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Andrey Tronin

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Papers by Andrey Tronin

Positional order and thermal expansion of surface crystalline N-alkane monolayers

Physical review. E, Statistical, nonlinear, and soft matter physics, 2001

We report a high-resolution synchrotron grazing incidence x-ray diffraction measurement of a surf... more We report a high-resolution synchrotron grazing incidence x-ray diffraction measurement of a surface crystalline monolayer at the liquid-vapor interface of the n-alkane eicosane (C20H42) just above its melting temperature. The peak width of the surface monolayer rotator phase is shown to be resolution limited and implies positional correlations of at least approximately 1 microm. The high resolution allowed determination of the temperature dependence of the peak position over the narrow (3 degrees C) temperature range of the surface crystal phase. The two-dimensional thermal expansion was determined to be (dA/dT)/A=1.8(+/-0.1)x10(-3) degrees C-1, which is comparable to the expansion in similar chain length bulk n-alkane rotator phases. Our data are consistent with the power-law shaped scattering tails expected from quasi-long-range order in two dimensions.

Direct Evidence of Conformational Changes Associated with Voltage Gating in a Voltage Sensor Protein by Time-Resolved X-ray/Neutron Interferometry

by Andrey Tronin, Ivan Kuzmenko, and V. Lauter

Langmuir, 2014

The voltage sensor domain (VSD) of voltage-gated cation (e.g., Na(+), K(+)) channels central to n... more The voltage sensor domain (VSD) of voltage-gated cation (e.g., Na(+), K(+)) channels central to neurological signal transmission can function as a distinct module. When linked to an otherwise voltage-insensitive, ion-selective membrane pore, the VSD imparts voltage sensitivity to the channel. Proteins homologous with the VSD have recently been found to function themselves as voltage-gated proton channels or to impart voltage sensitivity to enzymes. Determining the conformational changes associated with voltage gating in the VSD itself in the absence of a pore domain thereby gains importance. We report the direct measurement of changes in the scattering-length density (SLD) profile of the VSD protein, vectorially oriented within a reconstituted phospholipid bilayer membrane, as a function of the transmembrane electric potential by time-resolved X-ray and neutron interferometry. The changes in the experimental SLD profiles for both polarizing and depolarizing potentials with respect to zero potential were found to extend over the entire length of the isolated VSD&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s profile structure. The characteristics of the changes observed were in qualitative agreement with molecular dynamics simulations of a related membrane system, suggesting an initial interpretation of these changes in terms of the VSD&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s atomic-level 3-D structure.

Three-Dimensional Structure and Dynamics of a de Novo Designed, Amphiphilic, Metallo-Porphyrin-Binding Protein Maquette at Soft Interfaces by Molecular Dynamics Simulations

by Andrey Tronin and Hongling Zou

The Journal of Physical Chemistry B, 2007

The three-dimensional structure and dynamics of de novo designed, amphiphilic four-helix bundle p... more The three-dimensional structure and dynamics of de novo designed, amphiphilic four-helix bundle peptides (or "maquettes"), capable of binding metallo-porphyrin cofactors at selected locations along the length of the core of the bundle, are investigated via molecular dynamics simulations. The rapid evolution of the initial design to stable three-dimensional structures in the absence (apo-form) and presence (holo-form) of bound cofactors is described for the maquettes at two different soft interfaces between polar and nonpolar media. This comparison of the apo-versus holo-forms allows the investigation of the effects of cofactor incorporation on the structure of the four-helix bundle. The simulation results are in qualitative agreement with available experimental data describing the structures at lower resolution and limited dimension.

Portable UV-visible spectrometer for measuring absorbance and dichroism of Langmuir monolayers at air-water interfaces

by Andrey Tronin and Ivan Kuzmenko

Review of Scientific Instruments, 2009

An UV-visible spectrometer for measuring absorbance and dichroism of Langmuir monolayers under in... more An UV-visible spectrometer for measuring absorbance and dichroism of Langmuir monolayers under in situ conditions is described. The spectrometer utilizes a stand-alone multipass sensor, which is placed in a Langmuir trough and coupled with light source and spectrometer head via fiber optics. Implementation of the multipass scheme in the absorbance sensor makes it possible to obtain reliable quantitative spectroscopic data of the Langmuir monolayers with absorbance as low as 1 mOD. Such high sensitivity makes the developed sensor very useful for UV-visible spectral studies of a wide variety of chromophores. The new technique was applied to several model systems: fatty acid monolayers containing amphiphilic dyes DiI or BODIPY and also a monolayer of a synthetic amphiphilic porphyrin-binding peptide BBC16. Implementation of UV-visible absorbance spectroscopy measurements in situ together with x-ray scattering technique was used to confirm the bound state of the chromophore, and determine the exact position of the latter in the peptide matrix. Fiber optics design of the spectrometer provides portability and compatibility with other experimental techniques making it possible to study samples with a geometry unsuitable for conventional spectroscopic measurements and located in experimental environments with spatial limitations, such as synchrotron x-ray scattering stations.

Structural Studies of Amphiphilic 4-Helix Bundle Peptides Incorporating Designed Extended Chromophores for Nonlinear Optical Biomolecular Materials

by Andrey Tronin and Ivan Kuzmenko

Nano Letters, 2006

Extended conjugated chromophores containing (porphinato)zinc components that exhibit large optica... more Extended conjugated chromophores containing (porphinato)zinc components that exhibit large optical polarizabilities and hyperpolarizabiliites are incorporated into amphiphilic 4-helix bundle peptides via specific axial histidyl ligation of the metal. The bundle's designed amphiphilicity enables vectorial orientation of the chromophore/peptide complex in macroscopic monolayer ensembles. The 4-helix bundle structure is maintained upon incorporation of two different chromophores at stoichiometries of 1−2 per bundle. The axial ligation site appears to effectively control the position of the chromophore along the length of the bundle.

