Production of yeastolates for uniform stable isotope labelling in eukaryotic cell culture.

Preparation of stable isotope-labelled yeastolates opens up ways to establish more cost-effective stable isotope labelling of biomolecules in insect and mammalian cell lines and hence to employ higher eukaryotic cell lines for stable isotope labelling of complex recombinant proteins. Therefore, we evaluated several common yeast strains of the Saccharomycetoideae family as a source of high-quality, non-toxic yeastolates with the major aim to find a primary amino acid source for insect and mammalian cell culture that would allow cost-effective uniform stable isotope labelling (13C, 15N). Strains of the facultative methylotrophic yeasts Pichia pastoris and Hansenula polymorpha (Pichia angusta) as well as a strain of the baker's yeast Saccharomyces cerevisiae were compared as a source of yeastolate with respect to processing, recovery and ability to sustain growth of insect and mammalian cell lines. The best growth-supporting yeastolates were prepared via autolysis from yeast obtained from fed-batch cultures that were terminated at the end of the logarithmic growth phase. Yeastolates obtained from H. polymorpha performed well as a component of insect cell cultures, while yeastolates from S. cerevisiae and H. polymorpha both yielded good results in mammalian cell cultures. Growth of yeasts in Heine's medium without lactic acid allows relatively low concentrations of 13C and 15N sources, and this medium can be reused several times with supplementation of the 13C source only.

Differences in the pharmacological activation of visual opsins.

Opsins, like many other G-protein-coupled receptors, sustain constitutive activity in the absence of ligand. In partially bleached rods and cones, opsin's activity closes cGMP-gated channels and produces a state of "pigment adaptation" with reduced sensitivity to light and accelerated flash response kinetics. The truncated retinal analogue, beta-ionone, further desensitizes partially bleached green-sensitive salamander rods, but enables partially bleached red-sensitive cones to recover dark-adapted physiology. Structural differences between rod and cone opsins were proposed to explain the effect. Rods and cones, however, also contain different transducins, raising the possibility that G-protein type determines the photoreceptor-specific effects of beta-ionone. To test the two hypotheses, we applied beta-ionone to partially bleached blue-sensitive rods and cones of salamander, two cells that couple the same cone-like opsin to either rod or cone transducin, respectively. Immunocytochemistry confirmed that all salamander rods contain one form of transducin, whereas all cones contain another. beta-Ionone enhanced pigment adaptation in blue-sensitive rods, but it also did so in blue- and UV-sensitive cones. Furthermore, all recombinant salamander rod and cone opsins, with the exception of the red-sensitive cone opsin, activated rod transducin upon the addition of beta-ionone. Thus opsin structure determines the identity of beta-ionone as an agonist or an inverse agonist and in that respect distinguishes the red-sensitive cone opsin from all others.

The photoreceptive capacity of the developing pineal gland and eye of the golden hamster (Mesocricetus auratus).

Anatomical and physiological studies have suggested that the pineal gland of neonatal mammals has a photoreceptive capacity. Using the golden hamster (Mesocricetus auratus) as our model, we applied biochemical approaches to look for a functional photopigment within the pineal during early development. Immunocytochemistry and enzyme-linked immunosorbent assay (ELISA) were used to localize and quantify opsin, and high-performance liquid chromatography (HPLC) to identify photopigment chromophore (11-cis and all-trans retinaldehyde) in the developing eye and pineal. For HPLC analysis, retinaldehydes were converted to their corresponding retinoid oximes. Eluted retinoids were identified by comparison with standard vitamin A1 retinoid oxime isomers on the basis of relative elution sequence and characteristic absorbance spectra. Both immunocytochemistry and ELISA suggested an increase in the opsin content of the pineal during the first week of life. In the eye, 11-cis retinaldehyde was first detected between days 3 and 5 after birth. In three separate extractions, and using a considerable excess of pineal tissue, we failed to identify chromophore within the pineal during the first week of postnatal development. The appearance of 11-cis retinaldehyde within the eye between postnatal days 3-5 is consistent with the hypothesis that retinol isomerase activity is coordinated with outer segment development. The failure to identify chromophore within the neonatal pineal suggests that this gland lacks a functional opsin-based photopigment. These data contradict physiological evidence suggesting that the neonatal pineal of mammals contains photoreceptors.

