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

Evaluation of current tropospheric mapping functions by Deep Space Network very long baseline interferometry

To compare the validity of current algorithms that map zenith tropospheric delay to arbitrary elevation angles, 10 different tropospheric mapping functions are used to analyze the current data base of Deep Space Network Mark 3 intercontinental very long baseline interferometric (VLBI) data. This analysis serves as a stringent test because of the high proportion of low-elevation observations necessitated by the extremely long baselines. Postfit delay and delay-rate residuals are examined, as well as the scatter of baseline lengths about the time-linear model that characterizes tectonic motion. Among the functions that utilize surface meteorological data as input parameters, the Lanyi 1984 mapping shows the best performance both for residuals and baselines, through the 1985 Davis function is statistically nearly identical. The next best performance is shown by the recent function of Niell, which is based on an examination of global atmospheric characteristics as a function of season and uses no weather data at the time of the measurements. The Niell function shows a slight improvement in residuals relative to Lanyi, but also an increase in baseline scatter that is significant for the California-Spain baseline. Two variants of the Chao mapping function, as well as the Chao tables used with the interpolation algorithm employed in the Orbit Determination Program software, show substandard behavior for both VLBI residuals and baseline scatter. The length of the California-Australia baseline (10,600 km) in the VLBI solution can vary by as much as 5 to 10 cm for the 10 mapping functions

The Celestial Reference Frame at 24 and 43 GHz. II. Imaging

We have measured the sub-milli-arcsecond structure of 274 extragalactic sources at 24 and 43 GHz in order to assess their astrometric suitability for use in a high frequency celestial reference frame (CRF). Ten sessions of observations with the Very Long Baseline Array have been conducted over the course of $\sim$5 years, with a total of 1339 images produced for the 274 sources. There are several quantities that can be used to characterize the impact of intrinsic source structure on astrometric observations including the source flux density, the flux density variability, the source structure index, the source compactness, and the compactness variability. A detailed analysis of these imaging quantities shows that (1) our selection of compact sources from 8.4 GHz catalogs yielded sources with flux densities, averaged over the sessions in which each source was observed, of about 1 Jy at both 24 and 43 GHz, (2) on average the source flux densities at 24 GHz varied by 20%-25% relative to their mean values, with variations in the session-to-session flux density scale being less than 10%, (3) sources were found to be more compact with less intrinsic structure at higher frequencies, and (4) variations of the core radio emission relative to the total flux density of the source are less than 8% on average at 24 GHz. We conclude that the reduction in the effects due to source structure gained by observing at higher frequencies will result in an improved CRF and a pool of high-quality fiducial reference points for use in spacecraft navigation over the next decade.Comment: 63 pages, 18 figures, 6 tables, accepted by the Astronomical Journa

Progress in Measurements of the Gravitational Bending of Radio Waves Using the VLBA

We have used the Very Long Baseline Array (VLBA) at 43, 23 and 15 GHz to measure the solar gravitational deflection of radio waves among four radio sources during an 18-day period in October 2005. Using phase-referenced radio interferometry to fit the measured phase delay to the propagation equation of the parameterized post-Newtonian (PPN) formalism, we have determined the deflection parameter gamma = 0.9998 +/- 0.0003$ (68% confidence level), in agreement with General Relativity. The results come mainly from 43 GHz observations where the refraction effects of the solar corona were negligible beyond 3 degrees from the sun. The purpose of this experiment is three-fold: to improve on the previous results in the gravitational bending experiments near the solar limb; to examine and evaluate the accuracy limits of terrestrial VLBI techniques; and to determine the prospects and outcomes of future experiments. Our conclusion is that a series of improved designed experiments with the VLBA could increase the presented accuracy by at least a factor of 4.Comment: 22 pages, 3 figure

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

Excited-state reaction dynamics of bacteriorhodopsin studied by femtosecond spectroscopy

The photodynamics of bacteriorhodopsin were studied by transient absorption and gain measurements after excitation with femtosecond pulses at 620 nm. With probing pulses at longer wavelengths (λ > 770 nm) the previously reported formation of the J intermediate (with a time constant of 500±100 fs) was confirmed. With probing pulses around 700 nm, a faster process with a relaxation time of 200±70 fs was observed. The data analysis strongly suggests that this kinetic constant describes the reactive motion of the polyatomic molecule on its excited-state potential energy surface, i.e. one observes directly the incipient isomerization of the retinal molecule. The minimum of the S1 potential energy surface reached in 200 fs lies approximately 13300 cm−1 above the ground state of bacteriorhodopsin and from this minimum the intermediate J is formed with a time constant of 500 fs

Rotational Alignment Altered by Source Position Correlations

In the construction of modern Celestial Reference Frames (CRFs) the overall rotational alignment is only weakly constrained by the data. Therefore, common practice has been to apply a 3-dimensional No-Net-Rotation (NNR) constraint in order to align an under-construction frame to the ICRF. We present evidence that correlations amongst source position parameters must be accounted for in order to properly align a CRF at the 5-10 (mu)as level of uncertainty found in current work. Failure to do so creates errors at the 10-40 (mu)as level

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

Relocation of Advanced Water Vapor Radiometer 1 to Deep Space Station 55

In June of 2004, the Advanced Water Vapor Radiometer (AWVR) unit no. 1 was relocated to the Deep Space Station (DSS) 55 site in Madrid, Spain, from DSS 25 in Goldstone, California. This article summarizes the relocation activity and the subsequent operation and data acquisition. This activity also relocated the associated Microwave Temperature Profiler (MTP) and Surface Meteorology (SurfMET) package that collectively comprise the Cassini Media Calibration System (MCS)