Variable Acquisition Angle Total Internal Reflection Fluorescence: A New Technique for Orientation Distribution Studies of Ultrathin Films

Langmuir, 2001

ABSTRACT A new technique for the determination of the orientation distribution of a fluorophore w... more ABSTRACT A new technique for the determination of the orientation distribution of a fluorophore within an ultrathin film, in terms of the mean tilt angle relative to the monolayer normal and the distribution width, is described. The technique utilizes the measurement of polarized fluorescence, excited by the evanescent field appearing upon total internal reflection. The main difference between this and existing techniques is that the fluorescence intensity is measured as a function of the acquisition angle with low aperture detection. The potential of the technique was investigated with the help of computer modeling and experimentally utilizing a Langmuir−Blodgett film of arachidic acid doped with a fluorophore DiI. Both computer modeling and the measurements show that the acquisition of the angular dependence of the fluorescence dramatically improves the technique, increasing manyfold the accuracy and resolution of the determined orientation distribution parameters.

Orientational Distributions of the Di-α-helical Synthetic Peptide ZnPPIX-BBC16 in Langmuir Monolayers by X-ray Reflectivity and Polarized Epifluorescence

Langmuir, 2001

The vectorial orientation of the peptide ZnPPIX-BBC16 in a Langmuir monolayer was studied by mean... more The vectorial orientation of the peptide ZnPPIX-BBC16 in a Langmuir monolayer was studied by means of X-ray reflectivity and polarized epifluorescence. ZnPPIX-BBC16 consists of two R-helical 31-mers dimerized via a disulfide bridge between two N-terminal cysteines (R-S-S-R), so that two bis-his metalloporphyrin binding sites between positions 10,10′ and 24,24′ are formed. Zn(II) protoporphyrin IX was bound to the position 24. To enhance stability at the interface, a palmitoyl (C16) chain was bonded to each N-terminal cysteine. X-ray reflectivity measurements make it possible to infer the orientation of the R-helices by determining the electron density profile of the monolayer. Polarized epifluorescence provides the orientation distribution of the porphyrin with respect to the monolayer plane. Both X-ray and fluorescence measurements show that at low surface pressure (π ) 5 mN/m), the peptide R-helices lie in the plane of the water surface with a narrow orientational distribution of the porphyrin. At high pressures (π > 30 mN/m), the peptide R-helices are perpendicular to the water surface and the distribution is wider. At intermediate pressures the peptide R-helices in the monolayer undergo a transition from the parallel to perpendicular orientation with almost a complete loss of the orientational order of the porphyrin.

Assembly of a Vectorially Oriented Four-Helix Bundle at the Air/Water Interface via Directed Electrostatic Interactions †

Langmuir, 2003

Determination of the Porphyrin Orientation Distribution in Langmuir Monolayers by Polarized Epifluorescence

Langmuir, 2000

ABSTRACT A new technique for the determination of the orientation distribution of a porphyrin wit... more ABSTRACT A new technique for the determination of the orientation distribution of a porphyrin within a Langmuir monolayer, in terms of the mean tilt angle relative to the monolayer normal and the width, is described. The technique utilizes the measurement of polarized fluorescence, excited with the electric field both parallel and perpendicular to the monolayer plane. The main difference between this technique and the existing ones [Fraaije et al., Biophys. J. 1990, 57, 965; Edmiston et al., J. Am. Chem. Soc. 1997, 119, 560; Bos et al., Biophys. J. 1995, 68, 2573.] is that the fluorophores are excited directly by the incident beam rather than an evanescent field, which greatly facilitates its application to Langmuir monolayers at the air−water interface. The technique was applied to the study of two systems with different porphyrin orientations: a Langmuir monolayer of a dihelical synthetic peptide BBC16 containing Zn(II)protoporphyrinIX and a monolayer of tetrakis (N-methyl-4-pyridil) porphyne electrostatically bound to the Langmuir monolayer of dipalmitoylphosphatidic acid. The results for the mean tilt angle are in good agreement with other relevant data in the literature for both systems. It is also shown that both systems exhibit rather narrow distributions, which makes them attractive for biophysical studies.

Control of the Orientational Order and Nonlinear Optical Response of the “Push-Pull” Chromophore RuPZn via Specific Incorporation into Densely Packed Monolayer Ensembles of an Amphiphilic Four-Helix Bundle Peptide: Characterization of the Peptide−Chromophore Complexes

by V. Krishnan, Hai-lung Dai, Andrey Tronin, and Joseph Strzalka

Journal of the American Chemical Society, 2010

Push-pull" chromophores based on extended π-electron systems have been designed to exhibit except... more Push-pull" chromophores based on extended π-electron systems have been designed to exhibit exceptionally large molecular hyperpolarizabilities. We have engineered an amphiphilic four-helix bundle peptide to vectorially incorporate such hyperpolarizable chromophores having a metalloporphyrin moiety, with high specificity into the interior core of the bundle. The amphiphilic exterior of the bundle facilitates the formation of densely packed monolayer ensembles of the vectorially oriented peptide-chromophore complexes at the liquid-gas interface. Chemical specificity designed into the ends of the bundle facilitates the subsequent covalent attachment of these monolayer ensembles onto the surface of an inorganic substrate. In this article, we describe the structural characterization of these monolayer ensembles at each stage of their fabrication for one such peptide-chromophore complex designated as AP0-RuPZn. In the accompanying article, we describe the characterization of their macroscopic nonlinear optical properties.

