Chengshan Wang

Redox Reactions of the α-Synuclein-Cu2+ Complex and Their Effects on Neuronal Cell Viability

α-Synuclein (α-syn), a presynaptic protein believed to play an important role in neuropathology in Parkinsons disease (PD), is known to bind Cu(2+). Cu(2+) has been shown to accelerate the aggregation of α-syn to form various toxic aggregates in vitro. Copper is also a redox-active metal whose complexes with amyloidogenic proteins/peptides have been linked to oxidative stress in major neurodegenerative diseases. In this work, the formation of the Cu(2+) complex with α-syn or with an N-terminal peptide, α-syn(1-19), was confirmed with electrospray-mass spectrometry (ES-MS). The redox potentials of the Cu(2+) complex with α-syn (α-syn-Cu(2+)) and α-syn(1-19) were determined to be 0.018 and 0.053 V, respectively. Furthermore, the Cu(2+) center(s) can be readily reduced to Cu(+), and possible reactions of α-syn-Cu(2+) with cellular species (e.g., O(2), ascorbic acid, and dopamine) were investigated. The occurrence of a redox reaction can be rationalized by comparing the redox potential of the α-syn-Cu(2+) complex to that of the specific cellular species. For example, ascorbic acid can directly reduce α-syn-Cu(2+) to α-syn-Cu(+), setting up a redox cycle in which O(2) is reduced to H(2)O(2) and cellular redox species is continuously exhausted. In addition, the H(2)O(2) generated was demonstrated to reduce viability of the neuroblastoma SY-HY5Y cells. Although our results ruled out the direct oxidation of dopamine by α-syn-Cu(2+), the H(2)O(2) generated in the presence of α-syn-Cu(2+) can oxidize dopamine. Our results suggest that oxidative stress is at least partially responsible for the loss of dopaminergic cells in PD brain and reveal the multifaceted role of the α-syn-Cu(2+) complex in oxidative stress associated with PD symptoms.

Binding of α-Synuclein with Fe(III) and with Fe(II) and Biological Implications of the Resultant Complexes

Parkinsons disease (PD) is hallmarked by the abnormal intracellular inclusions (Lewy bodies or LBs) in dopaminergic cells. Amyloidogenic protein alpha-synuclein (alpha-syn) and iron (including both Fe(III) and Fe(II)) are both found to be present in LBs. The interaction between iron and alpha-syn might have important biological relevance to PD etiology. Previously, a moderate binding affinity between alpha-syn and Fe(II) (5.8x10(3)M(-1)) has been measured, but studies on the binding between alpha-syn and Fe(III) have not been reported. In this work, electrospray mass spectrometry (ES-MS), cyclic voltammetry (CV), and fluorescence spectroscopy were used to study the binding between alpha-syn and Fe(II) and the redox property of the resultant alpha-syn-Fe(II) complex. The complex is of a 1:1 stoichiometry and can be readily oxidized electrochemically and chemically (by O(2)) to the putative alpha-syn-Fe(III) complex, with H(2)O(2) as a co-product. The reduction potential was estimated to be 0.025V vs. Ag/AgCl, which represents a shift by -0.550V vs. the standard reduction potential of the free Fe(III)/Fe(II) couple. Such a shift allows a binding constant between alpha-syn and Fe(III), 1.2x10(13)M(-1), to be deduced. Despite the relatively high binding affinity, alpha-syn-Fe(III) generated from the oxidation of alpha-syn-Fe(II) still dissociates due to the stronger tendency of Fe(III) to hydrolyze to Fe(OH)(3) and/or ferrihydrite gel. The roles of alpha-syn and its interaction with Fe(III) and/or Fe(II) are discussed in the context of oxidative stress, metal-catalyzed alpha-syn aggregation, and iron transfer processes.