Characterization of calbindin-positive cones in primates.

The aim of this study is to characterize calbindin-positive photoreceptors and their opsin content in the retina of nocturnal prosimians (Microcebus murinus), New World monkeys (Callithrix jacchus), Old World monkeys (Macaca fascicularis), and humans. To identify the calbindin and opsin content of cones, combined multiple labeling with different fluorescent probes, antibodies directed against calbindin, short, and mid-long wavelength opsins, and lectin peanut agglutinin cytochemistry were used. With the exception of Microcebus, calbindin is present in the cones of all primates but is absent from rods. The distribution of calbindin is similar in human and macaque cones, with dense label in the inner segment, cell body, axon and cone pedicle. Cones in marmoset also show dense staining in the cell body, axon and pedicle but only light label in the inner segment. Primate cone outer segments do not contain calbindin. In the primates studied, three patterns of calbindin and opsin localization are observed. In macaque and marmoset all short and mid-long wavelength cones contain calbindin. In humans, all mid-long wavelength cones contain calbindin whereas all short wavelength cones are devoid of calbindin as confirmed by confocal microscopy. In the nocturnal prosimian Microcebus none of the mid-long or short wavelength cones contain calbindin. In addition to primates, calbindin is absent in cones of other nocturnal species but is present in cones of diurnal species suggesting a difference in the role of calbindin possibly related to the adaptational states or other photoreceptor properties.

A structural role for Asp83 in the photoactivation of rhodopsin.

Asp83 is a highly conserved residue in the second transmembrane domain of visual pigments and many members of other G protein-coupled receptor subfamilies. Upon illumination, the rod visual pigment rhodopsin proceeds through various intermediate states (Batho<-->BSI<-->Lumi<-->Meta I<-->Meta II). Meta II represents the active state of rhodopsin, which binds and activates the G protein transducin. Evidence has been presented that Asp83 participates in the formation of Meta II and undergoes a change in H-bonding. To investigate whether this role of Asp83 requires its proton-donating capacity and/or its H-bonding capability, we constructed the mutants D83C and D83N. Both mutants appear to effectively activate transducin, indicating that Asp83 is not essential for signal transduction. Differential effects of the mutations D83C and D83N are observed in the spectral properties and the pH sensitivity of the Meta I-->Meta II transition. In general, D83C behaves much more like wild-type than D83N. We conclude that the structural role of Asp83 also involves the acidic nature of its carboxyl group. In addition, the participation in Meta II formation of Cys83 in D83C manifests itself as a change in the vibrational properties of the sulfhydryl group, demonstrating that the -SH group can be used as a non-invasive probe for local structural changes.

Short and mid-wavelength cone distribution in a nocturnal Strepsirrhine primate (Microcebus murinus).

Strepsirrhines are of considerable interest for understanding the evolution of cone photoreceptors because they represent the most ancestral living primates. The retina of nocturnal Strepsirrhines is reported to contain a single population of medium/long wavelength (MW/LW) cones whereas short wavelength (SW) cones are totally absent. The area centralis of nocturnal Strepsirrhines also lacks the degree of central specialization seen in the fovea of diurnal primates. In this study of a nocturnal Strepsirrhine, the gray mouse lemur (Microcebus murinus), we used specific antibodies that recognize SW and MW/LW opsins to determine the presence of different cone subtypes and their distribution in relation to that of rods and ganglion cells. The results are compared to two diurnal Haplorhine species, a New World (Callithrix jacchus) and an Old World (Macaca fascicularis) monkey. In the mouse lemur, both antibodies to MW/LW cone opsin (COS-1 and CERN956) label the same population of cones. A small proportion of SW cones is only stained by the JH455 antiserum whereas the monoclonal OS-2 antibody shows negative staining. These two antibodies label the same SW cone population in other primates. The extracellular matrix of all cones is also labeled by the peanut agglutinin (PNA) lectin. In mouse lemur retinal wholemounts, peak cone density is localized at the area centralis and ranged from 7,500 to 8,000 cones/mm(2). SW cones represent less than 0.2 % of the total cone population and are mainly located in the nasal part of the retina. SW cones show an irregular distribution and densities never exceed 49 cones/mm(2). The distribution of neurons in the ganglion cell layer shows a distinct centroperipheral gradient with a peak of 28,000 cells/mm(2) at the area centralis. Rod distribution shows a centroperipheral gradient with the peak (850,000 rods/mm(2)) including and extending slightly dorsal to the area centralis. The theoretical spatial resolution of the mouse lemur (4.9 cycles/degree) is slightly lower to that of other nocturnal primates. The densities of rods, cones, and ganglion cell layer neurons represent a compromise between spatial resolution and sensitivity for both photopic and scotopic vision.