In situ determination of orientational distributions in Langmuir monolayers by total internal reflection fluorescence

Langmuir : the ACS journal of surfaces and colloids, 2005

A new application of the polarized total internal reflection fluorescence (PTIRF) technique to st... more A new application of the polarized total internal reflection fluorescence (PTIRF) technique to study the orientation distribution of a fluorophore within a Langmuir monolayer in situ on an aqueous subphase is described. The technique utilizes the measurement of polarized fluorescence, excited by the evanescent field appearing upon total internal reflection. The excitation by the evanescent field is achieved by launching the beam into a prism that is brought into contact with the monolayer from above. We also show here that a combination of PTIRF of monolayers on water and those freshly deposited onto the prism by horizontal lift in the same experiment provide enough data to determine the dielectric constant of the actual local environment of the fluorophore in the monolayer to eliminate the ambiguity of the orientation determination, arising from uncertainty in the normal component of excitation field. The new technique was applied to several model systems: fatty acid monolayers con...

Acentric 2-D ensembles of D-br-A electron-transfer chromophores via vectorial orientation within amphiphilic n-helix bundle peptides for photovoltaic device applications

by Andrey Tronin and Ivan Kuzmenko

Langmuir : the ACS journal of surfaces and colloids, Jan 14, 2012

We show that simply designed amphiphilic 4-helix bundle peptides can be utilized to vectorially o... more We show that simply designed amphiphilic 4-helix bundle peptides can be utilized to vectorially orient a linearly extended donor-bridge-acceptor (D-br-A) electron transfer (ET) chromophore within its core. The bundle's interior is shown to provide a unique solvation environment for the D-br-A assembly not accessible in conventional solvents and thereby control the magnitudes of both light-induced ET and thermal charge recombination rate constants. The amphiphilicity of the bundle's exterior was employed to vectorially orient the peptide-chromophore complex at a liquid-gas interface, and its ends were tailored for subsequent covalent attachment to an inorganic surface, via a "directed assembly" approach. Structural data, combined with evaluation of the excited state dynamics exhibited by these peptide-chromophore complexes, demonstrate that densely packed, acentrically ordered 2-D monolayer ensembles of such complexes at high in-plane chromophore densities approac...

Structural changes in single membranes in response to an applied transmembrane electric potential revealed by time-resolved neutron/X-ray interferometry

by Andrey Tronin and Ivan Kuzmenko

Chemical Physics, 2013

The profile structure of a hybrid lipid bilayer, tethered to the surface of an inorganic substrat... more The profile structure of a hybrid lipid bilayer, tethered to the surface of an inorganic substrate and fully hydrated with a bulk aqueous medium in an electrochemical cell, was investigated as a function of the applied transbilayer electric potential via time-resolved neutron reflectivity, enhanced by interferometry. Significant, and fully reversible structural changes were observed in the distal half (with respect to the substrate surface) of the hybrid bilayer comprised of a zwitterionic phospholipid in response to a +100mV potential with respect to 0mV. These arise presumably due to reorientation of the electric dipole present in the polar headgroup of the phospholipid and its resulting effect on the thickness of the phospholipid&#39;s hydrocarbon chain layer within the hybrid bilayer&#39;s profile structure. The profile structure of the voltage-sensor domain from a voltage-gated ion channel protein within a phospholipid bilayer membrane, tethered to the surface of an inorganic substrate and fully hydrated with a bulk aqueous medium in an electrochemical cell, was also investigated as a function of the applied transmembrane electric potential via time-resolved X-ray reflectivity, enhanced by interferometry. Significant, fully-reversible, and different structural changes in the protein were detected in response to ±100mV potentials with respect to 0mV. The approach employed is that typical of transient spectroscopy, shown here to be applicable to both neutron and X-ray reflectivity of thin films.

A New Approach to the Structural Investigation of the Voltage-Sensitive Domain of Voltage-Gated Cation Channels as a Function of the Transmembrane Voltage

Biophysical Journal, 2010

Structural Characterization of the Voltage Sensor Domain of the KvAP Channel Vectorially-Oriented within a Phospholipid Bilayer Membrane

