Sedated masculinity: the use of anaesthesia during vasectomy in Israel.

Vasectomy is considered a permanent form of contraception for men that can help reduce reproductive inequality. Its underutilisation in the context of the threats it may pose to perceptions of traditional masculinity has been closely investigated, but the lived experience of the procedure itself has been largely overlooked. This paper examines the experience of having a vasectomy in Israel, focusing on the recommended form of anaesthesia. Drawing on 15 semi-structured interviews with Israeli men who have experienced vasectomy, we found that the choice of anaesthesia has a significant impact on the meaning and experience of vasectomy. Unlike the widespread use of local anaesthesia during vasectomy in other countries, vasectomy in Israel is performed almost exclusively under twilight sedation administered intravenously. Based on our findings, we argue that framing vasectomy as an operation that requires sedation is related to views of masculinity and reproduction. Our data suggest that vasectomy in Israel is constituted as a potentially traumatic event, and anaesthesia is employed to protect patients from feelings of embarrassment and discomfort. We conclude that the prevailing method of sedation perpetuates the silencing and marginalisation of vasectomy as a contraceptive method, and consequently, its very limited accessibility in Israel.

Promoting crystallization of intrinsic membrane proteins with conjugated micelles.

A new technique for promoting nucleation and growth of membrane protein (MP) crystals from micellar environments is reported. It relies on the conjugation of micelles that sequester MPs in protein detergent complexes (PDCs). Conjugation via amphiphilic [metal:chelator] complexes presumably takes place at the micelle/water interface, thereby bringing the PDCs into proximity, promoting crystal nucleation and growth. We have successfully applied this approach to two light-driven proton pumps: bacteriorhodopsin (bR) and the recently discovered King Sejong 1-2 (KS1-2), using the amphiphilic 4,4'-dinonyl-2,2'-dipyridyl (Dinonyl) (0.7 mM) chelator in combination with Zn2+, Fe2+, or Ni2+ (0.1 mM). Crystal growth in the presence of the [metal-chelator] complexes leads to purple, hexagonal crystals (50-75 µm in size) of bR or pink, rectangular/square crystals (5-15 µm) of KS1-2. The effects of divalent cation identity and concentration, chelator structure and concentration, ionic strength and pH on crystal size, morphology and process kinetics, are described.

Conjugation of native membranes via linear oligo-amines.

We report the first observation of an efficient, native membrane conjugation mechanism via positively charged, linear oligo-amines. Clustering of membrane fragments relies on electrostatic interactions between the net negative charge of the membranes and the positively charged, water-soluble mediators. This conjugation principle is demonstrated with two different bacterial membranes in which are embedded either the intrinsic membrane protein (MP) bacteriorhodopsin (bR) or the more recently identified xanthorhodopsin (XR). As determined by their characteristic UV-vis absorption spectra and by circular dichroism, the MPs are not significantly perturbed by the oligo-amines carrying from +3 to +6 positive charges. Light microscopy and scanning electron microscope (SEM) imaging provide direct evidence for membrane conjugation. Process efficiency was found to be correlated with the net charge of the oligo-amine used. Membrane conjugation is accomplished within a wide range of pH values (7-2.5); is reversed by NaCl; and does not require the presence of a precipitant (e.g. PEG) nor Ca2+ ions. Some evidence for bilayer fusion is also observed, but only in the presence of the +6 oligo-amine analog.

A general platform for antibody purification utilizing engineered-micelles.

We introduce a new concept and potentially general platform for antibody (Ab) purification that does not rely on chromatography or specific ligands (e.g., Protein A); rather, it makes use of detergent aggregates capable of efficiently capturing Ab while rejecting hydrophilic impurities. Captured Ab are then extracted from the aggregates in pure form without co-extraction of hydrophobic impurities or aggregate dissolution. The aggregates studied consist of conjugated "Engineered-micelles" built from the nonionic detergent, Tween-20; bathophenanthroline, a hydrophobic metal chelator, and Fe2+ions. When tested in serum-free media with or without bovine serum albumin as additive, human or mouse IgGs were recovered with good overall yields (70-80%, by densitometry). Extraction of IgGs with 7 different buffers at pH 3.8 sheds light on possible interactions between captured Ab and their surrounding detergent matrix that lead to purity very similar to that obtained via Protein A or Protein G resins. Extracted Ab preserve their secondary structure, specificity and monomeric character as determined by circular dichroism, enzyme-linked immunosorbent assay and dynamic light scattering, respectively.

