Archaebacterial rhodopsin sequences: Implications for evolution

It was proposed over 10 years ago that the archaebacteria represent a separate kingdom which diverged very early from the eubacteria and eukaryotes. It follows that investigations of archaebacterial characteristics might reveal features of early evolution. So far, two genes, one for bacteriorhodopsin and another for halorhodopsin, both from Halobacterium halobium, have been sequenced. We cloned and sequenced the gene coding for the polypeptide of another one of these rhodopsins, a halorhodopsin in Natronobacterium pharaonis. Peptide sequencing of cyanogen bromide fragments, and immuno-reactions of the protein and synthetic peptides derived from the C-terminal gene sequence, confirmed that the open reading frame was the structural gene for the pharaonis halorhodopsin polypeptide. The flanking DNA sequences of this gene, as well as those of other bacterial rhodopsins, were compared to previously proposed archaebacterial consensus sequences. In pairwise comparisons of the open reading frame with DNA sequences for bacterio-opsin and halo-opsin from Halobacterium halobium, silent divergences were calculated. These indicate very considerable evolutionary distance between each pair of genes, even in the dame organism. In spite of this, three protein sequences show extensive similarities, indicating strong selective pressures

Halide binding by the purified halorhodopsin chromoprotein. II. New chloride-binding sites revealed by 35Cl NMR

Halorhodopsin is a light-driven chloride pump in the cell membrane of Halobacterium halobium. Recently, a polypeptide of apparent Mr = 20,000 has been purified that contains the halorhodopsin chromophore. Here we use 35Cl NMR to show that the purified chromoprotein possesses two previously unknown classes of chloride-binding sites. One class exhibits a low affinity (KD much greater than 1 M) for chloride and bromide. The second class exhibits a higher affinity (KD = 110 ± 50 mM) for chloride and also binds other anions according to the affinity series I-, SCN- greater than Br-, NO-3 greater than Cl- greater than F- , citrate. Both classes of NMR site remain intact at pH 11, indicating that the essential positive charges are provided by arginine. Also, both classes are unaffected by bleaching, suggesting that the sites are not in the immediate vicinity of the halorhodopsin chromophore. Although the chromoprotein also appears to contain the chloride- transport site (Steiner, M., Oesterhelt, D., Ariki, M., and Lanyi, J. K. (1984) J. Biol. Chem. 259, 2179-2184), this site was not detected by 35Cl NMR, suggesting that the transport site is in the interior of the protein where it is sampled slowly by chloride in the medium. It is proposed that the purified chromoprotein possesses a channel leading from the medium to the transport site and that the channel contains the high affinity NMR site which facilitates the migration of chloride between the medium and the transport site. We have also used 35Cl NMR to study chloride binding to purified monomeric bacteriorhodopsin; however, this protein contains no detectable chloride-binding sites

Anion binding to the chloride pump, halorhodopsin, and its implications for the transport mechanism

AbstractThe light-driven chloride pump, halorhodopsin, binds and transports chloride across the membrane, and to a lesser extent nitrate. Binding and transport kinetics, and resonance Raman spectra of the retinal Schiff base, with these anions suggest the existence of two mutually exclusive binding sites. One of these may be the uptake site, and the other the release site during the transport. Plausible locations can be suggested for these sites, because halorhodopsin is a small protein with few buried positively charged residues, and the primary structure of a second pigment with similar function has recently become available for comparison

Characterization of the Proton-Transporting Photocycle of Pharaonis Halorhodopsin

AbstractThe photocycle of pharaonis halorhodopsin was investigated in the presence of 100mM NaN3 and 1M Na2SO4. Recent observations established that the replacement of the chloride ion with azide transforms the photocycle from a chloride-transporting one into a proton-transporting one. Kinetic analysis proves that the photocycle is very similar to that of bacteriorhodopsin. After K and L, intermediate M appears, which is missing from the chloride-transporting photocycle. In this intermediate the retinal Schiff base deprotonates. The rise of M in halorhodopsin is in the microsecond range, but occurs later than in bacteriorhodopsin, and its decay is more accentuated multiphasic. Intermediate N cannot be detected, but a large amount of O accumulates. The multiphasic character of the last step of the photocycle could be explained by the existence of a HR′ state, as in the chloride photocycle. Upon replacement of chloride ion with azide, the fast electric signal changes its sign from positive to negative, and becomes similar to that detected in bacteriorhodopsin. The photocycle is enthalpy-driven, as is the chloride photocycle of halorhodopsin. These observations suggest that, while the basic charge translocation steps become identical to those in bacteriorhodopsin, the storage and utilization of energy during the photocycle remains unchanged by exchanging chloride with azide