Biophysical Journal, 2011

recorded voltage ramps in the interval 5200 mV in the presence of 2 M internal sucrose. In these ... more recorded voltage ramps in the interval 5200 mV in the presence of 2 M internal sucrose. In these conditions, outward currents become voltage independent at voltages >150 mV, providing size estimates consistent with the size of the internal mouth derived from the crystallographic structure. The ion density profile obtained from molecular dynamics simulations using an applied voltage reveals a high density of Kþ ions near P475D. Funded by Conicyt 190493. IV is a Conicyt fellow 3064-Pos Board B169 Calculating Conductance and Size of the Entrance to the Inner Cavity of BK Channels with Side-Chain Replacement and a Two-Resistor Model Yanyan Geng, Karl L. Magleby. BK channels have the largest conductance (~250 pS) of all Kþ selective channels. Previous studies suggest that residues E321/E324 in BK channels are located at the entrance to the inner cavity. We find that attachment of thiol reagents MPA and MTSET to E321C/E324C altered outward single-channel currents, suggesting that 321/324 face the ion conduction pathway. Therefore, substituting E321/E324 with different sized amino acids should change the size of the entrance to the inner cavity. We find that decreasing the size of the entrance decreases the conductance, whereas increasing the size of the entrance has little effect. Increasing [Kþ]i from 0.15 to 2.5 M negates differences in single-channel current associated with different side-chain volume. Plots of conductance vs. side-chain volume are approximated with a simple tworesistor model, where the ion conduction pathway is described by two resistors in series. R2 is a variable resistor, with resistance inversely proportional to the volume of the entrance to the inner cavity. R1 is a fixed resistor arising from the other parts of the conduction pathway including the selectivity filter. Fitting the data indicates that R1þR2 is~5.4 gigaohm for glycine substitution, with an R1/R2 ratio of~17, and effective radius and length of the entrance to the inner cavity of~9.0 and 5.4 Å , respectively. (The volume of Kþ and water are not taken into account.) The calculated size of the entrance to the inner cavity of BK channels is consistent with the crystal structure of large conductance bacterial MthK channels. These observations suggest that a large entrance to the inner cavity is required for the large conductance of BK channels, as decreasing the entrance size decreases the outward single-channel currents. Support: NIH-AR32805. Potassium channels containing the GYG motif often diverge in their selectivity for monovalent cations, but the molecular basis is unknown. Using the prokaryotic potassium channel KcsA as our model, we have investigated the role of the interaction between glutamate 71 and aspartate 80, located behind the selectivity filter, in determining the selectivity of the channel as well as its influence on the conformation of the filter. In E71A KcsA channels, Naþ permeates at higher rates in both the presence and absence of Kþ, as seen with 86Rbþ and 22Naþ flux measurements. Single channel recordings indicate that Naþ ''punches through'' E71A KcsA channels at lower voltages than wild-type KcsA, and in the punchthrough regime the Naþ-blocked current appears significantly larger. A crystal structure of E71A KcsA reveals that in contrast to what was seen for wild type KcsA, the selectivity filter does not collapse in the absence of Kþ, but instead assumes a ''flipped'' conformation. This flipped conformation is the same one observed in previous E71A KcsA structures in the presence of Kþ. The data reveal the importance of this E71-D80 interaction in both favoring inactivation and maintaining high Kþ selectivity. We propose a molecular mechanism by which inactivation and Kþ selectivity are linked, a mechanism that may also be at work in other channels containing the canonical GYG signature sequence.

Orientation Distributions for Cytochrome c on Polar and Nonpolar Interfaces by Total Internal Reflection Fluorescence

by Andrey Tronin and Jane Vanderkooi

Biophysical Journal, 2002

The formation of chemisorbed monolayers of yeast cytochrome c on both uncharged polar and nonpola... more The formation of chemisorbed monolayers of yeast cytochrome c on both uncharged polar and nonpolar soft surfaces of organic self-assembled monolayers (SAM) on solid inorganic substrates was followed in situ by polarized total internal reflection fluorescence. Two types of nonpolar surfaces and one type of uncharged polar surface were used. The first type of nonpolar surface contained only thiol endgroups, while the other was composed of a mixture of thiol and methyl endgroups. The uncharged polar surface was provided by the mixture of thiol and hydroxyl endgroups. The thiol endgroups were used to form a covalent disulfide bond with the unique surface-exposed cysteine residue 102 of the protein. The mean tilt angle of the protein's zinc-substituted porphyrin was found to be 41°and 50°for the adsorption onto the nonpolar and uncharged polar surfaces, respectively. The distribution widths for the pure thiol and the thiol/methyl and thiol/hydroxyl mixtures were 9°, 1°, and 18°, respectively. The high degree of the orientational order and good stability achieved for the protein monolayer on the mixed thiol/methyl endgroup SAM makes this system very attractive for studies of both intramolecular and intermolecular electron transfer processes.

Mechanism Of Interaction Between The Volatile Anesthetic Halothane And A Model Ion Channel Protein: Fluorescence And Infrared Spectroscopy Employing A Cyano-phenylalanine Probe And Molecular Dynamics Simulation

Biophysical Journal, 2009

Monolayers of a Model Anesthetic-Binding Membrane Protein: Formation, Characterization, and Halothane-Binding Affinity

by Andrey Tronin, Ivan Kuzmenko, and J. Blasie

Biophysical Journal, 2006

hbAP0 is a model membrane protein designed to possess an anesthetic-binding cavity in its hydroph... more hbAP0 is a model membrane protein designed to possess an anesthetic-binding cavity in its hydrophilic domain and a cation channel in its hydrophobic domain. Grazing incidence x-ray diffraction shows that hbAP0 forms four-helix bundles that are vectorially oriented within Langmuir monolayers at the air-water interface. Single monolayers of hbAP0 on alkylated solid substrates would provide an optimal system for detailed structural and dynamical studies of anesthetic-peptide interaction via x-ray and neutron scattering and polarized spectroscopic techniques. Langmuir-Blodgett and Langmuir-Schaeffer deposition and self-assembly techniques were used to form single monolayer films of the vectorially oriented peptide hbAP0 via both chemisorption and physisorption onto suitably alkylated solid substrates. The films were characterized by ultraviolet absorption, ellipsometry, circular dichroism, and polarized Fourier transform infrared spectroscopy. The a-helical secondary structure of the peptide was retained in the films. Under certain conditions, the average orientation of the helical axis was inclined relative to the plane of the substrate, approaching perpendicular in some cases. The halothane-binding affinity of the vectorially oriented hbAP0 peptide in the single monolayers, with the volatile anesthetic introduced into the moist vapor environment of the monolayer, was found to be similar to that for the detergent-solubilized peptide.

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

by Andrey Tronin and Jing Liu

Biophysical Journal, 2009

We previously reported the synthesis and structural characterization of a model membrane protein ... more 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.

