Semi-synthetic Rab proteins as tools for studying intermolecular interactions

Rab GTPases play a key role in the regulation of membrane traffic. Posttranslational geranylgeranylation is critical for their biological activity and is conferred by a Rab geranylgeranyl transferase (RabGGTase). To study the interactions between Rab proteins and RabGGTase, we used in vitro ligation methodology to generate a fluorescent semi-synthetic Rab7 protein. The obtained protein was functionally active and was used to demonstrate a micromolar affinity interaction of Rab7 with the RabGGTase in the absence of Rab escort protein (REP). This finding is consistent with an earlier proposed model according to which RabGGTase possesses two independent weak binding sites for REP and Rab proteins.     

Light‐induced pigment degradation in leaves and ripening fruits studied in situ with reflectance spectroscopy

Pigment breakdown mediated by activated oxygen species is a consequence and a general symptom of oxidative stress and injury to plants. We have attempted to estimate the patterns of pigment bleaching and follow pigment susceptibility to irradiation as related to the process of senescence/ripening. Light‐induced pigment breakdown was studied in situ in the leaves of a shade‐requiring plant, wax flower ( Hoya carnosa R. Br.), as well as in apple ( Malus domestica Borlh. cv. Zhigulevskoe) and lemon ( Citrus limon Burm. cv. Pavlovsky) fruits, using reflectance spectroscopy. It was found that the sensitivity of plant pigments to photobleaching increases as ripening progresses in lemon fruit. Kinetic analysis showed that in all systems a rapid breakdown of the pigment occurs after a lag‐phase. The signature analysis revealed a common pattern of chlorophyll and carotenoid changes, but degradation of the individual pigments was found to be inhomogeneous. Both in lemon and apple fruits a decrease in reflectance in the band of carotenoid absorption preceded pigment photodestruction. In the fruits, the bulk of chlorophyll b and the long‐wavelength chlorophyll a forms were degraded at early stages of the process whereas the breakdown of both chlorophylls in H. carnosa leaves was more synchronous. Prolonged irradiation induced bleaching of the main chlorophyll a band with maximum at 678 nm in the difference spectra, as well as carotenoids. Some features of reflectance spectra in the bands of chlorophyll and carotenoid absorption were found to be suitable for the differentiation of photo‐induced pigment breakdown from the transformation of the pigments taking place during senescence.     

Bright far-red fluorescent protein for whole-body imaging

For deep imaging of animal tissues, the optical window favorable for light penetration is in near-infrared wavelengths, which requires proteins with emission spectra in the far-red wavelengths. Here we report a far-red fluorescent protein, named Katushka, which is seven- to tenfold brighter compared to the spectrally close HcRed or mPlum, and is characterized by fast maturation as well as a high pH-stability and photostability. These unique characteristics make Katushka the protein of choice for visualization in living tissues. We demonstrate superiority of Katushka for whole-body imaging by direct comparison with other red and far-red fluorescent proteins. We also describe a monomeric version of Katushka, named mKate, which is characterized by high brightness and photostability, and should be an excellent fluorescent label for protein tagging in the far-red part of the spectrum.     

Channel-sizing experiments in multichannel bilayers

The possibility of obtaining information about the radius of high and low conductance states of channels in multichannel membranes was tested experimentally. In spite of the interference of non-electrolytes on the numbers of channels that appeared in the membrane, the non-electrolyte-exclusion method was successfully adapted to multichannel bilayers to estimate the radius of the larger opening of the low conductance state of the channel induced by Staphylococcus aureus alpha-toxin. At the pH used, the channel transition to a low conductance state was accompanied by a decrease of the opening radius from 1.3 +/- 0.2 nm to 0.9 +/- 0.1 nm. The determination criteria for maximum size of a channel opening when using the non-electrolyte exclusion method is discussed.     

A crystallographic study of bright far-red fluorescent protein mKate reveals pH-induced cis-trans isomerization of the chromophore

The far-red fluorescent protein mKate (lambda(ex), 588 nm; lambda(em), 635 nm; chromophore-forming triad Met(63)-Tyr(64)-Gly(65)), originating from wild-type red fluorescent progenitor eqFP578 (sea anemone Entacmaea quadricolor), is monomeric and characterized by the pronounced pH dependence of fluorescence, relatively high brightness, and high photostability. The protein has been crystallized at a pH ranging from 2 to 9 in three space groups, and four structures have been determined by x-ray crystallography at the resolution of 1.75-2.6 A. The pH-dependent fluorescence of mKate has been shown to be due to reversible cis-trans isomerization of the chromophore phenolic ring. In the non-fluorescent state at pH 2.0, the chromophore of mKate is in the trans-isomeric form. The weakly fluorescent state of the protein at pH 4.2 is characterized by a mixture of trans and cis isomers. The chromophore in a highly fluorescent state at pH 7.0/9.0 adopts the cis form. Three key residues, Ser(143), Leu(174), and Arg(197) residing in the vicinity of the chromophore, have been identified as being primarily responsible for the far-red shift in the spectra. A group of residues consisting of Val(93), Arg(122), Glu(155), Arg(157), Asp(159), His(169), Ile(171), Asn(173), Val(192), Tyr(194), and Val(216), are most likely responsible for the observed monomeric state of the protein in solution.     