Surface Chemistry and in Situ Spectroscopy of a Lysozyme Langmuir Monolayer

Surface pressure and surface potential-area isotherms were used to characterize a lysozyme Langmuir monolayer. The compression-decompression cycles and stability measurements showed a homogeneous and stable monolayer at the air-water interface. Salt concentration in the subphase and pH of the subphase were parameters controlling the homogeneity and stability of the Langmuir monolayer. In situ UV-vis and fluorescence spectroscopies were used to verify the homogeneity of the lysozyme monolayer and to identify the chromophore residues in the lysozyme. Optimal experimental conditions were determined to prepare a homogeneous and stable lysozyme Langmuir monolayer.

α-Synuclein in α-helical conformation at air–water interface: implication of conformation and orientation changes during its accumulation/aggregation

Alpha-synuclein, a natively unstructured protein important in the neuropathology of Parkinsons disease, was found to form a Langmuir monolayer in an alpha-helical conformation with its helical axis parallel to the air-water interface. This study sheds light on the role of vesicles in neuronal cells in the accumulation/aggregation of alpha-synuclein.

Infrared Reflection-Absorption Spectroscopy and Polarization-Modulated Infrared Reflection-Absorption Spectroscopy Studies of the Organophosphorus Acid Anhydrolase Langmuir Monolayer

The secondary structure of the organophosphorus acid anhydrolase (OPAA) Langmuir monolayer in the absence and presence of diisopropylfluorophosphate (DFP) in the subphase was studied by infrared reflection-absorption spectroscopy (IRRAS) and polarization-modulated IRRAS (PM-IRRAS). The results of both the IRRAS and the PM-IRRAS indicated that the alpha-helix and the beta-sheet conformations in OPAA were parallel to the air-water interface at a surface pressure of 0 mN.m-1 in the absence of DFP in the subphase. When the surface pressure increased, the alpha-helix and the beta-sheet conformations became tilted. When DFP was added to the subphase at a concentration of 1.1 x 10(-5) M, the alpha-helix conformation of OPAA was still parallel to the air-water interface, whereas the beta-sheet conformation was perpendicular at 0 mN.m-1. The orientations of both the alpha-helix and the beta-sheet conformations did not change with the increase of surface pressure. The shape of OPAA molecules is supposed to be elliptic, and the long axis of OPAA was parallel to the air-water interface in the absence of DFP in the subphase, whereas the long axis became perpendicular in the presence of DFP. This result explains the decrease of the limiting molecular area of the OPAA Langmuir monolayer when DFP was dissolved in the subphase.

Conversion of Natively Unstructured α-Synuclein to Its α-Helical Conformation Significantly Attenuates Production of Reactive Oxygen Species

The intracellular α-synuclein (α-syn) protein, whose conformational change and aggregation have been closely linked to the pathology of Parkingsons disease (PD), is highly populated at the presynaptic termini and remains there in the α-helical conformation. In this study, circular dichroism confirmed that natively unstructured α-syn in aqueous solution was transformed to its α-helical conformation upon addition of trifluoroethanol (TFE). Electrochemical and UV-visible spectroscopic experiments reveal that both Cu (I) and Cu (II) are stabilized, with the former being stabilized by about two orders of magnitude. Compared to unstructured α-syn (Binolfi et al., J. Am. Chem. Soc. 133 (2011) 194-196), α-helical α-syn stabilizes Cu (I) by more than three orders of magnitude. Through the measurements of H(2)O(2) and hydroxyl radicals (OH) in solutions containing different forms of Cu (II) (free and complexed by unstructured or α-helical α-syn), we demonstrate that the significantly enhanced Cu (I) binding affinity helps inhibit the production of highly toxic reactive oxygen species, especially the hydroxyl radicals. Our study provides strong evidence that, as a possible means to prevent neuronal cell damage, conversion of the natively unstructured α-syn to its α-helical conformation in vivo could significantly attenuate the copper-modulated ROS production.

Infrared reflection-absorption spectroscopy and polarization-modulated infrared reflection-absorption spectroscopy studies of the aequorin langmuir monolayer.