Design of an Amphiphilic Di-Helix and its Vectorial Orientation at the Air-Water Interface

Positional order and thermal expansion of surface crystalline N-alkane monolayers

Physical review. E, Statistical, nonlinear, and soft matter physics, 2001

We report a high-resolution synchrotron grazing incidence x-ray diffraction measurement of a surf... more We report a high-resolution synchrotron grazing incidence x-ray diffraction measurement of a surface crystalline monolayer at the liquid-vapor interface of the n-alkane eicosane (C20H42) just above its melting temperature. The peak width of the surface monolayer rotator phase is shown to be resolution limited and implies positional correlations of at least approximately 1 microm. The high resolution allowed determination of the temperature dependence of the peak position over the narrow (3 degrees C) temperature range of the surface crystal phase. The two-dimensional thermal expansion was determined to be (dA/dT)/A=1.8(+/-0.1)x10(-3) degrees C-1, which is comparable to the expansion in similar chain length bulk n-alkane rotator phases. Our data are consistent with the power-law shaped scattering tails expected from quasi-long-range order in two dimensions.

Direct Evidence of Conformational Changes Associated with Voltage Gating in a Voltage Sensor Protein by Time-Resolved X-ray/Neutron Interferometry

by Andrey Tronin, Ivan Kuzmenko, and V. Lauter

Langmuir, 2014

The voltage sensor domain (VSD) of voltage-gated cation (e.g., Na(+), K(+)) channels central to n... more The voltage sensor domain (VSD) of voltage-gated cation (e.g., Na(+), K(+)) channels central to neurological signal transmission can function as a distinct module. When linked to an otherwise voltage-insensitive, ion-selective membrane pore, the VSD imparts voltage sensitivity to the channel. Proteins homologous with the VSD have recently been found to function themselves as voltage-gated proton channels or to impart voltage sensitivity to enzymes. Determining the conformational changes associated with voltage gating in the VSD itself in the absence of a pore domain thereby gains importance. We report the direct measurement of changes in the scattering-length density (SLD) profile of the VSD protein, vectorially oriented within a reconstituted phospholipid bilayer membrane, as a function of the transmembrane electric potential by time-resolved X-ray and neutron interferometry. The changes in the experimental SLD profiles for both polarizing and depolarizing potentials with respect to zero potential were found to extend over the entire length of the isolated VSD&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s profile structure. The characteristics of the changes observed were in qualitative agreement with molecular dynamics simulations of a related membrane system, suggesting an initial interpretation of these changes in terms of the VSD&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;s atomic-level 3-D structure.

Three-Dimensional Structure and Dynamics of a de Novo Designed, Amphiphilic, Metallo-Porphyrin-Binding Protein Maquette at Soft Interfaces by Molecular Dynamics Simulations

by Andrey Tronin and Hongling Zou

The Journal of Physical Chemistry B, 2007

The three-dimensional structure and dynamics of de novo designed, amphiphilic four-helix bundle p... more The three-dimensional structure and dynamics of de novo designed, amphiphilic four-helix bundle peptides (or "maquettes"), capable of binding metallo-porphyrin cofactors at selected locations along the length of the core of the bundle, are investigated via molecular dynamics simulations. The rapid evolution of the initial design to stable three-dimensional structures in the absence (apo-form) and presence (holo-form) of bound cofactors is described for the maquettes at two different soft interfaces between polar and nonpolar media. This comparison of the apo-versus holo-forms allows the investigation of the effects of cofactor incorporation on the structure of the four-helix bundle. The simulation results are in qualitative agreement with available experimental data describing the structures at lower resolution and limited dimension.

Portable UV-visible spectrometer for measuring absorbance and dichroism of Langmuir monolayers at air-water interfaces

by Andrey Tronin and Ivan Kuzmenko

Review of Scientific Instruments, 2009

An UV-visible spectrometer for measuring absorbance and dichroism of Langmuir monolayers under in... more An UV-visible spectrometer for measuring absorbance and dichroism of Langmuir monolayers under in situ conditions is described. The spectrometer utilizes a stand-alone multipass sensor, which is placed in a Langmuir trough and coupled with light source and spectrometer head via fiber optics. Implementation of the multipass scheme in the absorbance sensor makes it possible to obtain reliable quantitative spectroscopic data of the Langmuir monolayers with absorbance as low as 1 mOD. Such high sensitivity makes the developed sensor very useful for UV-visible spectral studies of a wide variety of chromophores. The new technique was applied to several model systems: fatty acid monolayers containing amphiphilic dyes DiI or BODIPY and also a monolayer of a synthetic amphiphilic porphyrin-binding peptide BBC16. Implementation of UV-visible absorbance spectroscopy measurements in situ together with x-ray scattering technique was used to confirm the bound state of the chromophore, and determine the exact position of the latter in the peptide matrix. Fiber optics design of the spectrometer provides portability and compatibility with other experimental techniques making it possible to study samples with a geometry unsuitable for conventional spectroscopic measurements and located in experimental environments with spatial limitations, such as synchrotron x-ray scattering stations.

Structural Studies of Amphiphilic 4-Helix Bundle Peptides Incorporating Designed Extended Chromophores for Nonlinear Optical Biomolecular Materials

by Andrey Tronin and Ivan Kuzmenko

Nano Letters, 2006

Extended conjugated chromophores containing (porphinato)zinc components that exhibit large optica... more Extended conjugated chromophores containing (porphinato)zinc components that exhibit large optical polarizabilities and hyperpolarizabiliites are incorporated into amphiphilic 4-helix bundle peptides via specific axial histidyl ligation of the metal. The bundle's designed amphiphilicity enables vectorial orientation of the chromophore/peptide complex in macroscopic monolayer ensembles. The 4-helix bundle structure is maintained upon incorporation of two different chromophores at stoichiometries of 1−2 per bundle. The axial ligation site appears to effectively control the position of the chromophore along the length of the bundle.