Action and Ion Mobility Spectroscopy of a Shortened Retinal Derivative.

The development of tandem ion mobility spectroscopy (IMS) known as IMS-IMS has led to extensive research into isomerizations of isolated molecules. Many recent works have focused on the retinal chromophore which is the optical switch used in animal vision. Here, we study a shortened derivative of the chromophore, which exhibits a rich IM spectrum allowing for a detailed analysis of its isomerization pathways, and show that the longer the chromophore is, the lower the barrier energies for isomerization are. Graphical Abstract.

Ultrafast Carotenoid to Retinal Energy Transfer in Xanthorhodopsin Revealed by the Combination of Transient Absorption and Two-Dimensional Electronic Spectroscopy.

By comparing two-dimensional electronic spectroscopy (2DES) and Pump-Probe (PP) measurements on xanthorhodopsin (XR) and reduced-xanthorhodopsin (RXR) complexes, the ultrafast carotenoid-to-retinal energy transfer pathway is revealed, at very early times, by an excess of signal amplitude at the associated cross-peak and by the carotenoid bleaching reduction due to its ground state recovery. The combination of the measured 2DES and PP spectroscopic data with theoretical modelling allows a clear identification of the main experimental signals and a comprehensive interpretation of their origin and dynamics. The remarkable velocity of the energy transfer, despite the non-negligible energy separation between the two chromophores, and the analysis of the underlying transport mechanism, highlight the role played by the ground state carotenoid vibrations in assisting the process.

Bacteriorhodopsin based non-magnetic spin filters for biomolecular spintronics.

We discuss spin injection and spin valves, which are based on organic and biomolecules, that offer the possibility to overcome some of the limitations of solid-state devices, which are based on ferromagnetic metal electrodes. In particular, we discuss spin filtering through bacteriorhodopsin in a solid state biomolecular spin valve that is based on the chirality induced spin selectivity (CISS) effect and shows a magnetoresistance of ∼2% at room temperature. The device is fabricated using a layer of bacteriorhodopsin (treated with n-octyl-thioglucoside detergent: OTG-bR) that is adsorbed on a cysteamine functionalized gold electrode and capped with a magnesium oxide layer as a tunneling barrier, upon which a Ni top electrode film is placed and used as a spin analyzer. The bR based spin valves show an antisymmetric magnetoresistance response when a magnetic field is applied along the direction of the current flow, whereas they display a positive symmetric magnetoresistance curve when a magnetic field is applied perpendicular to the current direction.

Cation Binding to Xanthorhodopsin: Electron Paramagnetic Resonance and Magnetic Studies.

Xanthorhodopsin (xR) is a member of the retinal protein family and acts as a proton pump in the cell membranes of the extremely halophilic eubacterium Salinibacter ruber. In addition to the retinal chromophore, xR contains a carotenoid, which acts as a light-harvesting antenna as it transfers 40% of the quanta it absorbs to the retinal. Our previous studies have shown that the CD and absorption spectra of xR are dramatically affected due to the protonation of two different residues. It is still unclear whether xR can bind cations. Electron paramagnetic resonance (EPR) spectroscopy used in the present study revealed that xR can bind divalent cations, such as Mn2+ and Ca2+, to deionized xR (DI-xR). We also demonstrate that xR can bind 1 equiv of Mn2+ to a high-affinity binding site followed by binding of ∼40 equiv in cooperative manner and ∼100 equiv of Mn2+ that are weakly bound. SQUID magnetic studies suggest that the high cooperative binding of Mn2+ cations to xR is due to the formation of Mn2+ clusters. Our data demonstrate that Ca2+ cations bind to DI-xR with a lower affinity than Mn2+, supporting the assumption that binding of Mn2+ occurs through cluster formation, because Ca2+ cations cannot form clusters in contrast to Mn2+.

Membrane Independence of Ultrafast Photochemistry in Pharaonis Halorhodopsin: Testing the Role of Bacterioruberin.

Ultrafast photochemistry of pharaonis halorhodopsin (p-HR) in the intact membrane of Natronomonas pharaonis has been studied by photoselective femtosecond pump-hyperspectral probe spectroscopy with high time resolution. Two variants of this sample were studied, one with wild-type retinal prosthetic groups and another after shifting the retinal absorption deep into the blue range by reducing the Schiff base linkage, and the results were compared to a previous study on detergent-solubilized p-HR. This comparison shows that retinal photoisomerization dynamics is identical in the membrane and in the solubilized sample. Selective photoexcitation of bacterioruberin, which is associated with the protein in the native membrane, in wild-type and reduced samples, demonstrates conclusively that unlike the carotenoids associated with some bacterial retinal proteins the carrotenoid in p-HR does not act as a light-harvesting antenna.