A fully functional rod visual pigment in a blind mammal. A case for adaptive functional reorganization?

In the blind subterranean mole rat Spalax ehrenbergi superspecies complete ablation of the visual image-forming capability has been accompanied by an expansion of the bilateral projection from the retina to the suprachiasmatic nucleus. We have cloned the open reading frame of a visual pigment from Spalax that shows >90% homology with mammalian rod pigments. Baculovirus expression yields a membrane protein with all functional characteristics of a rod visual pigment (lambda(max) = 497 +/- 2 nm; pK(a) of meta I/meta II equilibrium = 6.5; rapid activation of transducin in the light). We not only provide evidence that this Spalax rod pigment is fully functional in vitro but also show that all requirements for a functional pigment are present in vivo. The physiological consequences of this unexpected finding are discussed. One attractive option is that during adaptation to a subterranean lifestyle, the visual system of this mammal has undergone mosaic reorganization, and the visual pigments have adapted to a function in circadian photoreception.

Photoactivation of rhodopsin: interplay between protein and chromophore.

Data in the literature suggest a finely tuned interaction between ligand (11-cis-retinal) and protein (opsin) in order to allow very efficient photoactivation of the ligand and highly vectorial rhodopsin activation with a huge increase in receptor activity. We have further investigated this interaction using ligand homologues, 13C-ligand labelling or 15N-protein labelling, in combination with Fourier transform infrared (FT-IR) and solid-state magic angle spinning (ss-MAS)-NMR spectroscopy. Using 1D rotational resonance (RR) or double-quantum heteronuclear local field (2Q-HLF) ss-MAS-NMR we report the first structure refinement of the rhodopsin chromophore in situ. These measurements yield a specification of the torsional strain in the for isomerization essential C10-C13 segment of the chromophore. This strain is thought to contribute to the high rate and stereospecificity of the photoisomerization reaction. In agreement with previous data, the C10-C13 segment region reaches a relaxed all-trans configuration at the lumirhodopsin photointermediate. MAS-NMR analysis of [15N]lysine-labelled rhodopsin reveals the presence of a 'soft' counterion, requiring intermediate water molecules for stabilization. FT-IR studies on [2H]tyrosine-labelled rhodopsin demonstrate participation of several tyrosin(at)e residues in receptor activation. One of these, probably Tyr268, is already active at the bathorhodopsin stage. Finally, the effect of ligands with single additional methyl substituents in the C10-C12 region has been investigated. They do not affect the general activation pathway, but perturb the activation kinetics of rhodopsin, suggesting steric interference with protein residues. Possible implications of these results for a structural role of water residues will be discussed, as well.

Probing intramolecular orientations in rhodopsin and metarhodopsin II by polarized infrared difference spectroscopy.

