Mechanism of Interaction between the General Anesthetic Halothane and a Model Ion Channel Protein, III: Molecular Dynamics Simulation Incorporating a Cyanophenylalanine Spectroscopic Probe

A nitrile-derived amino acid, Phe_CN, has been used as an internal spectroscopic probe to study the binding of an inhalational anesthetic to a model membrane protein. The infrared spectra from experiment showed a blue-shift of the nitrile vibrational frequency in the presence of the anesthetic halothane. To interpret the infrared results and explore the nature of the interaction between halothane and the model protein, all-atom molecular dynamics (MD) simulations have been used to probe the structural and dynamic properties of the protein in the presence and absence of one halothane molecule. The frequency shift analyzed from MD simulations agrees well with the experimental infrared results. Decomposition of the forces acting on the nitrile probes demonstrates an indirect impact on the probes from halothane, namely a change of the protein's electrostatic local environment around the probes induced by halothane. Although the halothane remains localized within the designed hydrophobic binding cavity, it undergoes a significant amount of translational and rotational motion, modulated by the interaction of the trifluorine end of halothane with backbone hydrogens of the residues forming the cavity. This dominant interaction between halothane and backbone hydrogens outweighs the direct interaction between halothane and the nitrile groups, making it a good “spectator” probe of the halothane-protein interaction. These MD simulations provide insight into action of anesthetic molecules on the model membrane protein, and also support the further development of nitrile-labeled amino acids as spectroscopic probes within the designed binding cavity.     

Mechanism of Interaction between the General Anesthetic Halothane and a Model Ion Channel Protein, I: Structural Investigations via X-Ray Reflectivity from Langmuir Monolayers

We previously reported the synthesis and structural characterization of a model membrane protein comprised of an amphiphilic 4-helix bundle peptide with a hydrophobic domain based on a synthetic ion channel and a hydrophilic domain with designed cavities for binding the general anesthetic halothane. In this work, we synthesized an improved version of this halothane-binding amphiphilic peptide with only a single cavity and an otherwise identical control peptide with no such cavity, and applied x-ray reflectivity to monolayers of these peptides to probe the distribution of halothane along the length of the core of the 4-helix bundle as a function of the concentration of halothane. At the moderate concentrations achieved in this study, approximately three molecules of halothane were found to be localized within a broad symmetric unimodal distribution centered about the designed cavity. At the lowest concentration achieved, of approximately one molecule per bundle, the halothane distribution became narrower and more peaked due to a component of ∼19Å width centered about the designed cavity. At higher concentrations, approximately six to seven molecules were found to be uniformly distributed along the length of the bundle, corresponding to approximately one molecule per heptad. Monolayers of the control peptide showed only the latter behavior, namely a uniform distribution along the length of the bundle irrespective of the halothane concentration over this range. The results provide insight into the nature of such weak binding when the dissociation constant is in the mM regime, relevant for clinical applications of anesthesia. They also demonstrate the suitability of both the model system and the experimental technique for additional work on the mechanism of general anesthesia, some of it presented in the companion parts II and III under this title.     

Study of Interaction of GM2 Activator Protein with GM2 Using Circular Dichroism and Fluorescence Spectroscopy

GM2 activator protein (GM2AP) is a cofactor for stimulating the enzymatic hydrolysis of the glycolipid GM2 by -hexosaminidase A to produce GM3. We have examined the conformation of GM2AP before and after its interaction with GM2, GM3, and GA2 using circular dichroism and fluorescence spectroscopy techniques. In the presence of GM2, a blue shift of the fluorescence emission maximum and a strong decrease of molar ellipticity values in circular dichroism spectra were observed only at pH 4.5 and at GM2/GM2AP molar ratio higher than 10:1 (up to 50:1). These results suggest that GM2AP assumed a more organized -helical conformation with the tryptophan residues moving from the polar medium toward the hydrophobic environment of the protein. The conformation of GM2AP in the presence of the downstream reaction product, GM3, or a less favorable substrate, GA2, clearly differed from that in the presence of GM2. The relationships between spectroscopic changes and enzymatic activity, herein discussed, strongly suggest that the specific conformation exhibited by GM2AP in the presence of GM2 is functional to serve as an activator for the enzymatic hydrolysis of GM2.     

