparable to that of fucoidan standard from F. vesiculosus. However, administration of fucoidan did not affect the phagocytic index and leukocyte differentiation. Injection of fucoidan from S. cristaefolium effectively increased innate immunity parameters of tilapia at doses of 0.4–0.6 mg/kg fish suggested that this fucoidan was potential use for immunostimulant to control fish diseases.

Starch sodium dodecenyl succinate prepared by one-step extrusion and its properties

One-step extrusion was developed to prepare starch sodium dodecenyl succinate (SSDS). Effects of screwing speed, reaction temperature, moisture content, sodium hydroxide amount (as catalyst), and dodecenly succinic anhydride (DDSA) amount on the degree of substitution (DS) were investigated. Optimum conditions were determined and found to be as follows: screwing speed, 110rpm; temperature, 120°C; moisture content, 30%; sodium hydroxide amount, 0.5%; DDSA amount, 3%. Under these conditions, the DS of SSDS was 0.014%, and the reaction efficiency was 78%. The structure of SSDS prepared by one-step extrusion was partially characterised. Infrared absorption spectra showed peaks of ester bond and carbonyl group at 1707 and 1564cm−1, respectively, indicating that dodecenyl succinic groups were introduced into starch molecule backbone by esterification agent. X-ray diffraction analysis showed that compared with native starch, the particle morphology of SSDS prepared by extrusion became irregular, and its crystallinity was partially destroyed.

(Invited) Water and Proton Dynamics in Perfluorinated Surfactants and Membranes by QENS

The proton transport in fuel cell ionomer membranes is intimately related to the dynamical properties of water molecules confined in the interconnected network of ionic domains. The confinement of a fluid at the nanoscale profoundly affects the molecular dynamics and results in important deviations with respect to the bulk. The rotational and translational motions can be uniquely investigated by means of Quasi Elastic Neutron Scattering experiments, especially in the case of protonated systems due to the high incoherent cross section of hydrogen. Insights into the nature of the motions, i.e. diffusive or confined, can be gained by analysing the scattering function S(Q,w) measured on the ps-ns time-scale, Q being the momentum transfer and w the energy transfer of the neutrons. We will present here a comprehensive QENS study of water/proton dynamics in PFSA membranes [1,2] and perfluorinated surfactants used as a model soft confining system [3.4]. The Gaussian model for localized translational diffusion [5] was used to obtain quantitative parameters as the relaxation times, confinement sizes and local/nanometric diffusion coefficients of protons and water molecules. The comparison between state-of-the art fuel cell membranes and self-assembled well-defined surfactant phases as host confining matrices will be used to highlight the subtle interplay between spatial confinement and local interactions, resulting in the presence of slow hydronium motions and faster water motions localized in nanometric droplets. Additionally, we will show that the QENS technique can be invaluably combined with PFG-NMR to obtain a multi-scale comprehensive description of the ionic diffusion and water behaviour [1,2]. These experimental results are also cross-fertilized by Molecular Dynamics simulations performed on the same materials using a coarse-grained backbone and explicit solvent molecules [6]. [1] J-C. Perrin, S. Lyonnard and F. Volino; Quasielastic neutron scattering study of water dynamics in hydrated nafion membranes, Journal of Physical Chemistry C, 111 (2007), 3393-3404.[2] S. Lyonnard and G. Gebel, Neutrons for Fuel Cells Membranes: structure, sorption and transport properties, European Physical Journal 213 (1) (2012), 195-211;S. Lyonnard, Structure and Transport Properties in Polymer Electrolyte Membranes Probed at Microscopic Scales, Springer-Verlag, New Energies, Ed. German Antonio Ferreira, 2013. [3] S. Lyonnard, Q. Berrod, B-A. Bruning, G. Gebel, A. Guillermo, H. Ftouni, J. Ollivier and B. Frick, Perfluorinated surfactants as model charged systems for understanding the effect of confinement on proton transport and water mobility in fuel cell membranes. A study by QENS., Eur. Phys. Journal Special Topics, 189 (1), 205-216 (2010). [4] Q. Berrod; S. Lyonnard ; A. Guillermo.;J.Ollivier.; B. Frick; G.Gebel, QENS investigation of proton confined motions in hydrated perfluorinated sulfonic membranes and self-assembled surfactants, The European Physical Journal 2014, in press. [5] F. Volino, J-C. Perrin and S. Lyonnard; Gaussian Model for Localized Translational Motion: Application to Incoherent Neutron Scattering, Journal of Physical Chemistry B, 110 (2006), 11217-11223. [6] S. Hanot, S. Lyonnard and S. Mossa, Water confined in self-assembled ionic surfactants nanostructures, under review (2014).

