Biomedical subjects

Joseph Strzalka

Publications and source records attributed to Joseph Strzalka.

8 recordsLinked to original sources

PubMed2006

Monolayers of a model anesthetic-binding membrane protein: formation, characterization, and halothane-binding affinity.

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 alpha-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.

Adsorption↗

PubMed2006

Structural studies of amphiphilic 4-helix bundle peptides incorporating designed extended chromophores for nonlinear optical biomolecular materials.

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.

Biocompatible Materials↗

PubMed2005

Design of amphiphilic protein maquettes: controlling assembly, membrane insertion, and cofactor interactions.

We have designed polypeptides combining selected lipophilic (LP) and hydrophilic (HP) sequences that assemble into amphiphilic (AP) alpha-helical bundles to reproduce key structure characteristics and functional elements of natural membrane proteins. The principal AP maquette (AP1) developed here joins 14 residues of a heme binding sequence from a structured diheme-four-alpha-helical bundle (HP1), with 24 residues of a membrane-spanning LP domain from the natural four-alpha-helical M2 channel of the influenza virus, through a flexible linking sequence (GGNG) to make a 42 amino acid peptide. The individual AP1 helices (without connecting loops) assemble in detergent into four-alpha-helical bundles as observed by analytical ultracentrifugation. The helices are oriented parallel as indicated by interactions typical of adjacent hemes. AP1 orients vectorially at nonpolar-polar interfaces and readily incorporates into phospholipid vesicles with >97% efficiency, although most probably without vectorial bias. Mono- and diheme-AP1 in membranes enhance functional elements well established in related HP analogues. These include strong redox charge coupling of heme with interior glutamates and internal electric field effects eliciting a remarkable 160 mV splitting of the redox potentials of adjacent hemes that leads to differential heme binding affinities. The AP maquette variants, AP2 and AP3, removed heme-ligating histidines from the HP domain and included heme-ligating histidines in LP domains by selecting the b(H) heme binding sequence from the membrane-spanning d-helix of respiratory cytochrome bc(1). These represent the first examples of AP maquettes with heme and bacteriochlorophyll binding sites located within the LP domains.

Binding Sites↗

PubMed2005

Amphiphilic four-helix bundle peptides designed for light-induced electron transfer across a soft interface.

A family of four-helix bundle peptides were designed to be amphiphilic, possessing distinct hydrophilic and hydrophobic domains along the length of the bundle's exterior. This facilitates their vectorial insertion across a soft interface between polar and nonpolar media. Their design also now provides for selective incorporation of electron donor and acceptor cofactors within each domain. This allows translation of the designed intramolecular electron transfer along the bundle axis into a macroscopic charge separation across the interface.

Amino Acid Sequence↗

PubMed2004

Specular neutron reflectivity and the structure of artificial protein maquettes vectorially oriented at interfaces.

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- alpha -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 alpha -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.

Lipid Bilayers↗

PubMed2004

Resonant x-ray reflectivity from a bromine-labeled fatty acid Langmuir monolayer.

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 bromo-stearic 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.

Algorithms↗

PubMed2004

A model membrane protein for binding volatile anesthetics.

Earlier work demonstrated that a water-soluble four-helix bundle protein designed with a cavity in its nonpolar core is capable of binding the volatile anesthetic halothane with near-physiological affinity (0.7 mM Kd). To create a more relevant, model membrane protein receptor for studying the physicochemical specificity of anesthetic binding, we have synthesized a new protein that builds on the anesthetic-binding, hydrophilic four-helix bundle and incorporates a hydrophobic domain capable of ion-channel activity, resulting in an amphiphilic four-helix bundle that forms stable monolayers at the air/water interface. The affinity of the cavity within the core of the bundle for volatile anesthetic binding is decreased by a factor of 4-3.1 mM Kd as compared to its water-soluble counterpart. Nevertheless, the absence of the cavity within the otherwise identical amphiphilic peptide significantly decreases its affinity for halothane similar to its water-soluble counterpart. Specular x-ray reflectivity shows that the amphiphilic protein orients vectorially in Langmuir monolayers at higher surface pressure with its long axis perpendicular to the interface, and that it possesses a length consistent with its design. This provides a successful starting template for probing the nature of the anesthetic-peptide interaction, as well as a potential model system in structure/function correlation for understanding the anesthetic binding mechanism.

Amino Acid Sequence↗

PubMed2003

Comparative structural studies of Vpu peptides in phospholipid monolayers by x-ray scattering.

Vpu is an 81-residue HIV-1 accessory protein, its transmembrane and cytoplasmic domains each responsible for one of its two functions. Langmuir monolayers of phospholipid incorporating a membrane protein with a unidirectional vectorial orientation, on a semiinfinite aqueous subphase, provide one "membranelike" environment for the protein. The cytoplasmic domain's interaction with the surface of the phospholipid monolayer in determining the tertiary structure of the peptide within the monolayer was investigated, employing a comparative structural study of Vpu with its submolecular fragments Tm and TmCy truncated to different extents in the cytoplasmic domain, via synchrotron x-ray scattering utilizing a new method of analysis. Localizations of the transmembrane and cytoplasmic domains within the monolayer profile structure were similar for all three proteins, the hydrophobic transmembrane helix within the hydrocarbon chain region tilted with respect to the monolayer plane and the helices of the cytoplasmic domains lying on the surface of the headgroups parallel to the monolayer plane. The thickness of the hydrocarbon chain region, determined by the tilt of the hydrocarbon chains and transmembrane domain with respect to the monolayer plane, was slightly different for Tm, TmCy, and Vpu systematically with protein/lipid mole ratio. Localization of the helices in the cytoplasmic domains of the three proteins relative to the headgroups depends on their extents and amphipathicities. Thus, the interaction of the cytoplasmic domain of Vpu on the surface may affect the tilt of the transmembrane helix within the hydrocarbon chain region in determining its tertiary structure in the membrane.

Amino Acid Sequence↗