Dynamic imaging of single DNA-protein interactions using atomic force microscopy

Atomic force microscopy (AFM) imaging of static DNA-protein complexes, in air and in liquid, can be used to directly obtain quantitative and qualitative information on the structure of different complexes. For example, DNA length, the location of preferential binding sites for proteins and bending of DNA as a result of the complexation can all be measured. Recording consecutive AFM images of DNA and protein molecules under conditions that they are still able to move and interact, or dynamic AFM imaging, however, can reveal information on the dynamic aspects of the interactions between these molecules. Here, an overview is given of the technical challenges that need to be considered for successful dynamic AFM imaging studies of individual DNA-protein interactions. Necessary technical improvements to the AFM set-up and the development of new sample preparation methods are described in this paper.     

Kinetic nature of the thermal destabilization of LHCII macroaggregates

The main light-harvesting chl a/b pigment-protein complex of photosystem II (LHCII) in isolated state forms macroaggregates with different ultrastructure and lipid content [I. Simidjiev, V. Barzda, L. Mustardy, G. Garab, Anal. Biochem. 250 (1997) 169-175]. The thermodynamic stability of highly delipidated tightly bound LHCII macroaggregates is studied by differential scanning calorimetry and fluorescence spectroscopy. The calorimetric profile of LHCII is asymmetric, the denaturation transition is taking place at around 72 degrees C. A shoulder, which overlaps with the main denaturation transition, appears around 58 degrees C. The denaturation temperature strongly depends on the scanning rate indicating the kinetic nature of the thermal destabilization of LHCII macroaggregates. The fluorescence data prove that the thermal denaturation of LHCII is an irreversible and kinetically controlled process.     

Molecularly-imprinted polypyrrole-modified stainless steel frits for selective solid phase preconcentration of ochratoxin A

A molecularly-imprinted polymer (MIP) was prepared by electropolymerization of pyrrole (Py) onto a stainless steel frit, using ochratoxin A (OTA) as the template, in order to make a micro solid phase preconcentration (SPP) device. The OTA template was removed with 1% triethylamine (TEA) in methanol. Compared to non-imprinted polypyrrole (PPy), the molecularly-imprinted polypyrrole (MIPPy) enhanced the selective binding of OTA. The percentage recovery improved from 0 to 40% when the OTA sample solution was acidified with 1 M HCl (1% by volume). At a flow rate of 0.2 mL/min, maximum OTA binding was reached in 6 min after a total loading of 3.2 ng OTA. Final elution of the OTA was analyzed by high performance liquid chromatography (HPLC) with fluorescence detection, using 20:80 v/v acetonitrile–ammonia buffer (NH₄Cl/NH₃, 20 mM, pH 9.2) as the mobile phase. The MIPPy-SPP-HPLC results clearly demonstrated that the MIPPy-SPP device afforded selective preconcentration of OTA from red wine samples, at OTA concentration levels as low as 0.05 ppb, prior to HPLC analysis.     

Electro-optic characteristics of aqueous beta-FeOOH particles

This paper presents a detailed analysis of the electro-optic behavior of suspensions of noninteracting monodisperse beta-FeOOH particles. The electro-optic parameters are determined for aqueous suspensions of the oxide particles and the influences of surface charge and Debye layer thickness are verified. Since the conventional method of frequency analysis is inconsistent in the low-frequency range, new electro-optic parameters are introduced to define the frequency variation of the effects. Electric polarizability is determined with precision to a constant, and its relative variations are followed. As reported for other oxides, electric polarizability correlates with charge variations in the diffuse part of the particle surface electric layer, and its relaxation frequency increases with surface charge density, indicating a Maxwell-Wagner type of surface polarization. The alternating component of the responses yields particle relaxation frequency and the phase shift of the responses at this frequency. For all studied samples the phase shift at particle relaxation frequency is 45 degrees. The relative changes in the steady component of the responses in the low-frequency range are followed by field intensity curves at characteristic frequencies of the samples. Electrophoretic rotation is the process consistent with our data for the low-frequency effect. The results show that it is enhanced by the combined actions of low or slowly relaxing polarizability and significant electrophoretic mobility.     