Iron-promoted C-C bond formation in the total synthesis of natural products and drugs.

Iron salts are inexpensive and almost innocuous; they are thus the promoters of choice, even in stoichiometric amounts, for the formation of carbon-carbon bonds in the backbone of complex molecules. This review encompasses the key role of iron complexes in the total synthesis of some natural products or pharmacologically important compounds.

The Orientational Order of the α-Helical Backbone of Poly(γ-Benzyl L-Glutamate) – a Molecular Dynamics Simulation

For a Poly(γ-benzyl L-glutamate) (PBLG) segment with 30 monomers, molecular dynamics simulation was carry out in the Amber forcefield, by the software package InsightII/Discover. The geometrical structure of the backbone of the model molecule in the minimized energy state is in close agreement with that in the solid polypeptides. In the dynamic equilibrium state, the structure becomes slightly flabby and has a potential ca. 4800kJ/mol higher than that in the minimized energy state for the model molecule. On the average, the α-helical backbone architecture of PBLG remains quite stable during the molecular dynamic process, though the fluctuation of the bond and dihedral angles. The observed D NMR spectra for the quadrupolar splittings are reproduced and the previous architecture analysis was verified by the simulation calculations.

Linker‐dependent Junction Formation Probability in Single‐Molecule Junctions

We compare the junction formation probabilities of single‐molecule junctions with different linker molecules by using a scanning tunneling microscope‐based break‐junction technique. We found that the junction formation probability varies as SH &gt; SMe &gt; NH2 for the benzene backbone molecule with different types of anchoring groups, through quantitative statistical analysis. These results are attributed to different bonding forces according to the linker groups formed with Au atoms in the electrodes, which is consistent with previous works. Our work allows a better understanding of the contact chemistry in the metal–molecule junction for future molecular electronic devices.

Investigation of the effect of large aromatic fusion in the small molecule backbone on the solar cell device fill factor

Molecules DR3TCz and DR2TDTCz were synthesized and DR2TDTCz exhibits a PCE up to 7.03% with a FF of 75%.

Single Gold Atom Containing Oligo(phenylene)ethynylene: Assembly into LB Films and Electrical Characterization

Monomolecular films of an oligo(phenylene)ethynylene, OPE, derivative [2-isocyano-1,3-dimethylbenzene][4-(4′-aminophenylethynyl)phenylethynyl]gold, 1, containing a gold atom in the molecule backbone have been prepared by the Langmuir–Blodgett (LB) method in order to study how the electrical properties can be modulated in monolayers of OPEs by incorporation of a gold center in their structures. UV–vis reflection spectra of Langmuir monolayers of 1 at the air–water interface reveal strong aurophilic interactions between neighboring molecules that increase upon compression. Monolayer Langmuir–Blodgett (LB) films were readily fabricated by the transfer of Langmuir films of 1 onto solid substrates. Quartz crystal microbalance (QCM) experiments conclusively demonstrate formation of monolayer LB films with a high surface coverage. The morphology of these films was analyzed by atomic force microscopy (AFM), revealing formation of homogeneous layers with an optimum surface pressure of transference of 6 mN·m–1. Fil...

Functionalized olefin cross-coupling to construct carbon-carbon bonds.

Carbon–carbon (C–C) bonds form the backbone of many important molecules, including polymers, dyes, and pharmaceutical agents. The development of new methods to create these essential connections in a rapid and practical fashion has been the focus of numerous organic chemists. This endeavor heavily relies on the ability to form C–C bonds in the presence of sensitive functional groups and congested structural environments. Here we report a fundamentally new chemical transformation that allows for the facile construction of highly substituted and uniquely functionalized C–C bonds. Using a simple iron catalyst, an inexpensive silane, and a benign solvent under an ambient atmosphere, heteroatom-substituted olefins are easily merged with electron-deficient olefins to create molecular architectures that were previously difficult or impossible to access. More than sixty examples are presented with a wide array of substrates, demonstrating the unique chemoselectivity and mildness of this simple reaction.

Spontaneous spin polarization in rubrene studied by density functional theory calculations

We theoretically studied the spontaneous spin polarization properties of organic molecule rubrene by using density functional theory calculations. Our investigations show that normally nonmagnetic molecule rubrene could be spin polarized by spinless-hole injection. Magnetic moment of the molecule increases linearly with the extra hole charge amount only when the injected hole charges reach a certain value. The spin density resides predominantly on the carbon atoms in the tetracene backbone of rubrene molecule and also the bond lengths change differently due to the injected charge. Spontaneous spin polarization can be explained as the preferably filling of the spin-splitted carbon pz orbitals near the Fermi energy for the injected charge.