The Langmuir monolayer of aequorin and apoaequorin was studied by infrared reflection-absorption spectroscopy (IRRAS) and polarization-modulated IRRAS techniques. The alpha-helices in the aequorin Langmuir monolayer were parallel to the air-water interface at zero surface pressure. When the surface pressure increased to 15 mN.(m-1), the alpha-helices became tilted and the turns became parallel to the air-water interface. As for apoaequorin, the alpha-helices were also parallel to the air-water interface at 0 mN.m(-1). However, the alpha-helix became tilted and the turns became parallel to the air-water interface quickly at 5 mN.m(-1). With further compression of the apoaequorin Langmuir monolayer, the orientation remained the same. The different behaviors of aequorin and apoaequorin at the air-water interface were explained by the fact that aequorin formed dimers at the air-water interface but apoaequorin was a monomer. It is more difficult for a dimer to be tilted by the compression of the Langmuir monolayer.

Self-assembly and Langmuir-Blodgett (LB) film of a novel hydrogen-bonded complex: a surface enhanced Raman scattering (SERS) study

Abstract Hydrogen-bonded complexes composed of a 1,2-bis(4-pyridyl)ethylene and two fatty acids were employed for the fabrication of organized monolayers. Surface enhanced Raman scattering (SERS) spectra demonstrated that monolayer Langmuir–Blodgett (LB) film could be directly obtained at surface pressure 5 mN m−1 by depositing the complex with one of its hexadecoic acid molecules lost due to decomposition of hydrogen bond. For the self-assembled monolayer, SERS spectra indicated that during self-assembling process, only one part of the complex was adsorbed on silver substrate, just similar to that occurring at air/water interface due to the same reason. Increasing the molar ratio of the hexadecoic acid to the mole of 1,2-bis(4-pyridyl)ethylene in the complex solution up to 16-fold, we successfully prepared a monolayer of the complex without decomposition. Under this condition the 1,2-bis(4-pyridyl)-ethylene was inferred to adopt the adsorption flat on the substrate.

Isotope-edited FTIR in H2O: determination of the conformation of specific residues in a model α-helix peptide by 13C labeled carbonyls

Isotope-edited FTIR spectroscopy has been shown to be able to determine peptides structure in the residue level in D2O, which is not a physiological solvent. Here, attenuated total reflection technique was utilized to successfully apply isotope-edited FTIR spectroscopy in H2O to determine the conformation of specific residues in a model peptide.

Surface chemistry and photophysical properties of a diacetylene-peptide derivative capped quantum dots Langmuir monolayer.

A diacetylene derivative, 10,12-pentacosadiynoic acid (PDA), was conjugated to a small peptide chain Cysteine-Cysteine-Glycine (CCG) through the solid-phase peptide synthesis. The (CdSe)ZnS core-shell quantum dots (QDs) capped with trioctylphosphine ligands were modified through a surface ligand reaction to prepare the PDA-CCG QDs conjugate. Both systems, PDA-CCG and PDA-CCG QDs, were investigated as Langmuir monolayer at the air-water interface through the surface pressure-area (pi-A) isotherms, compression-decompression cycles, stability measurements, and in situ UV-vis and fluorescence spectroscopy. Two different pi-A isotherms were observed for the systems investigated showing the importance of the peptide moiety in PDA-CCG to form a Langmuir monolayer up to a surface pressure of 50 m Nm(-1) compared with 15 m Nm(-1) for the PDA component alone. The compression-decompression cycles and stability measurements for both systems suggest the formation of a stable Langmuir monolayer over 1h time period. Although the in situ UV-vis spectroscopy of PDAA-CCG and PDA-CCG QDs does not show an absorption spectrum, we observed by in situ fluorescence spectroscopy the photoluminescence (PL) of the PDA-CCG QDs at 560 nm, with an intensity of the PL increasing linearly with the increase of the surface pressure. Irradiating the PDA-CCG QDs Langmuir monolayer at 254 nm, we observe the photopolymerization with two distinct bands at 575 (blue band) and 630 nm (red band) of the polymer.