Variable Acquisition Angle Total Internal Reflection Fluorescence: A New Technique for Orientation Distribution Studies of Ultrathin Films

Langmuir, 2001

ABSTRACT A new technique for the determination of the orientation distribution of a fluorophore w... more ABSTRACT A new technique for the determination of the orientation distribution of a fluorophore within an ultrathin film, in terms of the mean tilt angle relative to the monolayer normal and the distribution width, is described. The technique utilizes the measurement of polarized fluorescence, excited by the evanescent field appearing upon total internal reflection. The main difference between this and existing techniques is that the fluorescence intensity is measured as a function of the acquisition angle with low aperture detection. The potential of the technique was investigated with the help of computer modeling and experimentally utilizing a Langmuir−Blodgett film of arachidic acid doped with a fluorophore DiI. Both computer modeling and the measurements show that the acquisition of the angular dependence of the fluorescence dramatically improves the technique, increasing manyfold the accuracy and resolution of the determined orientation distribution parameters.

Orientational Distributions of the Di-α-helical Synthetic Peptide ZnPPIX-BBC16 in Langmuir Monolayers by X-ray Reflectivity and Polarized Epifluorescence

Langmuir, 2001

The vectorial orientation of the peptide ZnPPIX-BBC16 in a Langmuir monolayer was studied by mean... more The vectorial orientation of the peptide ZnPPIX-BBC16 in a Langmuir monolayer was studied by means of X-ray reflectivity and polarized epifluorescence. ZnPPIX-BBC16 consists of two R-helical 31-mers dimerized via a disulfide bridge between two N-terminal cysteines (R-S-S-R), so that two bis-his metalloporphyrin binding sites between positions 10,10′ and 24,24′ are formed. Zn(II) protoporphyrin IX was bound to the position 24. To enhance stability at the interface, a palmitoyl (C16) chain was bonded to each N-terminal cysteine. X-ray reflectivity measurements make it possible to infer the orientation of the R-helices by determining the electron density profile of the monolayer. Polarized epifluorescence provides the orientation distribution of the porphyrin with respect to the monolayer plane. Both X-ray and fluorescence measurements show that at low surface pressure (π ) 5 mN/m), the peptide R-helices lie in the plane of the water surface with a narrow orientational distribution of the porphyrin. At high pressures (π > 30 mN/m), the peptide R-helices are perpendicular to the water surface and the distribution is wider. At intermediate pressures the peptide R-helices in the monolayer undergo a transition from the parallel to perpendicular orientation with almost a complete loss of the orientational order of the porphyrin.

Assembly of a Vectorially Oriented Four-Helix Bundle at the Air/Water Interface via Directed Electrostatic Interactions †

Langmuir, 2003

Determination of the Porphyrin Orientation Distribution in Langmuir Monolayers by Polarized Epifluorescence

Langmuir, 2000

ABSTRACT A new technique for the determination of the orientation distribution of a porphyrin wit... more ABSTRACT A new technique for the determination of the orientation distribution of a porphyrin within a Langmuir monolayer, in terms of the mean tilt angle relative to the monolayer normal and the width, is described. The technique utilizes the measurement of polarized fluorescence, excited with the electric field both parallel and perpendicular to the monolayer plane. The main difference between this technique and the existing ones [Fraaije et al., Biophys. J. 1990, 57, 965; Edmiston et al., J. Am. Chem. Soc. 1997, 119, 560; Bos et al., Biophys. J. 1995, 68, 2573.] is that the fluorophores are excited directly by the incident beam rather than an evanescent field, which greatly facilitates its application to Langmuir monolayers at the air−water interface. The technique was applied to the study of two systems with different porphyrin orientations: a Langmuir monolayer of a dihelical synthetic peptide BBC16 containing Zn(II)protoporphyrinIX and a monolayer of tetrakis (N-methyl-4-pyridil) porphyne electrostatically bound to the Langmuir monolayer of dipalmitoylphosphatidic acid. The results for the mean tilt angle are in good agreement with other relevant data in the literature for both systems. It is also shown that both systems exhibit rather narrow distributions, which makes them attractive for biophysical studies.

Control of the Orientational Order and Nonlinear Optical Response of the “Push-Pull” Chromophore RuPZn via Specific Incorporation into Densely Packed Monolayer Ensembles of an Amphiphilic Four-Helix Bundle Peptide: Characterization of the Peptide−Chromophore Complexes

by V. Krishnan, Hai-lung Dai, Andrey Tronin, and Joseph Strzalka

Journal of the American Chemical Society, 2010

Push-pull" chromophores based on extended π-electron systems have been designed to exhibit except... more Push-pull" chromophores based on extended π-electron systems have been designed to exhibit exceptionally large molecular hyperpolarizabilities. We have engineered an amphiphilic four-helix bundle peptide to vectorially incorporate such hyperpolarizable chromophores having a metalloporphyrin moiety, with high specificity into the interior core of the bundle. The amphiphilic exterior of the bundle facilitates the formation of densely packed monolayer ensembles of the vectorially oriented peptide-chromophore complexes at the liquid-gas interface. Chemical specificity designed into the ends of the bundle facilitates the subsequent covalent attachment of these monolayer ensembles onto the surface of an inorganic substrate. In this article, we describe the structural characterization of these monolayer ensembles at each stage of their fabrication for one such peptide-chromophore complex designated as AP0-RuPZn. In the accompanying article, we describe the characterization of their macroscopic nonlinear optical properties.