Multisite Binding of a General Anesthetic to the Prokaryotic Pentameric Erwinia chrysanthemi Ligand-gated Ion Channel (ELIC)

Pentameric ligand-gated ion channels (pLGICs), such as nicotinic acetylcholine, glycine, γ-aminobutyric acid GABA_A/C receptors, and the Gloeobacter violaceus ligand-gated ion channel (GLIC), are receptors that contain multiple allosteric binding sites for a variety of therapeutics, including general anesthetics. Here, we report the x-ray crystal structure of the Erwinia chrysanthemi ligand-gated ion channel (ELIC) in complex with a derivative of chloroform, which reveals important features of anesthetic recognition, involving multiple binding at three different sites. One site is located in the channel pore and equates with a noncompetitive inhibitor site found in many pLGICs. A second transmembrane site is novel and is located in the lower part of the transmembrane domain, at an interface formed between adjacent subunits. A third site is also novel and is located in the extracellular domain in a hydrophobic pocket between the β7–β10 strands. Together, these results extend our understanding of pLGIC modulation and reveal several specific binding interactions that may contribute to modulator recognition, further substantiating a multisite model of allosteric modulation in this family of ion channels.     

Binding of the Activating Ion Co(II) to myo-Inositol Monophosphatase Monitored by Fluorescence and Phosphorescence Spectroscopy

Two extrinsic probes, pyrene-maleimide and eosin-maleimide, were used to label specific SH groups of the enzyme myo-inositol monophosphatase. The fluorescence of pyrene-monophosphatase is enhanced upon addition of the activating metal ions Co(II) and Mg(II). Co(II) ions bind with a dissociation constant of 4 μM, whereas the apparent activation constant K _a is 0.4 mM. Energy transfer measurements demonstrated that the pyrene chromophore, covalently linked to Cys-218, is within 9 Å of the metal ion Tb(III) coordinated to the metal-binding site. The phosphorescence emitted by eosin covalently linked to the protein is quenched by the addition of the activating cations Co(II) and Mg(II). Phosphorescence titrations conducted under anaerobic conditions were used to determine a dissociation constant of approximately 3 μM for the binding of Co(II) ions. The results are consistent with the hypothesis that two activating ions per monomeric subunit participate in the catalytic mechanism. The affinity of the tightly bound ion is at least 100-fold greater than the affinity of the weakly bound ion.     

Structure and Inhibition of the SARS Coronavirus Envelope Protein Ion Channel

The envelope (E) protein from coronaviruses is a small polypeptide that contains at least one α-helical transmembrane domain. Absence, or inactivation, of E protein results in attenuated viruses, due to alterations in either virion morphology or tropism. Apart from its morphogenetic properties, protein E has been reported to have membrane permeabilizing activity. Further, the drug hexamethylene amiloride (HMA), but not amiloride, inhibited in vitro ion channel activity of some synthetic coronavirus E proteins, and also viral replication. We have previously shown for the coronavirus species responsible for severe acute respiratory syndrome (SARS-CoV) that the transmembrane domain of E protein (ETM) forms pentameric α-helical bundles that are likely responsible for the observed channel activity. Herein, using solution NMR in dodecylphosphatidylcholine micelles and energy minimization, we have obtained a model of this channel which features regular α-helices that form a pentameric left-handed parallel bundle. The drug HMA was found to bind inside the lumen of the channel, at both the C-terminal and the N-terminal openings, and, in contrast to amiloride, induced additional chemical shifts in ETM. Full length SARS-CoV E displayed channel activity when transiently expressed in human embryonic kidney 293 (HEK-293) cells in a whole-cell patch clamp set-up. This activity was significantly reduced by hexamethylene amiloride (HMA), but not by amiloride. The channel structure presented herein provides a possible rationale for inhibition, and a platform for future structure-based drug design of this potential pharmacological target.     

Binding of the Activating Ion Co(II) to myo-Inositol Monophosphatase Monitored by Fluorescence and Phosphorescence Spectroscopy

Two extrinsic probes, pyrene-maleimide and eosin-maleimide, were used to label specific SH groups of the enzyme myo-inositol monophosphatase. The fluorescence of pyrene-monophosphatase is enhanced upon addition of the activating metal ions Co(II) and Mg(II). Co(II) ions bind with a dissociation constant of 4 M, whereas the apparent activation constant K _a is 0.4 mM. Energy transfer measurements demonstrated that the pyrene chromophore, covalently linked to Cys-218, is within 9 Å of the metal ion Tb(III) coordinated to the metal-binding site. The phosphorescence emitted by eosin covalently linked to the protein is quenched by the addition of the activating cations Co(II) and Mg(II). Phosphorescence titrations conducted under anaerobic conditions were used to determine a dissociation constant of approximately 3 M for the binding of Co(II) ions. The results are consistent with the hypothesis that two activating ions per monomeric subunit participate in the catalytic mechanism. The affinity of the tightly bound ion is at least 100-fold greater than the affinity of the weakly bound ion.     