Effects of light and nitrogen starvation on the content and composition of carotenoids of the green microalga <Emphasis Type="Italic">Parietochloris incisa</Emphasis>

The changes in pigment content and composition of the unicellular alga Parietochloris incisa comb. nov (Trebouxiophyceae, Chlorophyta) were studied. This alga is unique in its ability to accumulate high amounts of arachidonic acid in the cell during cultivation under different irradiances and nitrogen availability in the medium. Under low irradiance of 35 μE/(m₂ s) photosynthetically active radiation the P. incisa cultures possessed slow growth and a relatively low carotenoid-to-chlorophyll ratio. At higher irradiances (200 and 400 μE/(m₂ s)) on complete medium, the alga displayed higher growth rate and an increase in the carotenoid content, especially that of β-carotene and lutein. Both on nitrogen-free (regardless of illumination intensity) and nitrogen-replete medium (under high light), a considerable increase in the ratio of carotenoid and chlorophyll contents was recorded. Predominant accumulation of xanthophylls took place in thylakoid membranes, whereas β-carotene deposition occurred mainly in the cytoplasmic lipid globules (oil bodies); lower amounts of carotenoids were accumulated in the absence of nitrogen. Under high light and nitrogen-deficiency conditions, an increase in violaxanthin de-epoxidation and nonphotochemical quenching was recorded together with a decline in variable chlorophyll fluorescence (F _v/F _m) level. A possible photoprotective role of carotenoids in adaptation of P. incisa to high light under nitrogen starvation conditions is discussed.     

Destruction of Pigments and Ultrastructural Changes in Cyanobacteria during Photodamage

Sequential stages of pigment degradation and ultrastructural changes were examined in cyanobacteria Anabaena variabilis ATCC 29413, Synechococcus sp. PCC 6301 (Anacystis nidulans) and S. elongatus B-267 during irradiation of cell suspensions with high-intensity light. Early manifestations of photooxidative destruction were evident as profound changes in ultrastructure of thylakoids; in A. variabilis these changes appeared even before bleaching of pigments. Concomitant to these alterations, the cytoplasmic matrix turned homogenous and the nucleoid was subject to degradation, while ultrastructural changes of cytoplasmic membrane and cell walls became evident in some species. In A. variabilis these changes were related to a subsequent autolysis of cells. Synechococcus strains demonstrated comparatively high resistance to irradiation. The experimental data were compared with previously described behavior of the same species of cyanobacteria cultured under photooxidative conditions. This comparison revealed principal similarity and species-specific features in the destructive changes of thylakoids and other cell components of cyanobacterial cells.     

Does a leaf absorb radiation in the near infrared (780–900 nm) region? A new approach to quantifying optical reflection,absorption and transmission of leaves

Photosynthesis Research - The following question is addressed here: do healthy leaves absorb, as the spectra published over the last 50 years indicate, some 5–20% of incident radiation in the...     

Diversity and phylogeny of insect trypanosomatids based on small subunit rRNA genes: polyphyly of Leptomonas and Blastocrithidia

With the aim of further investigating phylogenetic relationships in insect trypanosomatids, we have determined the sequences of small subunit rRNA genes from ten isolates, which were originally classified as Leptomonas, Blastocrithidia, and Wallaceina based on their morphology in the hosts. The inferred maximum likelihood, parsimony, and distance trees indicate that the Leptomonas and Blastocrithidia are polyphyletic, and confirm the polyphyly of Herpetomonas and Crithidia. Blastocrithidia triatoma and Leptomonas collosoma were among the earliest branching lineages among the insect trypanosomatids, while most other isolates were found within a closely related terminal clade, which also included Crithidia fasciculata. This analysis has clearly demonstrated that the morphological classification system of insect trypanosomatids does not always reflect their genetic affinities warranting its revision in the future.     

Lumen geometry of ion channels formed by Vibrio cholerae EL Tor cytolysin elucidated by nonelectrolyte exclusion

Vibrio cholerae EL Tor cytolysin, a water-soluble protein with a molecular mass of 63 kDa, forms small pores in target cell membranes. In this communication, planar lipid bilayers under voltage clamp conditions were used to investigate the geometric properties of the pores. It was established that all cytolysin channels were inserted into membranes with the same orientation. Sharp asymmetry in the I-V curve of fully open cytolysin channels persisting at high electrolyte concentrations indicated asymmetry in the geometry of the channel lumen. Using the nonelectrolyte exclusion method, evidence was obtained that the cis opening of the channel had a larger diameter (< or = 1.9 nm) than the trans opening (< or = 1.6 nm). The channel lumen appeared constricted, with a diameter of < or = 1.2 nm. Cup-shaped lumen geometry was deduced for both channel openings, which appeared to be connected to each other via a central narrow part. The latter contributed significantly to the total electrical resistance and determined the discontinuous character of channel filling with nonelectrolytes. Comparisons of the properties of pores formed by cytolysins of two V. cholerae biotypes (EL Tor and non-O1) indicated that the two ion channels possessed a similar geometry.