Membrane protein crystallization in micelles conjugated by nucleoside base-pairing: A different concept.

The dearth of high quality, three dimensional crystals of membrane proteins, suitable for X-ray diffraction analysis, constitutes a serious barrier to progress in structural biology. To address this challenge, we have developed a new crystallization medium that relies on the conjugation of surfactant micelles via base-pairing of complementary hydrophobic nucleosides. Base-pairs formed at the interface between micelles bring them into proximity with each other; and when the conjugated micelles contain a membrane protein, crystal nucleation centers can be stabilized, thereby promoting crystal growth. Accordingly, two hydrophobic nucleoside derivatives - deoxyguanosine (G) and deoxycytidine (C), each covalently bonded to a 10 carbon chain were synthesized and added to an aqueous solution containing octyl ß-d-thioglucopyranoside micelles. These hydrophobic nucleosides induced the formation of oil-rich globules after 2days incubation at 19°C or after a few hours in the presence of ammonium sulfate; however, phase separation was inhibited by 100mM GMP. The presence of the membrane protein bacteriorhodopsin in the conjugated - micellar dispersion resulted in the growth within the colorless globules of a variety of purple crystals, the color indicating a functional protein. On this basis, we suggest that conjugation of micelles via base-pair complementarity may provide significant assistance to the structural determination of integral membrane proteins.

Isotope Labeling Study of Retinal Chromophore Fragmentation.

Previous studies have shown that the gas-phase fragmentation of the retinal chromophore after S0-S1 photoexcitation results in a prominent fragment of mass 248 which cannot be explained by the cleavage of any single bond along the polyene chain. It was therefore theorized that the fragmentation mechanism involves a series of isomerizations and cyclization processes, and two mechanisms for these processes were suggested. Here we used isotope labeling MS-MS to provide conclusive support for the fragmentation mechanism suggested by Coughlan et al. (J. Phys. Chem. Lett. 2014, 5, 3195).

Spin-Controlled Photoluminescence in Hybrid Nanoparticles Purple Membrane System.

Spin-dependent photoluminescence (PL) quenching of CdSe nanoparticles (NPs) has been explored in the hybrid system of CdSe NP purple membrane, wild-type bacteriorhodopsin (bR) thin film on a ferromagnetic (Ni-alloy) substrate. A significant change in the PL intensity from the CdSe NPs has been observed when spin-specific charge transfer occurs between the retinal and the magnetic substrate. This feature completely disappears in a bR apo membrane (wild-type bacteriorhodopsin in which the retinal protein covalent bond was cleaved), a bacteriorhodopsin mutant (D96N), and a bacteriorhodopsin bearing a locked retinal chromophore (isomerization of the crucial C13═C14 retinal double bond was prevented by inserting a ring spanning this bond). The extent of spin-dependent PL quenching of the CdSe NPs depends on the absorption of the retinal, embedded in wild-type bacteriorhodopsin. Our result suggests that spin-dependent charge transfer between the retinal and the substrate controls the PL intensity from the NPs.

Light-controlled spin filtering in bacteriorhodopsin.

The role of the electron spin in chemistry and biology has received much attention recently owing to to the possible electromagnetic field effects on living organisms and the prospect of using molecules in the emerging field of spintronics. Recently the chiral-induced spin selectivity effect was observed by electron transmission through organic molecules. In the present study, we demonstrated the ability to control the spin filtering of electrons by light transmitted through purple membranes containing bacteriorhodopsin (bR) and its D96N mutant. The spin-dependent electrochemical cyclic voltammetry (CV) and chronoamperometric measurements were performed with the membranes deposited on nickel substrates. High spin-dependent electron transmission through the membranes was observed; however, after the samples were illuminated by 532 nm light, the spin filtering in the D96N mutant was dramatically reduced whereas the light did not have any effect on the wild-type bR. Beyond demonstrating spin-dependent electron transmission, this work also provides an interesting insight into the relationship between the structure of proteins and spin filtering by conducting electrons.

Engineered-membranes and engineered-micelles as efficient tools for purification of halorhodopsin and bacteriorhodopsin.