Analysis of Binding Interaction of Curcumin and Diacetylcurcumin with Human and Bovine Serum Albumin Using Fluorescence and Circular Dichroism Spectroscopy

The current study reports the binding of curcumin (CUR) as the main pharmacologically active ingredient of turmeric and diacetylcurcumin (DAC) as a bioactive derivative of curcumin to human serum albumin (HSA) and bovine serum albumin (BSA). The apparent binding constants and number of substantive binding sites have been evaluated by fluorescence quenching method. The distance (r) between donor (HSA and BSA) and acceptor (CUR and DAC) was obtained on the basis of the Förster’s theory of non-radiative energy transfer. The minor changes on the far-UV circular dichroism spectra resulted in partial changes in the calculated secondary structure contents of HSA and BSA. The negligible alteration in the secondary structure of both albumin proteins indicated that ligand-induced conformational changes are localized to the binding site and do not involve considerable changes in protein folding. The visible CD spectra indicated that the optical activity observed during the ligand binding due to induced-protein chirality. All of the achieved results suggested the important role of the phenolic OH group of CUR in the binding process.     

Study of the Spectral Characteristics of L-Lysine and L-Arginine Using UV-VIS Spectroscopy and Steady-State and Synchronous Fluorescence Spectroscopy

Biophysics - The recently discovered luminescent properties of nonaromatic amino acids with excitation-dependent photoluminescence in the absence of conjugation within individual molecules have...     

Towards a Mechanism of Function of the Viral Ion Channel Vpu from HIV-1

Abstract

Vpu, an integral membrane protein encoded in HIV-1, is implicated in the release of new virus particles from infected cells, presumably mediated by ion channel activity of homo- oligomeric Vpu bundles.

Reconstitution of both full length Vpu_1–81 and a short, the transmembrane (TM) domain comprising peptide Vpu_1-32 into bilayers under a constant electric field results in an asymmetric orientation of those channels. For both cases, channel activity with similar kinetics is observed. Channels can open and remain open within a broad series of conductance states even if a small or no electric potential is applied.

The mean open time for Vpu peptide channels is voltage-independent. The rate of channel opening shows a biphasic voltage activation, implicating that the gating is influenced by the interaction of the dipole moments of the TM helices with an electric field.     

Study of Interaction of Fluorescent Cytochrome C with Liposomes,Mitochondria, and Mitoplasts by Fluorescence Correlation Spectroscopy

Russian Journal of Bioorganic Chemistry - Here, we studied the interaction of Cys-substituted (G56C) cytochrome c labeled with sulfocyanin-3 fluorescent dye (fCyt) with artificial and natural lipid...     

Tilt and Rotation Angles of a Transmembrane Model Peptide as Studied by Fluorescence Spectroscopy

In this study the membrane orientation of a tryptophan-flanked model peptide, WALP23, was determined by using peptides that were labeled at different positions along the sequence with the environmentally sensitive fluorescent label BADAN. The fluorescence properties, reflecting the local polarity, were used to determine the tilt and rotation angles of the peptide based on an ideal α-helix model. For WALP23 inserted in dioleoylphosphatidylcholine (DOPC), an estimated tilt angle of the helix with respect to the bilayer normal of 24° ± 5° was obtained. When the peptides were inserted into bilayers with different acyl chain lengths or containing different concentrations of cholesterol, small changes in tilt angle were observed as response to hydrophobic mismatch, whereas the rotation angle appeared to be independent of lipid composition. In all cases, the tilt angles were significantly larger than those previously determined from ²H NMR experiments, supporting recent suggestions that the relatively long timescale of ²H NMR measurements may result in an underestimation of tilt angles due to partial motional averaging. It is concluded that although the fluorescence technique has a rather low resolution and limited accuracy, it can be used to resolve the discrepancies observed between previous ²H NMR experiments and molecular-dynamics simulations.     

Mechanism of the Interaction of Superoxide Ion and Ascorbate with Anthracycline Antibiotics

The interaction of superoxide ion and ascorbate anion with anthracycline antibiotics (adriamycin and aclacinimycin A) as well as with their Fe³⁺ complexes has been studied in aprotic and protic media. It was found that both superoxide and ascorbate reduce anthracyclines to deoxyaglycons via a one-electron transfer mechanism under all conditions studied. The reaction of ascorbate anion with adriamycin and aclacinomycin A in aqueous solution proceeded only in the presence of Fe³⁺ ions; it is supposed that an active catalytic species was Fe³⁺ adriamycin. It is also supposed that the reduction of anthracycline antibiotics by O,⁷ and ascorbate in cells may increase their anticancer effect.     