Quantifying Surfactant Alkyl Chain Orientation and Conformational Order from Sum Frequency Generation Spectra of CH Modes at the Surfactant-Water Interface.

We combine second-order nonlinear vibrational spectroscopy and quantum-chemical calculations to quantify the molecular tilt angle and the structural variation of a decanoic acid surfactant monolayer on water. We demonstrate that there is a remarkable degree of delocalization of the vibrational modes along the backbone of the amphiphilic molecule. A simulation-based on modeled sum frequency generation (SFG) spectra offers quantitative insights into the disorder of surfactant monolayers at the water-air interface. It is shown that an average of one gauche defect in the alkyl chain suffices to give rise to the methylene stretch intensity similar in magnitude to the methyl stretch.

Change in molecular structure and dynamics of protein in milk protein concentrate powder upon ageing by solid-state carbon NMR

Instability of proteins in dry form causes solubility loss of milk protein concentrate (MPC) powder upon ageing. High resolution solid state NMR techniques were used to investigate the changes in molecular structure and dynamics of proteins in MPC with varying moisture content (5.5–16.5% w/w) and storage period. The results indicate a slight higher rigidity of molecular domains of protein molecules of non-aged MPC compared to that of the long aged (at 25 °C) MPC. It could be suggested from this observation that long-term storage at high relative humidity (RH) may reduce rigidity of the molecular domains due to interaction with water rather than short-term storage at high RH. This may indicate increased molecular mobility of backbone and side chains of protein molecules due to plasticization during ageing which could facilitate protein–protein interaction and protein denaturation.

Re-evaluation of biotin-streptavidin conjugation in Förster resonance energy transfer applications.

Bioaffinity conjugation between streptavidin (SA) and biotin has been widely used to link donors and acceptors for investigating the distance-dependent Förster resonance energy transfer (FRET). When studying a commonly used FRET system of (QD-SA)-(biotin-DNA-dye) [donor: quantum dot (QD); acceptor: small organic fluorescent dye; and linker: deoxyribose nucleic acid (DNA) molecule via SA-biotin conjugation], however, a contradictory finding was recently reported in the literature. It was found that the FRET lost its dependence on the number of DNA base pairs when using a phosphate-buffered saline (PBS) solution. We found that the conflicted results were caused by the ionic strength of the adopted buffer solutions. Our results suggest that the dependent FRET on the number of DNA bases is favorable in a low-ionic-strength buffer, whereas in relatively high-ionic-strength buffers, the FRET loses the DNA length dependence. We propose that the independence is mainly caused by the conformational change of DNA molecules from a stretched to a coiled mode when the cations in the high-ionic-strength buffer neutralize the negatively charged backbone of DNA molecules, thereby bringing the acceptors close to the donors.

Influence of cross-link structure, density and mechanical properties in the mesoscale deformation mechanisms of collagen fibrils

Collagen is a ubiquitous protein with remarkable mechanical properties. It is highly elastic, shows large fracture strength and enables substantial energy dissipation during deformation. Most of the connective tissue in humans consists of collagen fibrils composed of a staggered array of tropocollagen molecules, which are connected by intermolecular cross-links. In this study, we report a three-dimensional coarse-grained model of collagen and analyze the influence of enzymatic cross-links on the mechanics of collagen fibrils. Two representatives immature and mature cross-links are implemented in the mesoscale model using a bottom-up approach. By varying the number, type and mechanical properties of cross-links in the fibrils and performing tensile test on the models, we systematically investigate the deformation mechanisms of cross-linked collagen fibrils. We find that cross-linked fibrils exhibit a three phase behavior, which agrees closer with experimental results than what was obtained using previous models. The fibril mechanical response is characterized by: (i) an initial elastic deformation corresponding to the collagen molecule uncoiling, (ii) a linear regime dominated by molecule sliding and (iii) the second stiffer elastic regime related to the stretching of the backbone of the tropocollagen molecules until the fibril ruptures. Our results suggest that both cross-link density and type dictate the stiffness of large deformation regime by increasing the number of interconnected molecules while cross-links mechanical properties determine the failure strain and strength of the fibril. These findings reveal that cross-links play an essential role in creating an interconnected fibrillar material of tunable toughness and strength.