We describe two alternative and complementary purification methods for halorhodopsin and bacteriorhodopsin. The first relies on a unique form of detergent micelles which we have called engineered-micelles. These are specifically conjugated in the presence of [hydrophobic chelator:Fe(2+)] complexes and form detergent aggregates into which membrane proteins partition, but hydrophilic water-soluble proteins do not. The approach was tested on the membrane protein, bacteriorhodopsin (bR), with five non-ionic detergents (OG, OTG, NG, DM, and DDM), commonly used in purification and crystallization of membrane proteins, in combination with the commercially available bathophenanthroline or with one of the three synthesized phenanthroline derivatives (Phen-C10, Phen-C8 and Phen-C6). Our results show that bR is extracted efficiently (60-86%) and directly from its native membrane into diverse detergent aggregates with preservation of its native conformation, while 90-95% of an artificial contaminating background is excluded. For implementation of the second method, based on engineered-membranes, the use of detergents, which in some cases may produce protein denaturation, is not required at all. Protein-containing membranes are conjugated via the same hydrophobic [chelator:metal ion] complexes but maintain the membrane protein in its native bilayer environment throughout the process. This method is demonstrated on the membrane protein halorhodopsin from Natronomonas pharaonis (phR) and leads to good recovery yields (74-89%) and removal of >85% of artificial background impurities while preserving the native state of phR. The detailed structure of the hydrophobic chelator used has been found to have a marked effect on the purity and yield of both methods.

Origin of circular dichroism of xanthorhodopsin. A study with artificial pigments.

Xanthorhodopsin (xR) is a retinal protein that contains, in addition to the retinal moiety, a salinixanthin chromophore absorbing at 456, 486, and 520 nm [Balashov, S. P.; Science 2005, 309, 2061]. The CD spectrum of xR is very unique with a "conservative" character, containing negative and positive lobes and resembling the first derivative of the absorption spectrum [Balashov, S. P.; Biochemistry 2006, 45, 10998]. It was suggested that the CD spectrum is likely to be composed of several components and that the salinixanthin interacts closely with the retinal chromophore [Balashov, S. P.; Biochemistry 2006, 45, 10998; Imasheva, E. S.; Photochem. Photobiol. 2008, 84, 977; Lanyi, J. K.; Acta Bioenerg. 2008, 1777, 684; Smolensky, E.; Biochemistry 2009, 48, 8179; Smolensky Koganov, E.; Biochemistry 2013, 52, 1290]. In this work, we aim to further explore the nature and origin of the unique CD spectrum of xR. We follow the absorption and CD spectra at different pHs of wild-type (wt) xR and of artificial xR pigments, characterized by a shifted absorption maximum of the retinal chromophore, as well as their corresponding reduced retinal protonated Schiff base pigments. Our results revealed a protein residue (other than the protonated Schiff base counterion), for which protonation affects the CD spectrum by decreasing the negative lobe at ∼530 nm and the positive lobes at 478 and 455 nm, which might be due to elimination of excitonic coupling between the salinixanthin chromophores, although other possibilities cannot be completely excluded. This spectrum change occurs by the pH decreasing, even in artificial pigment where the absorption of the retinal pigment is significantly shifted from 570 to about 450 nm. The possible excitonic coupling between the salinixanthin chromophores and its contribution to the CD spectrum of xR were supported by a good fitting of the CD spectrum to conservative (excitonic) bands [Zsila, F.; Tetrahedron: Asymmetry 2001, 12, 3125; Zsila, F.; Tetrahedron: Asymmetry 2002, 13, 273]. We propose that the CD spectrum of xR consists of contributions from an excitonic coupling interaction between the salinixanthins chromophores located in different subunits of the 3D structure of xR, the chiral conformation of the salinixanthin within its binding site, and the contribution of the retinal chromophore to the negative lobe at around 550 nm.

Nanoscale electron transport and photodynamics enhancement in lipid-depleted bacteriorhodopsin monomers.

Potential future use of bacteriorhodopsin (bR) as a solid-state electron transport (ETp) material requires the highest possible active protein concentration. To that end we prepared stable monolayers of protein-enriched bR on a conducting HOPG substrate by lipid depletion of the native bR. The ETp properties of this construct were then investigated using conducting probe atomic force microscopy at low bias, both in the ground dark state and in the M-like intermediate configuration, formed upon excitation by green light. Photoconductance modulation was observed upon green and blue light excitation, demonstrating the potential of these monolayers as optoelectronic building blocks. To correlate protein structural changes with the observed behavior, measurements were made as a function of pressure under the AFM tip, as well as humidity. The junction conductance is reversible under pressure changes up to ∼300 MPa, but above this pressure the conductance drops irreversibly. ETp efficiency is enhanced significantly at >60% relative humidity, without changing the relative photoactivity significantly. These observations are ascribed to changes in protein conformation and flexibility and suggest that improved electron transport pathways can be generated through formation of a hydrogen-bonding network.

