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 determi...
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.
Relative electron density profiles derived from high-resolution meridional x-ray diffraction data... more Relative electron density profiles derived from high-resolution meridional x-ray diffraction data for ultrathin multilayers, fabricated by repeating from one to five bilayers of arachidic acid deposited on alkylated glass substrates via the Langmuir-Blodgett technique, have ...
ABSTRACT The phase problem for specular x-ray and neutron reflectivity from liquid surfaces and t... more ABSTRACT The phase problem for specular x-ray and neutron reflectivity from liquid surfaces and thin films on liquid surfaces can be solved in the distorted-wave Born approximation. The gradient of the scattering-length density (SLD) profile normal to the plane of the surface is bounded in these cases. This provides a powerful constraint allowing the phase problem to be solved with no a priori assumptions via an iterative Fourier refinement procedure applied to the Fresnel-normalized reflectivity. The critical boundary condition can be determined experimentally from the autocorrelation of the gradient profile obtained via an inverse Fourier transform of the Fresnel-normalized reflectivity without phase information. The phase solution and the resulting gradient SLD profile can be shown to be unique, and therefore unambiguously determined, when all of phase space is systematically explored for particular cases, especially for thin films on liquid surfaces. This gradient SLD profile can then be integrated either numerically, or better, analytically to provide the scattering-length density profile itself on an absolute scale.
... Figure 1(b) shows the corrected meridional intensity functions for one-, two-, three-, and fi... more ... Figure 1(b) shows the corrected meridional intensity functions for one-, two-, three-, and five-bilayer arachidic acid specimens. Note that ... maxima. As N decreases, dif-fraction orders two and three, four and five, shift togeth-er. As ...
High-resolution, meridional x-ray diffraction data were recorded from ultrathin Langmuir-Blodgett... more High-resolution, meridional x-ray diffraction data were recorded from ultrathin Langmuir-Blodgett multilayer films containing five bilayers of either arachidic acid (AA), pentacosa-10, 12-diynoic acid (a disubstituted diacetylene monomer, DAM), the blue polymeric form of ...
... Alan S. Haas, Denis L. Pilloud, Konda S. Reddy ... For comparison, the rate for cyt c bou... more ... Alan S. Haas, Denis L. Pilloud, Konda S. Reddy ... For comparison, the rate for cyt c bound to much longer carboxy-terminated linkers (gray triangles, 11-mercaptoundecanoic acid) is only slightly slower (X. Chen, personal communication). Binding of CcO to Cyt c Films. ...
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.
Proceedings of the National Academy of Sciences, 1975
Lamellar neutron diffraction from oriented multilayers of hydrated dipalmitoyl lecithin was phase... more Lamellar neutron diffraction from oriented multilayers of hydrated dipalmitoyl lecithin was phased by isomorphous H20-D20 exchange and swelling techniques. Bound water sites were located in the polar head group region of the bilayer profile. A 6-A resolution structure based on the neutron scattering density profile is proposed for the bilayer. It is consistent with the electron density profile from x-ray diffraction.
Artificial peptides can be designed to possess a variety of functionalities. If these peptides ca... more Artificial peptides can be designed to possess a variety of functionalities. If these peptides can be ordered in an ensemble, the functionality can impart macroscopic material properties to the ensemble. Neutron reflectivity is shown to be an effective probe of the intramolecular structures of such peptides vectorially oriented at an interface, key to ensuring that the designed molecular structures translate into the desired material properties of the interface. A model-independent method is utilized to analyze the neutron reflectivity from an alkylated, di-␣-helical peptide, containing perdeuterated leucine residues at one or two pre-selected positions, in mixed Langmuir monolayers with a phospholipid. The results presented here are more definitive than prior work employing x-ray reflectivity. They show explicitly that the di-helical peptide retains its designed ␣-helical secondary structure at the interface, when oriented perpendicular to the interface at high surface pressure, with the helices projecting into the aqueous subphase without penetrating the layer of phospholipid headgroups.
Resonant x-ray reflectivity exploits the energy dependence of atomic scattering factors to locate... more Resonant x-ray reflectivity exploits the energy dependence of atomic scattering factors to locate resonant atoms within the electron density distribution of thin films. We apply the technique to a monolayer of bromostearic acid at the air/water interface. The data collection protocol employed cycles through several energies in the vicinity of the bromine K absorption edge and verifies that the energy dependencies observed are indeed resonant effects. The analysis specifies the location of the Br atom with sub-angstrom precision and must consider both the real and imaginary parts of the changes in the scattering factor to be consistent with the known structure and stoichiometry of this test case.