The light-induced conformational changes of rhodopsin, which lead to the formation of the G-protein activating metarhodopsin II intermediate, are studied by polarized attenuated total reflectance infrared difference spectroscopy. Orientations of protein groups as well as the retinylidene chromophore were calculated from the linear dichroism of infrared difference bands. These bands correspond to changes in the vibrational modes of individual molecular groups that are structurally active during receptor activation, i.e., during the rhodopsin to metarhodopsin II transition. The orientation of the transition dipole moments of bands previously assigned to the carboxyl (C=O) groups of Asp83 and Glu113 has been determined. The orientation of specific groups in the retinylidene chromophore has been inferred from the dichroism of the bands associated with the polyene C-C, C=C, and hydrogen-out-of-plane vibrations. Interestingly, the use of polarized infrared light reveals several difference bands in the rhodopsin to metarhodopsin II difference spectrum which were previously undetected, e.g., at 1736 and 939 cm(-1). The latter is tentatively assigned to the hydrogen-out-of-plane mode of the HC(11)=C(12)H segment of the chromophore. Our data suggest a significant change in orientation of this group in the late phase of rhodopsin activation. On the basis of available site-directed mutagenesis data, bands at 1406, 1583, and 1736 cm(-1) are tentatively assigned to Glu134. The main features in the amide regions in the dichroic difference spectrum are discussed in terms of a slight reorientation of helical segments upon receptor activation.

Large-scale production and purification of functional recombinant bovine rhodopsin with the use of the baculovirus expression system.

Here we describe a generic procedure for the expression and purification of milligram quantities of functional recombinant eukaryotic integral membrane proteins, exemplified by hexahistidine-tagged bovine rhodopsin. These quantities were obtained with the recombinant baculovirus/Sf9 insect cell-based expression system in large-scale bioreactor cultures with the use of a serum-free and protein-free growth medium. After optimization procedures, expression levels up to 4 mg/l were established. The recombinant rhodopsin could be purified with high overall yield by using immobilized-metal-affinity chromatography on Ni(2+)-agarose. After reconstitution into a native lipid environment, the purified protein was functionally indistinguishable from native rhodopsin with regard to the following parameters: spectral absorbance band, structural changes after photoactivation, and G-protein activation. The procedures developed can be adapted to other membrane proteins. The ability to produce and purify tens of milligrams of functional recombinant eukaryotic membrane protein meets the ever-increasing demand of material necessary to perform detailed biochemical and structural biophysical studies that are essential in unravelling their working mechanism at a molecular level.

Identification and distribution of photoreceptor subtypes in the neotenic tiger salamander retina.

The neotenic tiger salamander retina is a major model system for the study of retinal physiology and circuitry, yet there are unresolved issues regarding the organization of the photoreceptors and the photoreceptor mosaic. The rod and cone subtypes in the salamander retina were identified using a combination of morphological and immunocytochemical markers for specific rod and cone opsin epitopes. Because the visual pigment mechanisms present in the tiger salamander retina are well characterized and the antibodies employed in these studies are specific for particular rod and cone opsin epitopes, we also were able to identify the spectral class of the various rod and cone subtypes. Two classes of rods corresponding to the "red" and "green" rods previously reported in amphibian retinas were identified. In serial semithin section analyses, rods and cones comprised 62.4+/-1.4% and 37.6+/-1.4% of all photoreceptors, respectively. One rod type comprising 98.0+/-0.7% of all rods showed the immunological and morphological characteristics of "red" rods, which are maximally sensitive to middle wavelengths. The second rod subtype comprised 2.0+/-0.7% of all rods and possessed the immunological and morphological characteristics of "green" rods, which are maximally sensitive to short wavelengths. By morphology four cone types were identified, showing three distinct immunological signatures. Most cones (84.8+/-1.5% of all cones), including most large single cones, the accessory and principal members of the double cone, and some small single cones, showed immunolabeling by antisera that recognize long wavelength-sensitive cone opsins. A subpopulation of small single cones (8.4+/-1.7% of all cones) showed immunolabeling for short wavelength-sensitive cone opsin. A separate subpopulation of single cones which included both large and small types (6.8+/-1.4% of all cones) was identified as the UV-Cone population and showed immunolabeling by antibodies that recognize rod opsin epitopes. Analysis of flatmounted retinas yielded similar results. All photoreceptor types appeared to be distributed in all retinal regions. There was no obvious crystalline organization of the various photoreceptor subtypes in the photoreceptor mosaic.

