Ulrike Alexiev

Elucidation of the Nature of the Conformational Changes of the EF-interhelical Loop in Bacteriorhodopsin and of the Helix VIII on the Cytoplasmic Surface of Bovine Rhodopsin: A Time-resolved Fluorescence Depolarization Study

The conformation of the AB-loop and EF-loop of bacteriorhodopsin and of the fourth cytoplasmic loop (helix VIII) of bovine rhodopsin were assessed by a combination of time-resolved fluorescence depolarization and site-directed fluorescence labeling. The fluorescence anisotropy decays were measured employing a tunable Ti:sapphire laser/microchannel plate based single-photon counting apparatus with picosecond time resolution. This method allows measurement of the diffusional dynamics of the loops directly on a nanosecond time-scale. We implemented the method to study model peptides and two-helix systems representing sequences of bacteriorhodopsin. Thus, we systematically analyzed the anisotropic behavior of four different fluorescent dyes covalently bound to a single cysteine residue on the protein surface and assigned the anisotropy decay components to the modes of motion of the protein and its segments. We have identified two mechanisms of loop conformational changes in the functionally intact proteins bacteriorhodopsin and bovine rhodopsin. First, we found a surface potential-dependent transition between two conformational states of the EF-loop of bacteriorhodopsin, detected with the fluorescent dye bound to position 160. A transition between the two conformational states at 150mM KCl and 20 degrees C requires a surface potential change that corresponds to Deltasigma approximately -1.0e(-)/bacteriorhodopsin molecule. We suggest, that the surface potential-based switch of the EF-loop is the missing link between the movement of helix F and the transient surface potential change detected during the photocycle of bacteriorhodopsin. Second, in the visual pigment rhodopsin, with the fluorescent dye bound to position 316, a particularly striking pH-dependent conformational change of the fourth loop on the cytoplasmic surface was analyzed. The loop mobility increased from pH 5 to 8. The midpoint of this transition is at pH 6.2 and correlates with the midpoint of the pH-dependent equilibrium between the active metarhodopsin II and the inactive metarhodopsin I state.

Conformational dynamics of helix 8 in the GPCR rhodopsin controls arrestin activation in the desensitization process

Arrestins are regulatory molecules for G-protein coupled receptor function. In visual rhodopsin, selective binding of arrestin to the cytoplasmic side of light-activated, phosphorylated rhodopsin (P-Rh*) terminates signaling via the G-protein transducin. While the “phosphate-sensor” of arrestin for the recognition of receptor-attached phosphates is identified, the molecular mechanism of arrestin binding and the involvement of receptor conformations in this process are still largely hypothetic. Here we used fluorescence pump-probe and time-resolved fluorescence depolarization measurements to investigate the kinetics of arrestin conformational changes and the corresponding nanosecond dynamical changes at the receptor surface. We show that at least two sequential conformational changes of arrestin occur upon interaction with P-Rh*, thus providing a kinetic proof for the suggested multistep nature of arrestin binding. At the cytoplasmic surface of P-Rh*, the structural dynamics of the amphipathic helix 8 (H8), connecting transmembrane helix 7 and the phosphorylated C-terminal tail, depends on the arrestin interaction state. We find that a high mobility of H8 is required in the low-affinity (prebinding) but not in the high-affinity binding state. High-affinity arrestin binding is inhibited when a bulky, inflexible group is bound to H8, indicating close interaction. We further show that this close steric interaction of H8 with arrestin is mandatory for the transition from prebinding to high-affinity binding; i.e., for arrestin activation. This finding implies a regulatory role for H8 in activation of visual arrestin, which shows high selectivity to P-Rh* in contrast to the broad receptor specificity displayed by the two nonvisual arrestins.

Penetration of normal, damaged and diseased skin — An in vitro study on dendritic core–multishell nanotransporters

A growing intended or accidental exposure to nanoparticles asks for the elucidation of potential toxicity linked to the penetration of normal and lesional skin. We studied the skin penetration of dye-tagged dendritic core-multishell (CMS) nanotransporters and of Nile red loaded CMS nanotransporters using fluorescence microscopy. Normal and stripped human skin ex vivo as well as normal reconstructed human skin and in vitro skin disease models served as test platforms. Nile red was delivered rapidly into the viable epidermis and dermis of normal skin, whereas the highly flexible CMS nanotransporters remained solely in the stratum corneum after 6h but penetrated into deeper skin layers after 24h exposure. Fluorescence lifetime imaging microscopy proved a stable dye-tag and revealed striking nanotransporter-skin interactions. The viable layers of stripped skin were penetrated more efficiently by dye-tagged CMS nanotransporters and the cargo compared to normal skin. Normal reconstructed human skin reflected the penetration of Nile red and CMS nanotransporters in human skin and both, the non-hyperkeratotic non-melanoma skin cancer and hyperkeratotic peeling skin disease models come along with altered absorption in the skin diseases.

Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin.

BackgroundProteorhodopsin (pR) is a light-activated proton pump homologous to bacteriorhodopsin and recently discovered in oceanic γ-proteobacteria. One perplexing difference between these two proteins is the absence in pR of homologues of bR residues Glu-194 and Glu-204. These two residues, along with Arg-82, have been implicated in light-activated fast H+ release to the extracellular medium in bR. It is therefore uncertain that pR carries out its physiological activity using a mechanism that is completely homologous to that of bR.ResultsA pR purification procedure is described that utilizes Phenylsepharose™ and hydroxylapatite columns and yields 85% (w/w) purity. Through SDS-PAGE of the pure protein, the molecular weight of E.-coli-produced pR was determined to be 36,000, approximately 9,000 more than the 27,000 predicted by the DNA sequence. Post-translational modification of one or more of the cysteine residues accounts for 5 kDa of the weight difference as measured on a cys-less pR mutant. At pH 9.5 and in the presence of octylglucoside and diheptanoylphosphotidylcholine, flash photolysis results in fast H+ release and a 400-nm absorbing (M-like) photoproduct. Both of these occur with a similar rise time (4–10 μs) as reported for monomeric bR in detergent.ConclusionsThe presence of fast H+ release in pR indicates that either different groups are responsible for fast H+ release in pR and bR (i.e. that the H+ release group is not highly conserved); or, that the H+ release group is conserved and is therefore likely Arg-94 itself in pR (and Arg-82 in bR, correspondingly).

Penetration of silver nanoparticles into porcine skin ex vivo using fluorescence lifetime imaging microscopy, Raman microscopy, and surface-enhanced Raman scattering microscopy

Abstract. In order to investigate the penetration depth of silver nanoparticles (Ag NPs) inside the skin, porcine ears treated with Ag NPs are measured by two-photon tomography with a fluorescence lifetime imaging microscopy (TPT-FLIM) technique, confocal Raman microscopy (CRM), and surface-enhanced Raman scattering (SERS) microscopy. Ag NPs are coated with poly-N-vinylpyrrolidone and dispersed in pure water solutions. After the application of Ag NPs, porcine ears are stored in the incubator for 24 h at a temperature of 37°C. The TPT-FLIM measurement results show a dramatic decrease of the Ag NPs’ signal intensity from the skin surface to a depth of 4  μm. Below 4  μm, the Ag NPs’ signal continues to decline, having completely disappeared at 12 to 14  μm depth. CRM shows that the penetration depth of Ag NPs is 11.1±2.1  μm. The penetration depth measured with a highly sensitive SERS microscopy reaches 15.6±8.3  μm. Several results obtained with SERS show that the penetration depth of Ag NPs can exceed the stratum corneum (SC) thickness, which can be explained by both penetration of trace amounts of Ag NPs through the SC barrier and by the measurements inside the hair follicle, which cannot be excluded in the experiment.

Fluorescence spectroscopy of rhodopsins: insights and approaches.

Fluorescence spectroscopy has become an established tool at the interface of biology, chemistry and physics because of its exquisite sensitivity and recent technical advancements. However, rhodopsin proteins present the fluorescence spectroscopist with a unique set of challenges and opportunities due to the presence of the light-sensitive retinal chromophore. This review briefly summarizes some approaches that have successfully met these challenges and the novel insights they have yielded about rhodopsin structure and function. We start with a brief overview of fluorescence fundamentals and experimental methodologies, followed by more specific discussions of technical challenges rhodopsin proteins present to fluorescence studies. Finally, we end by discussing some of the unique insights that have been gained specifically about visual rhodopsin and its interactions with affiliate proteins through the use of fluorescence spectroscopy. This article is part of a Special Issue entitled: Retinal Proteins - You can teach an old dog new tricks.

Characterization of membrane protein non-native states. 2. The SDS-unfolded states of rhodopsin

Little is known about the molecular nature of residual structure in unfolded states of membrane proteins. A screen of chemical denaturants to maximally unfold the mammalian membrane protein and prototypic G protein coupled receptor rhodopsin, without interference from aggregation, described in an accompanying paper (DOI 10.1021/bi100338e ), identified sodium dodecyl sulfate (SDS), alone or in combination with other chemicals, as the most suitable denaturant. Here, we initiate the biophysical characterization of SDS-denatured states of rhodopsin. Using absorption, steady-state and time-resolved fluorescence spectroscopy, dynamic light scattering, and cysteine accessibility studies, tertiary structure of denatured states was characterized. In agreement with the pattern of secondary structure changes detected by circular dichroism described in the accompanying paper (DOI 10.1021/bi100338e ), tertiary structure changes are distinct over four SDS concentration ranges based on the expected predominant micellar structures. Dodecyl maltoside (DM)/SDS mixed micelle spheres (0.05-0.3% SDS) turn into SDS spheres (0.3-3% SDS) that gradually (3-15% SDS) become cylindrical (above 15% SDS). Denatured states in SDS spheres and cylinders show a relatively greater burial of cysteine and tryptophan residues and are more compact as compared to the states observed in mixed micellar structures. Protein structural changes at the membrane/water interface region are most prominent at very low SDS concentrations but reach transient stability in the compact conformations in SDS spheres. This is the first experimental evidence for the formation of a compact unfolding intermediate state with flexible surface elements in a membrane protein.