One subunit of the prokaryotic voltage-gated potassium ion channel from Aeropyrum pernix (KvAP) i... more One subunit of the prokaryotic voltage-gated potassium ion channel from Aeropyrum pernix (KvAP) is comprised of six transmembrane α-helices, of which S1-S4 form the voltagesensor domain (VSD) and S5-S6 contribute to the pore domain (PD) of the functional homotetramer. However, the mechanism of electromechanical coupling interconverting the closed-toopen (i.e. non-conducting to K + conducting) states remains undetermined. Here, we have vectorially-oriented the detergent (OG)-solubilized VSD in single monolayers by two independent approaches, namely "directed-assembly" and "self-assembly" to achieve a high inplane density. Both utilize Ni coordination chemistry to tether the protein to an alkylated inorganic surface via its C-terminal His 6-tag. Subsequently, the detergent is replaced by phospholipid (POPC) via exchange, intended to reconstitute a phospholipid bilayer environment for the protein. X-ray interferometry, in which interference with a multilayer reference structure is used to both enhance and phase the specular X-ray reflectivity from the tethered single membrane, was used to determine directly the electron density profile structures of the VSD protein solvated by detergent versus phospholipid, and with either a moist He (moderate hydration) or bulk aqueous buffer (high hydration) environment to preserve a native structure conformation. Difference electron density profiles, with respect to the multilayer substrate itself, for the VSD:OG monolayer and VSD:POPC membranes at both the solid-vapor and solid-liquid interfaces, reveal the profile structures of the VSD protein dominating these profiles and further indicate a successful reconstitution of a lipid bilayer environment. The self-assembly approach was similarly extended to the intact full-length KvAP channel for comparison. The spatial extent and asymmetry in the profile structures of both proteins confirm their uni-directional vectorial orientation within the reconstituted membrane and indicate retention of the protein's folded 3-D tertiary structure upon completion of membrane bilayer reconstitution. Moreover, the resulting high in-plane density of vectorially-oriented protein within a fully-hydrated single phospholipid bilayer membrane at the solid-liquid interface will enable investigation of their conformational states as a function of the transmembrane electric potential. .
Langmuir monolayers provide an important system for the investigation of the intramolecular struc... more Langmuir monolayers provide an important system for the investigation of the intramolecular structure and intermolecular ordering of organic and bio-organic macromolecular amphiphiles at an interface between polar and nonpolar media, e.g., the liquid-gas interface. Specular x-ray and neutron reflectivity have contributed substantially to these investigations. However, these reflectivity techniques are generally limited by the absence of crucial phase information, the relatively small contribution of the amphiphile to the scattering-length density contrast across the interface, and the relatively limited range of momentum transfer available perpendicular to the interface. Although several procedures have been developed to provide model-independent solutions to the phase problem, there remains a limited ability to distinguish features of slightly differing contrast (i.e., the "sensitivity") as well as their minimum allowable separation (i.e., the "spatial resolution") along the length of the scattering-length density profile derived from the reflectivity data via solution to the phase problem. Here, we demonstrate how the well-known interferometric approach can be extended to the structural investigation of otherwise unperturbed Langmuir monolayers of these amphiphiles to provide a direct solution to the phase problem and importantly, substantially enhance both the sensitivity and the spatial resolution in the derived profiles.
General formulas for the polarized fluorescence intensities from a linear transition dipole in th... more General formulas for the polarized fluorescence intensities from a linear transition dipole in the case of low aperture collection, which is used in the present study, were derived in [1], and a similar analysis for the case of a porphyrin ring was given in [2]. A typographic error that occurred in equations (12) of our previous paper [2] has been corrected in [3]. Here we will show only the final working formulae for the linear transition dipole and the porphyrin ring orientation distribution determination extended to the case of a bimodal distribution, which implies that the flourophores can be oriented at two different tilt angles, (θ 1 and θ 2), and the goal is to find the fractions of each population. In PTIRF or PEF, utilization of two polarization modes of the excitation wave, TE and TM (electric field respectively perpendicular and parallel to the incidence plane) and measuring emitted light intensity with two polarizations, X and Y (respectively parallel and perpendicular to the incidence plane of the excitation beam), provide four measurable intensities: I TEX , I TEY , I TMX , I TMY. In the case of linear transition dipole, these are (taken directly from [3]): fluorescence yield, detector sensitivity, etc., ψ and δ are the differences in the tilt and precession angles for the absorbing and emitting dipoles. A x,y,z are the evanescent field components. To account for possible fluorophore screening due to the embedding into a monolayer with effective refractive index n f , the normal component, A z , calculated for the prism-water or prism-air interface was divided by n f 2 , which is valid for thin film
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 determi...