Spin-dependent electron transmission through bacteriorhodopsin embedded in purple membrane.

Spin-dependent photoelectron transmission and spin-dependent electrochemical studies were conducted on purple membrane containing bacteriorhodopsin (bR) deposited on gold, aluminum/aluminum-oxide, and nickel substrates. The result indicates spin selectivity in electron transmission through the membrane. Although the chiral bR occupies only about 10% of the volume of the membrane, the spin polarization found is on the order of 15%. The electrochemical studies indicate a strong dependence of the conduction on the protein's structure. Denaturation of the protein causes a sharp drop in the conduction through the membrane.

Purification of a membrane protein with conjugated engineered micelles.

A novel method for purifying membrane proteins is presented. The approach makes use of engineered micelles composed of a nonionic detergent, ß-octylglucoside, and a hydrophobic metal chelator, bathophenanthroline. Via the chelators, the micelles are specifically conjugated, i.e., tethered, in the presence of Fe(2+) ions, thereby forming micellar aggregates which provide the environment for separation of lipid-soluble membrane proteins from water-soluble proteins. The micellar aggregates (here imaged by cryo-transmission electron microscopy) successfully purify the light driven proton pump, bacteriorhodopsin (bR), from E. coli lysate. Purification takes place within 15 min and can be performed both at room temperature and at 4 °C. More than 94% of the water-soluble macromolecules in the lysate are excluded, with recovery yields of the membrane protein ranging between 74% and 85%. Since this approach does not require precipitants, high concentrations of detergent to induce micellar aggregates, high temperature, or changes in pH, it is suggested that it may be applied to the purification of a wide variety of membrane proteins.

Probing ultrafast photochemistry of retinal proteins in the near-IR: bacteriorhodopsin and anabaena sensory rhodopsin vs retinal protonated Schiff base in solution.

Photochemistry of bacteriorhodopsin (bR), anabaena sensory rhodopsin (ASR), and all-trans retinal protonated Schiff base (RPSB) in ethanol is followed with femtosecond pump-hyperspectral near-IR (NIR) probe spectroscopy. This is the first systematic probing of retinal protein photochemistry in this spectral range. Stimulated emission of the proteins is demonstrated to extend deep into the NIR, and to decay on the same characteristic time scales previously determined by visible probing. No signs of a transient NIR absorption band above λpr > 1.3 µm, which was recently reported and is verified here for the RPSB in solution, is observed in either protein. This discrepancy demonstrates that the protein surroundings change photochemical traits of the chromophore significantly, inducing changes either in the energies or couplings of photochemically relevant electronic excited states. In addition, low-frequency and heavily damped spectral modulations are observed in the NIR signals of all three systems up to 1.4 µm. By background subtraction and Fourier analysis they are shown to resemble wave packet signatures in the visible, stemming from multiple vibrational modes and by analogy are assigned to torsional wave packets in the excited state of the retinal chromophore. Differences in the vibrational frequencies between the three samples and the said discrepancy in transient spectra are discussed in terms of opsin effects on the RPSB electronic structure.

Engineered-membranes: a novel concept for clustering of native lipid bilayers.

A strategy for clustering of native lipid membranes is presented. It relies on the formation of complexes between hydrophobic chelators embedded within the lipid bilayer and metal cations in the aqueous phase, capable of binding two (or more) chelators simultaneously Fig. 1. We used this approach with purple membranes containing the light driven proton pump protein bacteriorhodopsin (bR) and showed that patches of purple membranes cluster into mm sized aggregates and that these are stable for months when incubated at 19°C in the dark. The strategy may be general since four different hydrophobic chelators (1,10-phenanthroline, bathophenanthroline, Phen-C10, and 8-hydroxyquinoline) and various divalent cations (Ni(2+), Zn(2+), Cd(2+), Mn(2+), and Cu(2+)) induced formation of membrane clusters. Moreover, the absolute requirement for a hydrophobic chelator and the appropriate metal cations was demonstrated with light and atomic force microscopy (AFM); the presence of the metal does not appear to affect the functional state of the protein. The potential utility of the approach as an alternative to assembled lipid bilayers is suggested.