Skin barrier disruptions in tape stripped and allergic dermatitis models have no effect on dermal penetration and systemic distribution of AHAPS-functionalized silica nanoparticles

The skin is a potential site of entry for nanoparticles (NP) but the role of disease-associated barrier disturbances on the path and extent of skin penetration of NP remains to be characterized. Silica nanoparticles (SiO2-NP) possess promising potential for various medical applications. Here, effects of different skin barrier disruptions on the penetration of N-(6-aminohexyl)-aminopropyltrimethoxysilane (AHAPS) functionalized SiO2-NP were studied. AHAPS-SiO2-NP (55±6 nm diameter) were topically applied on intact, tape stripped or on inflamed skin of SKH1 mice with induced allergic contact dermatitis for one or five consecutive days, respectively. Penetration of AHAPS-SiO2-NP through the skin was not observed regardless of the kind of barrier disruption. However, only after subcutaneous injection, AHAPS-SiO2-NP were incorporated by macrophages and transported to the regional lymph node only. Adverse effects on cells or tissues were not observed. In conclusion, AHAPS-SiO2-NP seem to not cross the normal or perturbed mouse skin. From the clinical editor: Skin is a potential site of entry for nanoparticles; however, it is poorly understood how skin diseases may alter this process. In tape-stripped skin and allergic contact dermatitis models the delivery properties of AHAPS-SiO2 nanoparticles remained unchanged, and in neither case were these NP-s able to penetrate the skin. No adverse effects were noted on the skin in these models and control mice.

Overview about the localization of nanoparticles in tissue and cellular context by different imaging techniques

Summary The increasing interest and recent developments in nanotechnology pose previously unparalleled challenges in understanding the effects of nanoparticles on living tissues. Despite significant progress in in vitro cell and tissue culture technologies, observations on particle distribution and tissue responses in whole organisms are still indispensable. In addition to a thorough understanding of complex tissue responses which is the domain of expert pathologists, the localization of particles at their sites of interaction with living structures is essential to complete the picture. In this review we will describe and compare different imaging techniques for localizing inorganic as well as organic nanoparticles in tissues, cells and subcellular compartments. The visualization techniques include well-established methods, such as standard light, fluorescence, transmission electron and scanning electron microscopy as well as more recent developments, such as light and electron microscopic autoradiography, fluorescence lifetime imaging, spectral imaging and linear unmixing, superresolution structured illumination, Raman microspectroscopy and X-ray microscopy. Importantly, all methodologies described allow for the simultaneous visualization of nanoparticles and evaluation of cell and tissue changes that are of prime interest for toxicopathologic studies. However, the different approaches vary in terms of applicability for specific particles, sensitivity, optical resolution, technical requirements and thus availability, and effects of labeling on particle properties. Specific bottle necks of each technology are discussed in detail. Interpretation of particle localization data from any of these techniques should therefore respect their specific merits and limitations as no single approach combines all desired properties.

Nanodynamics of dendritic core-multishell nanocarriers.

The molecular dynamics of polymeric nanocarriers is an important parameter for controlling the interaction of nanocarrier branches with cargo. Understanding the interplay of dendritic polymer dynamics, temperature, and cargo molecule interactions should provide valuable new insight for tailoring the dendritic architecture to specific needs in nanomedicine, drug, dye, and gene delivery. Here, we have investigated polyglycerol-based core-multishell (CMS) nanotransporters with incorporated Nile Red as a fluorescent drug mimetic and CMS nanotransporters with a covalently bound fluorophore (Indocarbocyanine) using fluorescence spectroscopy methods. From time-resolved fluorescence depolarization we have obtained the rotational diffusion dynamics of the incorporated dye, the nanocarrier, and its branches as a function of temperature. UV/vis and fluorescence lifetime measurements provided additional information on the local dye environment. Our results show a distribution of the cargo Nile Red within the nanotransporter shells that depends on solvent and temperature. In particular, we show that the flexibility of the polymer branches in the unimolecular state of the nanotransporter undergoes a temperature-dependent transition which correlates with a larger space for the mobility of the incorporated hydrophobic drug mimetic Nile Red and a higher probability of cargo-solvent interactions at temperatures above 31 °C. The measurements have further revealed that a loss of the cargo molecule Nile Red occurred neither upon dilution of the CMS nanotransporters nor upon heating. Thus, the unimolecular preloaded CMS nanotransporters retain their cargo and are capable to transport and respond to temperature, thereby fulfilling important requirements for biomedical applications.