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.
Relative electron density profiles derived from high-resolution meridional x-ray diffraction data... more Relative electron density profiles derived from high-resolution meridional x-ray diffraction data for ultrathin multilayers, fabricated by repeating from one to five bilayers of arachidic acid deposited on alkylated glass substrates via the Langmuir-Blodgett technique, have ...
ABSTRACT The phase problem for specular x-ray and neutron reflectivity from liquid surfaces and t... more ABSTRACT The phase problem for specular x-ray and neutron reflectivity from liquid surfaces and thin films on liquid surfaces can be solved in the distorted-wave Born approximation. The gradient of the scattering-length density (SLD) profile normal to the plane of the surface is bounded in these cases. This provides a powerful constraint allowing the phase problem to be solved with no a priori assumptions via an iterative Fourier refinement procedure applied to the Fresnel-normalized reflectivity. The critical boundary condition can be determined experimentally from the autocorrelation of the gradient profile obtained via an inverse Fourier transform of the Fresnel-normalized reflectivity without phase information. The phase solution and the resulting gradient SLD profile can be shown to be unique, and therefore unambiguously determined, when all of phase space is systematically explored for particular cases, especially for thin films on liquid surfaces. This gradient SLD profile can then be integrated either numerically, or better, analytically to provide the scattering-length density profile itself on an absolute scale.
... Figure 1(b) shows the corrected meridional intensity functions for one-, two-, three-, and fi... more ... Figure 1(b) shows the corrected meridional intensity functions for one-, two-, three-, and five-bilayer arachidic acid specimens. Note that ... maxima. As N decreases, dif-fraction orders two and three, four and five, shift togeth-er. As ...
High-resolution, meridional x-ray diffraction data were recorded from ultrathin Langmuir-Blodgett... more High-resolution, meridional x-ray diffraction data were recorded from ultrathin Langmuir-Blodgett multilayer films containing five bilayers of either arachidic acid (AA), pentacosa-10, 12-diynoic acid (a disubstituted diacetylene monomer, DAM), the blue polymeric form of ...
... Alan S. Haas, Denis L. Pilloud, Konda S. Reddy ... For comparison, the rate for cyt c bou... more ... Alan S. Haas, Denis L. Pilloud, Konda S. Reddy ... For comparison, the rate for cyt c bound to much longer carboxy-terminated linkers (gray triangles, 11-mercaptoundecanoic acid) is only slightly slower (X. Chen, personal communication). Binding of CcO to Cyt c Films. ...
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.
Proceedings of the National Academy of Sciences, 1975
Lamellar neutron diffraction from oriented multilayers of hydrated dipalmitoyl lecithin was phase... more Lamellar neutron diffraction from oriented multilayers of hydrated dipalmitoyl lecithin was phased by isomorphous H20-D20 exchange and swelling techniques. Bound water sites were located in the polar head group region of the bilayer profile. A 6-A resolution structure based on the neutron scattering density profile is proposed for the bilayer. It is consistent with the electron density profile from x-ray diffraction.
Artificial peptides can be designed to possess a variety of functionalities. If these peptides ca... more Artificial peptides can be designed to possess a variety of functionalities. If these peptides can be ordered in an ensemble, the functionality can impart macroscopic material properties to the ensemble. Neutron reflectivity is shown to be an effective probe of the intramolecular structures of such peptides vectorially oriented at an interface, key to ensuring that the designed molecular structures translate into the desired material properties of the interface. A model-independent method is utilized to analyze the neutron reflectivity from an alkylated, di-␣-helical peptide, containing perdeuterated leucine residues at one or two pre-selected positions, in mixed Langmuir monolayers with a phospholipid. The results presented here are more definitive than prior work employing x-ray reflectivity. They show explicitly that the di-helical peptide retains its designed ␣-helical secondary structure at the interface, when oriented perpendicular to the interface at high surface pressure, with the helices projecting into the aqueous subphase without penetrating the layer of phospholipid headgroups.
Resonant x-ray reflectivity exploits the energy dependence of atomic scattering factors to locate... more Resonant x-ray reflectivity exploits the energy dependence of atomic scattering factors to locate resonant atoms within the electron density distribution of thin films. We apply the technique to a monolayer of bromostearic acid at the air/water interface. The data collection protocol employed cycles through several energies in the vicinity of the bromine K absorption edge and verifies that the energy dependencies observed are indeed resonant effects. The analysis specifies the location of the Br atom with sub-angstrom precision and must consider both the real and imaginary parts of the changes in the scattering factor to be consistent with the known structure and stoichiometry of this test case.