Trimeric mutant bacteriorhodopsin, D85N, shows a monophasic CD spectrum

AbstractThe structure of mutant bacteriorhodopsin (bR), D85N, was examined by CD and X-ray diffraction at pH 7. The absorption maximum of D85N at pH 7 is located at 605 nm, which is similar to the acid-blue form of wild-type bR. D85N shows a monophasic CD band, the maximum of which is at 575 nm, although the crystalline arrangement and the trimeric structure is maintained. The acid-blue form of wild-type bR shows a biphasic CD despite the similarity in absorption spectra

Protein Conformational Changes in the Bacteriorhodopsin Photocycle: Comparison of Findings from Electron and X-Ray Crystallographic Analyses

Light-driven conformational changes in the membrane protein bacteriorhodopsin have been studied extensively using X-ray and electron crystallography, resulting in the deposition of >30 sets of coordinates describing structural changes at various stages of proton transport. Using projection difference Fourier maps, we show that coordinates reported by different groups for the same photocycle intermediates vary considerably in the extent and nature of conformational changes. The different structures reported for the same intermediate cannot be reconciled in terms of differing extents of change on a single conformational trajectory. New measurements of image phases obtained by cryo-electron microscopy of the D96G/F171C/F219L triple mutant provide independent validation for the description of the large protein conformational change derived at 3.2 Å resolution by electron crystallography of 2D crystals, but do not support atomic models for light-driven conformational changes derived using X-ray crystallography of 3D crystals. Our findings suggest that independent determination of phase information from 2D crystals can be an important tool for testing the accuracy of atomic models for membrane protein conformational changes

High-precision Measurements of Ionospheric TEC Gradients with the Very Large Array VHF System

We have used a relatively long, contiguous VHF observation of a bright cosmic radio source (Cygnus A) with the Very Large Array (VLA) to demonstrate the capability of this instrument to study the ionosphere. This interferometer, and others like it, can observe ionospheric total electron content (TEC) fluctuations on a much wider range of scales than is possible with many other instruments. We have shown that with a bright source, the VLA can measure differential TEC values between pairs of antennas (delta-TEC) with an precision of 0.0003 TECU. Here, we detail the data reduction and processing techniques used to achieve this level of precision. In addition, we demonstrate techniques for exploiting these high-precision delta-TEC measurements to compute the TEC gradient observed by the array as well as small-scale fluctuations within the TEC gradient surface. A companion paper details specialized spectral analysis techniques used to characterize the properties of wave-like fluctuations within this data.Comment: accepted for publication in Radio Scienc

Membrán fehérjék szerkezete és működése közti kapcsolat vizsgálata = The study of the connection between the structure and function of membrane proteins