Mechanism of the Interaction of Superoxide Ion and Ascorbate with Anthracycline Antibiotics

The interaction of superoxide ion and ascorbate anion with anthracycline antibiotics (adriamycin and aclacinimycin A) as well as with their Fe³⁺ complexes has been studied in aprotic and protic media. It was found that both superoxide and ascorbate reduce anthracyclines to deoxyaglycons via a one-electron transfer mechanism under all conditions studied. The reaction of ascorbate anion with adriamycin and aclacinomycin A in aqueous solution proceeded only in the presence of Fe³⁺ ions; it is supposed that an active catalytic species was Fe³⁺ adriamycin. It is also supposed that the reduction of anthracycline antibiotics by O,⁷ and ascorbate in cells may increase their anticancer effect.     

罗丹明类荧光分子探针与血清白蛋白之间相互作用的研究

本研究旨在对罗丹明类荧光探针ZM-6与人血清白蛋白(HSA)的相互作用进行研究。采用了荧光光谱法、三维荧光光谱法、同步荧光光谱法以及CD光谱法在模拟生理条件下对二者的相互作用以及HSA的构象进行了研究。研究结果表明,探针与ZM-6之间的猝灭机理主要是静态猝灭方式。根据热力学数据确定了二者之间的作用力,类型为范德华力和氢键。二者之间的结合距离为4.45 nm。同时得出,ZM-6对HSA的构象产生了影响。此研究对于探针分子的设计以及修饰提供有效的数据以及理论支持。     

Fluorescence Spectroscopy as a Tool to Unravel the Dynamics of Protein Nanoparticle Formation by Liquid Antisolvent Precipitation

Protein-based particles are very promising colloidal systems for protection and controlled release applications in the food, cosmetics and pharmaceutical sector. One technique to produce these protein colloidal particles is liquid antisolvent precipitation (LAS). Despite the simplicity and versatility of LAS, not much is known about the protein conformational changes and interactions that are at the basis of the particle formation process. In this study, steady state fluorescence experiments using intrinsic fluorophores were evaluated as a tool to unravel the dynamics of the protein nanoparticle formation. Colloidal whey protein isolate and gliadin particles were produced by LAS. Changes in particle diameter (distribution), polydispersity index and photophysical properties of intrinsic fluorophores were monitored as a function of antisolvent concentration. By combining dynamic light scattering with photophysical data, a model of the changes occurring during particle formation and disintegration could be proposed. The results suggest that particle formation and disintegration are fully reversible processes during which the main changes in protein conformation (around the fluorescent probes) occur at the same antisolvent concentrations. In principle, steady state fluorescence measurements using intrinsic probes can indeed be used to effectively report on (part of the) conformational changes for both protein systems under study.     

Binding Site and Inhibitory Mechanism of the Mambalgin-2 Pain-relieving Peptide on Acid-sensing Ion Channel 1a

Acid-sensing ion channels (ASICs) are neuronal proton-gated cation channels associated with nociception, fear, depression, seizure, and neuronal degeneration, suggesting roles in pain and neurological and psychiatric disorders. We have recently discovered black mamba venom peptides called mambalgin-1 and mambalgin-2, which are new three-finger toxins that specifically inhibit with the same pharmacological profile ASIC channels to exert strong analgesic effects in vivo. We now combined bioinformatics and functional approaches to uncover the molecular mechanism of channel inhibition by the mambalgin-2 pain-relieving peptide. Mambalgin-2 binds mainly in a region of ASIC1a involving the upper part of the thumb domain (residues Asp-349 and Phe-350), the palm domain of an adjacent subunit, and the β-ball domain (residues Arg-190, Asp-258, and Gln-259). This region overlaps with the acidic pocket (pH sensor) of the channel. The peptide exerts both stimulatory and inhibitory effects on ASIC1a, and we propose a model where mambalgin-2 traps the channel in a closed conformation by precluding the conformational change of the palm and β-ball domains that follows proton activation. These data help to understand inhibition by mambalgins and provide clues for the development of new optimized blockers of ASIC channels.     

Thermodynamic Framework of the Interaction between Protein and Solvent Drives Protein Folding

Abstract

We use internal coordinate molecular mechanics calculations to study the impact of abasic sites on the conformation and the mechanics of the DNA double helix. Abasic sites, which are common mutagenic lesions, are shown to locally modify both the groove geometry and the curvature of DNA in a sequence dependent manner. By controlled twisting and bending, it is also shown that these lesions modify the deformability of the duplex, generally increasing its flexibility, but again to an extent which depends on the nature of the abasic site and on the surrounding base sequence. Both the conformational and mechanical influence of this type of DNA damage may be significant for recognition and repair mechanisms.