In situ determination of orientational distributions in Langmuir monolayers by total internal reflection fluorescence

Langmuir : the ACS journal of surfaces and colloids, 2005

A new application of the polarized total internal reflection fluorescence (PTIRF) technique to st... more A new application of the polarized total internal reflection fluorescence (PTIRF) technique to study the orientation distribution of a fluorophore within a Langmuir monolayer in situ on an aqueous subphase is described. The technique utilizes the measurement of polarized fluorescence, excited by the evanescent field appearing upon total internal reflection. The excitation by the evanescent field is achieved by launching the beam into a prism that is brought into contact with the monolayer from above. We also show here that a combination of PTIRF of monolayers on water and those freshly deposited onto the prism by horizontal lift in the same experiment provide enough data to determine the dielectric constant of the actual local environment of the fluorophore in the monolayer to eliminate the ambiguity of the orientation determination, arising from uncertainty in the normal component of excitation field. The new technique was applied to several model systems: fatty acid monolayers con...

Acentric 2-D ensembles of D-br-A electron-transfer chromophores via vectorial orientation within amphiphilic n-helix bundle peptides for photovoltaic device applications

by Andrey Tronin and Ivan Kuzmenko

Langmuir : the ACS journal of surfaces and colloids, Jan 14, 2012

We show that simply designed amphiphilic 4-helix bundle peptides can be utilized to vectorially o... more We show that simply designed amphiphilic 4-helix bundle peptides can be utilized to vectorially orient a linearly extended donor-bridge-acceptor (D-br-A) electron transfer (ET) chromophore within its core. The bundle's interior is shown to provide a unique solvation environment for the D-br-A assembly not accessible in conventional solvents and thereby control the magnitudes of both light-induced ET and thermal charge recombination rate constants. The amphiphilicity of the bundle's exterior was employed to vectorially orient the peptide-chromophore complex at a liquid-gas interface, and its ends were tailored for subsequent covalent attachment to an inorganic surface, via a "directed assembly" approach. Structural data, combined with evaluation of the excited state dynamics exhibited by these peptide-chromophore complexes, demonstrate that densely packed, acentrically ordered 2-D monolayer ensembles of such complexes at high in-plane chromophore densities approac...

Structural changes in single membranes in response to an applied transmembrane electric potential revealed by time-resolved neutron/X-ray interferometry

by Andrey Tronin and Ivan Kuzmenko

Chemical Physics, 2013

The profile structure of a hybrid lipid bilayer, tethered to the surface of an inorganic substrat... more The profile structure of a hybrid lipid bilayer, tethered to the surface of an inorganic substrate and fully hydrated with a bulk aqueous medium in an electrochemical cell, was investigated as a function of the applied transbilayer electric potential via time-resolved neutron reflectivity, enhanced by interferometry. Significant, and fully reversible structural changes were observed in the distal half (with respect to the substrate surface) of the hybrid bilayer comprised of a zwitterionic phospholipid in response to a +100mV potential with respect to 0mV. These arise presumably due to reorientation of the electric dipole present in the polar headgroup of the phospholipid and its resulting effect on the thickness of the phospholipid&#39;s hydrocarbon chain layer within the hybrid bilayer&#39;s profile structure. The profile structure of the voltage-sensor domain from a voltage-gated ion channel protein within a phospholipid bilayer membrane, tethered to the surface of an inorganic substrate and fully hydrated with a bulk aqueous medium in an electrochemical cell, was also investigated as a function of the applied transmembrane electric potential via time-resolved X-ray reflectivity, enhanced by interferometry. Significant, fully-reversible, and different structural changes in the protein were detected in response to ±100mV potentials with respect to 0mV. The approach employed is that typical of transient spectroscopy, shown here to be applicable to both neutron and X-ray reflectivity of thin films.

A New Approach to the Structural Investigation of the Voltage-Sensitive Domain of Voltage-Gated Cation Channels as a Function of the Transmembrane Voltage

Biophysical Journal, 2010

Structural Characterization of the Voltage Sensor Domain of the KvAP Channel Vectorially-Oriented within a Phospholipid Bilayer Membrane