A 2005 januárjában kezdődőtt négy éves OTKA támogatással a membrán fehérjék szerkezete és működése közti kapcsolatok tisztázására folytatott kutatás két fő irányba haladt. Az egyik a membrán fehérjék és itt főleg a fényhajtotta retinál fehérjék működésével kapcsolatos kutatásokra összpontosított. Ennek során nemcsak a jól ismert retinál fehérjék a bakteriorodopszin, vagy halorodopszin, hanem újonnan felfedezett fehérjék, mint például a xantorodopsin és leptospheria rodopszin működését is tanulmányoztuk, abszorpciókinetikai mérésekkel. A retinál fehérjék kutatásával kapcsolatosan, az OTKA pályázat támogatásával, nyolc cikket közöltünk. Hasonló kinetikai méréseket végeztünk fotoszintetikus rekciócentrumok és szén nanocsövek keverékén, ami ígéretes biotechnologiai anyagnak bizonyúl. A reakciócentrumokkal kapcsolatban két OTKA támogatta cikkünk jelent meg A másik fő kutatási irány a fehérjék, membránok és élő sejtek morfológiai és mechanikai vizsgálata atomerő mikroszkóppal. Természetes és mesterséges membránokon is végeztünk méréseket. Amíg különböző letapogatási technikákkal nagyfelbontású képet alkottunk a tanulmányozott objektumok felszínéről, addig erőméréssel sikerült meghatározni a tanulmányozott minta Young moduluszát és ennek a változását különböző külső körülmények között. Egy teljesen új technikát dolgoztunk ki retinál fehérjék konformációváltozásának atomerőmikroszkópos megfigyelésére. Az elmúlt négy évben ezekkel a mérésekkel kapcsolatban nyolc OTKA által támogatott cikkelt közöltünk. | Beginning in 2005 the four years OTKA supported study of the relation between the structure and function of membrane proteins was conducted in two main directions. One research direction concentrates on the function of the membrane proteins and mainly on the function of the light activated retinal proteins. In this study not only the well known bacteriorhodopsin and halorhodopsin, but some newly discovered proteins, such as the xantorhodopsin and leptospheria rhodopsin were studied by absorption kinetic measurements. Related to this research, eight papers supported by the OTKA were published. Similar kinetic measurements were performed on the mixture of photosynthetic reaction centers and carbon nanotubes. This material is considered as a promising biotechnological material. The results of the reaction center research were published in two OTKA supported papers. The second main research direction was the morphological and mechanical study of the proteins, membranes and living cells by atomic force microscopy. We performed measurements on both artificial and natural membranes. With different scanning techniques high resolution images of the studied object surface were recorded. With force measurement it was possible to determine the Young?s module of the sample and its dependence from the external conditions. A new technique was developed to study the conformational change of the retinal proteins with the atomic force microscope. During the OTKA supported four years, related to this field, eight papers were published

What works to support carers of older people and older carers? an international evidence map of interventions and outcomes

\ua9 The Author(s) 2024.Background: Unpaid carers of older people, and older unpaid carers, experience a range of adverse outcomes. Supporting carers should therefore be a public health priority. Our understanding of what works to support carers could be enhanced if future evaluations prioritise under-researched interventions and outcomes. To support this, we aimed to: map evidence about interventions to support carers, and the outcomes evaluated; and identify key gaps in current evidence. Methods: Evidence gap map review methods were used. Searches were carried out in three bibliographic databases for quantitative evaluations of carer interventions published in OECD high-income countries between 2013 and 2023. Interventions were eligible if they supported older carers (50 + years) of any aged recipient, or any aged carers of older people (50 + years). Findings: 205 studies reported across 208 publications were included in the evidence map. The majority evaluated the impact of therapeutic and educational interventions on carer burden and carers’ mental health. Some studies reported evidence about physical exercise interventions and befriending and peer support for carers, but these considered a limited range of outcomes. Few studies evaluated interventions that focused on delivering financial information and advice, pain management, and physical skills training for carers. Evaluations rarely considered the impact of interventions on carers’ physical health, quality of life, and social and financial wellbeing. Very few studies considered whether interventions delivered equitable outcomes. Conclusion: Evidence on what works best to support carers is extensive but limited in scope. A disproportionate focus on mental health and burden outcomes neglects other important areas where carers may need support. Given the impact of caring on carers’ physical health, financial and social wellbeing, future research could evaluate interventions that aim to support these outcomes. Appraisal of whether interventions deliver equitable outcomes across diverse carer populations is critical

Directed evolution of a far-red fluorescent rhodopsin

Microbial rhodopsins are a diverse group of photoactive transmembrane proteins found in all three domains of life. A member of this protein family, Archaerhodopsin-3 (Arch) of halobacterium Halorubrum sodomense, was recently shown to function as a fluorescent indicator of membrane potential when expressed in mammalian neurons. Arch fluorescence, however, is very dim and is not optimal for applications in live-cell imaging. We used directed evolution to identify mutations that dramatically improve the absolute brightness of Arch, as confirmed biochemically and with live-cell imaging (in Escherichia coli and human embryonic kidney 293 cells). In some fluorescent Arch variants, the pK_a of the protonated Schiff-base linkage to retinal is near neutral pH, a useful feature for voltage-sensing applications. These bright Arch variants enable labeling of biological membranes in the far-red/infrared and exhibit the furthest red-shifted fluorescence emission thus far reported for a fluorescent protein (maximal excitation/emission at ∼620 nm/730 nm)