Molecular dynamics study of changes in physico-chemical properties of DMPC lipid bilayers by addition of nonionic surfactant C12E10

Changes in physico-chemical properties of dimyristoyl phosphatidylcholine (DMPC) lipid bilayers caused by the addition of 9.4 mol% nonionic surfactant decaoxyethylene monododecyl ethers (C12E10) have been investigated by molecular dynamics calculations. In spite of addition of single chain C12E10, the lipid bilayers showed an increase of membrane area. Isothermal area compressibility, which is a measure of membrane softness in lateral direction, also increased by 50% for DMPC/C12E10 mixed bilayers. Furthermore, the order parameter of C–H vector for DMPC acyl tails decreased. We found that these changes are caused by the hydrophilic head groups of C12E10 which are located near the glycerol backbone of the DMPC molecules and have bulky random coil conformation without any preferential ordered structures.

Probe Dependent Solvation Dynamics Study in a Microscopically Immiscible Dimethyl Sulfoxide-Glycerol Binary Solvent.

Excited state dipole solvation of three coumarin dyes with different hydrophobicities was studied in DMSO-glycerol binary solvent. The solvation times obtained from the three dyes are remarkably different. The highly hydrophilic dye coumarin 343 (C343) exhibits the slowest solvation time (>12 ns) among all the dyes we used. This is in contrast to the most hydrophobic dye coumarin 153 (C153), where the solvated state is reached just within ∼104 ps. However, the moderately hydrophobic dye coumarin 480 (C480) demonstrates an intermediate (∼396 ps) solvation time. Unprecedented slowdown of solvation time of C343 is probably due to the slow diffusion of solvent molecules in the glycerol-rich first solvation shell followed by hydrogen bond rearrangements around the solute dipole. On the other hand, fast solvation of hydrophobic dye C153 is most likely caused by the fast reorganization dynamics of hydrophobic -CH3 groups of DMSO or the carbon backbone of the glycerol molecule around the solute dipole. Interestingly, a remarkable probe dependency in solvation dynamics was not observed in the case of DMSO-water binary solvent or in a neat solvent isopropanol. Probe dependent solvation in a DMSO-glycerol mixture is attributed to the microscopic phase segregation and different locations of coumarin dyes within this binary solvent.

False asymmetry, pseudosymmetry, disorder, polymorphism and atomic displacement parameters

Two similar sugars, with chemical formulas differing only by the presence of a methyl group connected to the molecule backbones in different positions, crystallize in the monoclinic P21 space group giving rise to Z′=2 structures. They both bear an azide side chain which is the principal responsible for the lack of a higher symmetry for one compound only. We analyzed their most relevant features by means of X-ray single crystal diffraction coupled with a quantitative estimation of their potential tendency to crystallize in a different space group with higher symmetry. The latter tendency of the most promising of the two compounds is commented in the light of the anisotropic behaviour of the atomic displacement parameters.

Resonance Raman and Low Temperature FTIR Characterization of the Red Shifted Channelrhodopsin 1 from Chlamydomonas Augustae

Channelrhodopsins (ChRs) control phototaxis in green algae and function as light-gated cation channels when expressed in animal cells. Because ChRs can be functionally expressed in neuronal membranes, this distinct family of microbial rhodopsins have rapidly become an important tool in neuroscience. While the light-activated molecular changes occurring in channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) have been extensively studied, little is known about such changes in the diverse groups of other ChRs including the major class of channelrhodopsin-1 (ChR1). Here, we have characterized the structure and molecular changes in ChR1 from Chlamydomonas augustae (CaChR1). This ChR has properties advantageous for light modulated neuronal control including a red-shifted λmax and slow light inactivation compared to CrChR2 (Hou, S. et al. (2012) Photochem Photobiol 88, 119-128). Near-infrared confocal resonance Raman spectroscopy (RRS) reveals that in contrast to CrChR2, which contains a mixture of retinal isomers, the retinal chromophore structure of CaChR1 is almost completely all-trans in the light-adapted state similar to many microbial rhodopsins including bacteriorhodopsin. In addition, unlike other ChR1s, such as CrChR1 (ChR1 from Chlamydomonas reinhardtii), which exhibit significant shifts in λmax at different pHs, the RRS of CaChR1 including the ethylenic frequency which reflects λmax remains largely unaltered over a wide pH range. This insensitivity to pH is despite the presence of the residue Glu87 (CrChR1 sequence numbering) which has been previously associated with pH sensitivity. Low-temperature FITR difference-spectroscopy reveals that the primary phototransition of CaChR1 to the K photointermediate involves an all-trans to 13-cis retinal isomerization and significant changes in the protein structure including structural changes in the peptide backbone, Asp/Glu residues, Cys residues and internal water molecules. These results are discussed in terms of possible differences in the molecular mechanism of various ChRs.