Tyrosine structural changes detected during the photoactivation of rhodopsin.

We present the first Fourier transform infrared (FTIR) analysis of an isotope-labeled eukaryotic membrane protein. A combination of isotope labeling and FTIR difference spectroscopy was used to investigate the possible involvement of tyrosines in the photoactivation of rhodopsin (Rho). Rho --> MII difference spectra were obtained at 10 degrees C for unlabeled recombinant Rho and isotope-labeled L-[ring-2H4]Tyr-Rho expressed in Spodoptera frugiperda cells grown on a stringent culture medium containing enriched L-[ring-2H4]Tyr and isolated using a His6 tag. A comparison of these difference spectra revealed reproducible changes in bands that correspond to tyrosine and tyrosinate vibrational modes. A similar pattern of tyrosine/tyrosinate bands has also been observed in the bR --> M transition in bacteriorhodopsin, although the sign of the bands is reversed. In bacteriorhodopsin, these bands were assigned to Tyr-185, which along with Pro-186 in the F-helix, may form a hinge that facilitates alpha-helix movement.

Large-scale production and purification of the human green cone pigment: characterization of late photo-intermediates.

We present the first characterization of the late photo-intermediates (Meta I, Meta II and Meta III) of a vertebrate cone pigment in a lipid environment. Marked differences from the same pathway in the rod pigment were observed. The histidine-tagged human green cone pigment was functionally expressed in large-scale suspension cultures in Sf9 insect cells using recombinant baculovirus. The recombinant pigment was extensively purified in a single step by immobilized metal affinity chromatography and displays the expected spectral characteristics. The purified pigment was able to activate the rod G-protein transducin at about half the rate of the rod pigment. Following reconstitution into bovine retina lipid proteoliposomes, identification and analysis of the photo-intermediates Meta I, Meta II and Meta III was accomplished. Similar to the rod pigment, our results indicate the existence of a Meta I-Meta II equilibrium, but we find no evidence for pH dependence. Replacement of native Cl- by NO3- in the anion-binding site of the cone pigment affected the spectral position of the pigment itself and of the Meta I intermediate, but not that of Meta II and Meta III. The decay rate of the 'active' intermediate Meta II did not differ for the Cl- and NO3- state. However, in qualitative agreement with results reported before for chicken cone pigments, the rate of Meta II decay was significantly higher in the human cone pigment than in the rod pigment.

The molecular basis for UV vision in birds: spectral characteristics, cDNA sequence and retinal localization of the UV-sensitive visual pigment of the budgerigar (Melopsittacus undulatus).

Microspectrophotometric (msp) studies have shown that the colour-vision system of many bird species is based on four pigments with absorption peaks in the red, green, blue and UV regions of the spectrum. The existence of a fourth pigment (UV) is the major difference between the trichromacy of humans and the tetrachromacy of such birds, and recent studies have shown that it may play a determining role in such diverse aspects of behaviour as mate selection and detection of food. Avian visual pigments are composed of an opsin protein covalently bound via a Schiff-base linkage to the chromophore 11-cis-retinal. Here we report the cDNA sequence of a UV opsin isolated from an avian species, Melopsittacus undulatus (budgerigar or small parakeet). This sequence has been expressed using the recombinant baculovirus system; the pigment generated from the expressed protein on addition of 11-cis-retinal yielded an absorption spectrum typical of a UV photopigment, with lambdamax 365+/-3 nm. This is the first UV opsin from an avian species to be sequenced and expressed in a heterologous system. In situ hybridization of this sequence to budgerigar retinas selectively labelled a sub-set of UV cones, representing approx. 9% of the total cone population, that are distributed in a semi-regular pattern across the entire retina.

Selective detergent-extraction from mixed detergent/lipid/protein micelles, using cyclodextrin inclusion compounds: a novel generic approach for the preparation of proteoliposomes.