One subunit of the prokaryotic voltage-gated potassium ion channel from Aeropyrum pernix (KvAP) i... more One subunit of the prokaryotic voltage-gated potassium ion channel from Aeropyrum pernix (KvAP) is comprised of six transmembrane α-helices, of which S1-S4 form the voltagesensor domain (VSD) and S5-S6 contribute to the pore domain (PD) of the functional homotetramer. However, the mechanism of electromechanical coupling interconverting the closed-toopen (i.e. non-conducting to K + conducting) states remains undetermined. Here, we have vectorially-oriented the detergent (OG)-solubilized VSD in single monolayers by two independent approaches, namely "directed-assembly" and "self-assembly" to achieve a high inplane density. Both utilize Ni coordination chemistry to tether the protein to an alkylated inorganic surface via its C-terminal His 6-tag. Subsequently, the detergent is replaced by phospholipid (POPC) via exchange, intended to reconstitute a phospholipid bilayer environment for the protein. X-ray interferometry, in which interference with a multilayer reference structure is used to both enhance and phase the specular X-ray reflectivity from the tethered single membrane, was used to determine directly the electron density profile structures of the VSD protein solvated by detergent versus phospholipid, and with either a moist He (moderate hydration) or bulk aqueous buffer (high hydration) environment to preserve a native structure conformation. Difference electron density profiles, with respect to the multilayer substrate itself, for the VSD:OG monolayer and VSD:POPC membranes at both the solid-vapor and solid-liquid interfaces, reveal the profile structures of the VSD protein dominating these profiles and further indicate a successful reconstitution of a lipid bilayer environment. The self-assembly approach was similarly extended to the intact full-length KvAP channel for comparison. The spatial extent and asymmetry in the profile structures of both proteins confirm their uni-directional vectorial orientation within the reconstituted membrane and indicate retention of the protein's folded 3-D tertiary structure upon completion of membrane bilayer reconstitution. Moreover, the resulting high in-plane density of vectorially-oriented protein within a fully-hydrated single phospholipid bilayer membrane at the solid-liquid interface will enable investigation of their conformational states as a function of the transmembrane electric potential. .
Langmuir monolayers provide an important system for the investigation of the intramolecular struc... more Langmuir monolayers provide an important system for the investigation of the intramolecular structure and intermolecular ordering of organic and bio-organic macromolecular amphiphiles at an interface between polar and nonpolar media, e.g., the liquid-gas interface. Specular x-ray and neutron reflectivity have contributed substantially to these investigations. However, these reflectivity techniques are generally limited by the absence of crucial phase information, the relatively small contribution of the amphiphile to the scattering-length density contrast across the interface, and the relatively limited range of momentum transfer available perpendicular to the interface. Although several procedures have been developed to provide model-independent solutions to the phase problem, there remains a limited ability to distinguish features of slightly differing contrast (i.e., the "sensitivity") as well as their minimum allowable separation (i.e., the "spatial resolution") along the length of the scattering-length density profile derived from the reflectivity data via solution to the phase problem. Here, we demonstrate how the well-known interferometric approach can be extended to the structural investigation of otherwise unperturbed Langmuir monolayers of these amphiphiles to provide a direct solution to the phase problem and importantly, substantially enhance both the sensitivity and the spatial resolution in the derived profiles.
General formulas for the polarized fluorescence intensities from a linear transition dipole in th... more General formulas for the polarized fluorescence intensities from a linear transition dipole in the case of low aperture collection, which is used in the present study, were derived in [1], and a similar analysis for the case of a porphyrin ring was given in [2]. A typographic error that occurred in equations (12) of our previous paper [2] has been corrected in [3]. Here we will show only the final working formulae for the linear transition dipole and the porphyrin ring orientation distribution determination extended to the case of a bimodal distribution, which implies that the flourophores can be oriented at two different tilt angles, (θ 1 and θ 2), and the goal is to find the fractions of each population. In PTIRF or PEF, utilization of two polarization modes of the excitation wave, TE and TM (electric field respectively perpendicular and parallel to the incidence plane) and measuring emitted light intensity with two polarizations, X and Y (respectively parallel and perpendicular to the incidence plane of the excitation beam), provide four measurable intensities: I TEX , I TEY , I TMX , I TMY. In the case of linear transition dipole, these are (taken directly from [3]): fluorescence yield, detector sensitivity, etc., ψ and δ are the differences in the tilt and precession angles for the absorbing and emitting dipoles. A x,y,z are the evanescent field components. To account for possible fluorophore screening due to the embedding into a monolayer with effective refractive index n f , the normal component, A z , calculated for the prism-water or prism-air interface was divided by n f 2 , which is valid for thin film
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Papers by J. Blasie