Amphipathic helices as mediators of the membrane interaction of amphitropic proteins, and as modulators of bilayer physical properties

The amphipathic helix (AH) motif is used by a subset of amphitropic proteins to accomplish reversible and controlled association with the interfacial zone of membranes. Functioning as more than mere membrane anchoring domains, amphipathic helices can serve as autoinhibitory domains to suppress the protein activity in its soluble form, and as sensors or modulators of membrane curvature. Thus amphipathic helices can both respond to and modulate membrane physical properties. These and other features are illustrated by the behavior of CTP: phosphocholine cytidylyltransferase (CCT), a key regulatory enzyme in PC synthesis. A comparison of the physico-chemical features of CCT's AH motif and 10 others reveals similarities and several differences. The importance of these parameters to the particulars of the membrane interaction and to functional consequences requires more systematic exploration. The membrane partitioning of amphitropic proteins with AH motifs can be regulated by various strategies including changes in membrane lipid composition, phosphorylation, ligand-induced conformational changes, and membrane curvature. Several amphitropic proteins that control budding or tubule formation in cells have AH motifs. The insertion of the hydrophobic face of these amphipathic helices generates an asymmetry in the lateral pressure of the two leaflets resulting in an induction of positive curvature. Curvature induction or stabilization may be a universal property of AHA proteins, not just those involved in budding, but this possibility requires further demonstration.     

van der Waals interactions of polycyclic aromatic hydrocarbon dimers

Density functional theory is in principle exact and includes also long-range interactions, such as the van der Waals interactions. These are, however, part of the exchange-correlation energy functional that needs to be approximated, and are absent in the local and semilocal standard implementations. Recently a density functional which includes van der Waals interactions for planar systems has been developed, which we show can be extended to provide a treatment of planar molecules. We use this functional to calculate binding distances and energies for dimers of three of the smallest polycyclic aromatic hydrocarbons (PAHs)--naphthalene, anthracene, and pyrene.     

Projection structure of photosystem II In vivo determined by cryo-electron crystallography

Photosystem II (PSII) is a protein-pigment complex situated in the thylakoid membranes of plants and cyanobacteria where it catalyses the conversion of light into chemical energy. This energy is used to extract electrons from water, during which process oxygen is evolved. Owing to its extreme fragility and the large number of polypeptides (>20) it is composed of, the complex has so far proven recalcitrant to high-resolution structural studies. Cryo-electron crystallography of 2-D crystals (a = 15.4 nm, b = 23.1 nm, γ = 97.2°, p1) comprising in situ PSII revealed the first projection structure of the native complex. The unit cell contain one monomeric complex in which three central domains straddle an elongated intramolecular cavity. In conjunction with earlier data, these central domains were assigned to the reaction centre core subunits of PSII consisting of CP43, CP47, the reaction centre heterodimer D1/D2 and cytochrome b-559. The data are discussed in view of the evolution of reaction centres from anoxygenic to oxygenic photosynthesis.     

Cholesterol Alters the Phase Separation in Model Membranes Containing hBest1

Human retinal pigment epithelial (RPE) cells express the transmembrane Ca²⁺-dependent Cl⁻ channel bestrophin-1 (hBest1) of the plasma membrane. Mutations in the hBest1 protein are associated with the development of distinct pathological conditions known as bestrophinopathies. The interactions between hBest1 and plasma membrane lipids (cholesterol (Chol), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and sphingomyelin (SM)) determine its lateral organization and surface dynamics, i.e., their miscibility or phase separation. Using the surface pressure/mean molecular area (π/A) isotherms, hysteresis and compressibility moduli (C_s⁻¹) of hBest1/POPC/Chol and hBest1/SM/Chol composite Langmuir monolayers, we established that the films are in an LE (liquid-expanded) or LE-LC (liquid-condensed) state, the components are well-mixed and the Ca²⁺ ions have a condensing effect on the surface molecular organization. Cholesterol causes a decrease in the elasticity of both films and a decrease in the ΔG_mix^π values (reduction of phase separation) of hBest1/POPC/Chol films. For the hBest1/SM/Chol monolayers, the negative values of ΔG_mix^π are retained and equalized with the values of ΔG_mix^π in the hBest1/POPC/Chol films. Shifts in phase separation/miscibility by cholesterol can lead to changes in the structure and localization of hBest1 in the lipid rafts and its channel functions.