A novel generic approach is described for the selective extraction of detergents from mixed detergent/lipid/protein micelles for the preparation of proteoliposomes of defined lipid-protein ratio. The approach is based on the much higher affinity of inclusion compounds of the cyclodextrin type for detergents in comparison with bilayer-forming lipids. This approach has distinct advantages over other procedures currently in use. It produces good results with all detergents tested, independent of type and critical micelle concentration, and appears to be generally applicable. It yields nearly quantitative recovery of membrane protein in the proteoliposome fraction. Finally, no large excess of lipid is required; a molar ratio of lipid to protein of 100 to 1 already produces proteoliposomes with functional membrane protein, but higher ratios are well tolerated. The size of the vesicles thus obtained depends on the detergent used. Separation of the resulting proteoliposomes from the detergent-cyclodextrin complexes was most easily achieved by centrifugation through a discontinuous sucrose gradient. A variety of detergents was tested in this procedure on the bovine rod visual pigment rhodopsin in combination with retina lipids. In all cases good yields of proteoliposomes were obtained, which contained fully functional rhodopsin.

An additional methyl group at the 10-position of retinal dramatically slows down the kinetics of the rhodopsin photocascade.

The present study focuses on ligand-protein interactions in a rhodopsin analogue generated from bovine opsin and the 10-methyl homologue of 11-cis-retinal. The analogue pigment displays a reduced alpha-band at 506 +/- 2 and a stronger beta-band at 325 nm. Remarkably, the rotational strength of these bands observed in visible circular dichroism spectra was found to be similar for both native and 10-methyl rhodopsin. The quantum yield of the analogue pigment was determined to be 0.55. All photointermediates were analyzed by Fourier transform infrared difference spectroscopy. At the batho stage, strong hydrogen-out-of-plane vibrations were observed, indicating that the 10-methyl chromophore also adopts a distorted all-trans conformation at this stage. In contrast to native rhodopsin, the batho intermediate of the 10-methyl pigment is stable up to 180 K and only slowly decays to the next intermediate between 180 and 210 K. As in native rhodopsin, the 10-methyl metarhodopsin I intermediate is generated at about 220 K, but its transition to the metarhodopsin II state is again shifted to a much higher temperature (> 293 K) than for the native pigment (> 260 K). Infrared analysis, nevertheless, shows that the conformational changes in the photointermediates of the 10-methyl pigment are basically identical with those observed in the native pigment. This is supported by a signal function assay, showing that the analogue pigment is able to activate transducin. The dual effect of the 10-methyl group on the photocascade is attributed to steric interactions which, initially, hamper the relaxation of strain in the polyene chain of the chromophore and, eventually, interfere with the conformational rearrangements of the protein moiety required to adopt the active conformation of the receptor. Our data provide direct support for the concept that the relaxation of strain in the retinal polyene chain acts as the major driving force of the photocascade dark reaction.

Hydrogen bonding changes of internal water molecules in rhodopsin during metarhodopsin I and metarhodopsin II formation.

Rhodopsin is a 7-helix, integral membrane protein found in the rod outer segments, which serves as the light receptor in vision. Light absorption by the retinylidene chromophore of rhodopsin triggers an 11-cis-->all-trans isomerization, followed by a series of protein conformational changes, which culminate in the binding and activation of the G-protein transducin by the metarhodopsin II (Meta II) intermediate. Fourier transform IR difference spectroscopy has been used to investigate the structural changes that water, as well as other OH- and NH-containing groups, undergo during the formation of the metarhodopsin I (Meta I) and Meta II intermediates. Bands associated with the OH stretch modes of water are identified by characteristic downshifts upon substitution of H2(18)O for H2O. Compared with earlier work, several negative bands associated with water molecules in unphotolysed rhodopsin were detected, which shift to lower frequencies upon formation of the Meta I and Meta II intermediates. These data indicate that at least one water molecule undergoes an increase in hydrogen bonding upon formation of the Meta I intermediate, while at least one other increases its hydrogen bonding during Meta II formation. Amino acid residue Asp-83, which undergoes a change in its hydrogen bonding during Meta II formation, does not appear to interact with any of the structurally active water molecules. Several NH and/or OH groups, which are inaccessible to hydrogen/deuterium exchange, also undergo alterations during Meta I and Meta II formation.