Biophysical Journal, 2011

recorded voltage ramps in the interval 5200 mV in the presence of 2 M internal sucrose. In these ... more recorded voltage ramps in the interval 5200 mV in the presence of 2 M internal sucrose. In these conditions, outward currents become voltage independent at voltages >150 mV, providing size estimates consistent with the size of the internal mouth derived from the crystallographic structure. The ion density profile obtained from molecular dynamics simulations using an applied voltage reveals a high density of Kþ ions near P475D. Funded by Conicyt 190493. IV is a Conicyt fellow 3064-Pos Board B169 Calculating Conductance and Size of the Entrance to the Inner Cavity of BK Channels with Side-Chain Replacement and a Two-Resistor Model Yanyan Geng, Karl L. Magleby. BK channels have the largest conductance (~250 pS) of all Kþ selective channels. Previous studies suggest that residues E321/E324 in BK channels are located at the entrance to the inner cavity. We find that attachment of thiol reagents MPA and MTSET to E321C/E324C altered outward single-channel currents, suggesting that 321/324 face the ion conduction pathway. Therefore, substituting E321/E324 with different sized amino acids should change the size of the entrance to the inner cavity. We find that decreasing the size of the entrance decreases the conductance, whereas increasing the size of the entrance has little effect. Increasing [Kþ]i from 0.15 to 2.5 M negates differences in single-channel current associated with different side-chain volume. Plots of conductance vs. side-chain volume are approximated with a simple tworesistor model, where the ion conduction pathway is described by two resistors in series. R2 is a variable resistor, with resistance inversely proportional to the volume of the entrance to the inner cavity. R1 is a fixed resistor arising from the other parts of the conduction pathway including the selectivity filter. Fitting the data indicates that R1þR2 is~5.4 gigaohm for glycine substitution, with an R1/R2 ratio of~17, and effective radius and length of the entrance to the inner cavity of~9.0 and 5.4 Å , respectively. (The volume of Kþ and water are not taken into account.) The calculated size of the entrance to the inner cavity of BK channels is consistent with the crystal structure of large conductance bacterial MthK channels. These observations suggest that a large entrance to the inner cavity is required for the large conductance of BK channels, as decreasing the entrance size decreases the outward single-channel currents. Support: NIH-AR32805. Potassium channels containing the GYG motif often diverge in their selectivity for monovalent cations, but the molecular basis is unknown. Using the prokaryotic potassium channel KcsA as our model, we have investigated the role of the interaction between glutamate 71 and aspartate 80, located behind the selectivity filter, in determining the selectivity of the channel as well as its influence on the conformation of the filter. In E71A KcsA channels, Naþ permeates at higher rates in both the presence and absence of Kþ, as seen with 86Rbþ and 22Naþ flux measurements. Single channel recordings indicate that Naþ ''punches through'' E71A KcsA channels at lower voltages than wild-type KcsA, and in the punchthrough regime the Naþ-blocked current appears significantly larger. A crystal structure of E71A KcsA reveals that in contrast to what was seen for wild type KcsA, the selectivity filter does not collapse in the absence of Kþ, but instead assumes a ''flipped'' conformation. This flipped conformation is the same one observed in previous E71A KcsA structures in the presence of Kþ. The data reveal the importance of this E71-D80 interaction in both favoring inactivation and maintaining high Kþ selectivity. We propose a molecular mechanism by which inactivation and Kþ selectivity are linked, a mechanism that may also be at work in other channels containing the canonical GYG signature sequence.

Orientation Distributions for Cytochrome c on Polar and Nonpolar Interfaces by Total Internal Reflection Fluorescence

by Andrey Tronin and Jane Vanderkooi

Biophysical Journal, 2002

The formation of chemisorbed monolayers of yeast cytochrome c on both uncharged polar and nonpola... more The formation of chemisorbed monolayers of yeast cytochrome c on both uncharged polar and nonpolar soft surfaces of organic self-assembled monolayers (SAM) on solid inorganic substrates was followed in situ by polarized total internal reflection fluorescence. Two types of nonpolar surfaces and one type of uncharged polar surface were used. The first type of nonpolar surface contained only thiol endgroups, while the other was composed of a mixture of thiol and methyl endgroups. The uncharged polar surface was provided by the mixture of thiol and hydroxyl endgroups. The thiol endgroups were used to form a covalent disulfide bond with the unique surface-exposed cysteine residue 102 of the protein. The mean tilt angle of the protein's zinc-substituted porphyrin was found to be 41°and 50°for the adsorption onto the nonpolar and uncharged polar surfaces, respectively. The distribution widths for the pure thiol and the thiol/methyl and thiol/hydroxyl mixtures were 9°, 1°, and 18°, respectively. The high degree of the orientational order and good stability achieved for the protein monolayer on the mixed thiol/methyl endgroup SAM makes this system very attractive for studies of both intramolecular and intermolecular electron transfer processes.

Mechanism Of Interaction Between The Volatile Anesthetic Halothane And A Model Ion Channel Protein: Fluorescence And Infrared Spectroscopy Employing A Cyano-phenylalanine Probe And Molecular Dynamics Simulation

Biophysical Journal, 2009

Monolayers of a Model Anesthetic-Binding Membrane Protein: Formation, Characterization, and Halothane-Binding Affinity

by Andrey Tronin, Ivan Kuzmenko, and J. Blasie

Biophysical Journal, 2006

hbAP0 is a model membrane protein designed to possess an anesthetic-binding cavity in its hydroph... more hbAP0 is a model membrane protein designed to possess an anesthetic-binding cavity in its hydrophilic domain and a cation channel in its hydrophobic domain. Grazing incidence x-ray diffraction shows that hbAP0 forms four-helix bundles that are vectorially oriented within Langmuir monolayers at the air-water interface. Single monolayers of hbAP0 on alkylated solid substrates would provide an optimal system for detailed structural and dynamical studies of anesthetic-peptide interaction via x-ray and neutron scattering and polarized spectroscopic techniques. Langmuir-Blodgett and Langmuir-Schaeffer deposition and self-assembly techniques were used to form single monolayer films of the vectorially oriented peptide hbAP0 via both chemisorption and physisorption onto suitably alkylated solid substrates. The films were characterized by ultraviolet absorption, ellipsometry, circular dichroism, and polarized Fourier transform infrared spectroscopy. The a-helical secondary structure of the peptide was retained in the films. Under certain conditions, the average orientation of the helical axis was inclined relative to the plane of the substrate, approaching perpendicular in some cases. The halothane-binding affinity of the vectorially oriented hbAP0 peptide in the single monolayers, with the volatile anesthetic introduced into the moist vapor environment of the monolayer, was found to be similar to that for the detergent-solubilized peptide.

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

by Andrey Tronin and Jing Liu

Biophysical Journal, 2009

We previously reported the synthesis and structural characterization of a model membrane protein ... more 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.

Design of an Amphiphilic Di-Helix and its Vectorial Orientation at the Air-Water Interface