Photosensory Proteins and Methods for Their Investigation

Nature evolved a very limited number of photoreceptive molecules in the different evolutionary branches of the tree of life because of the closely similar needs of organisms for the detection of the external world. Two photoreceptor molecules are considered as underlying “vision” in algae: rhodopsin-like proteins and flavoproteins. Photoreceptor proteins should occur only in small amounts in algal structures, because they are used to detect light, not to collect it. They are concentrated in photoreceptive structures and do not contribute to the color of the organism. This is an important consideration because a primary, historical criterion for algal taxonomy and phylogeny has been their bulk pigment composition. Sensory pigments have played no role at all in these determinations, because it is generally believed that different algal groups have different photosensory pigments; furthermore, the association of particular sensory pigments with particular algal groups has been based largely upon the examination of only one or a few species.

For a long time the behavior of algae in response to light stimuli has attracted the attention of scientists, who hoped that the simplicity of the material would make this phenomenon easy to investigate. On the contrary, the undertaking turned out to be more difficult than expected and to date a clear understanding of the phenomenon has still to be attained, while comprehension of the visual process in humans is extensive. Initial ideas on the chemical nature of receptors for photobehavioral responses were gained by measurements of their spectral sensitivity. Action spectroscopy results were then screened and supported by other methods such as biochemistry, absorption spectroscopy, electrophysiology, and molecular biology, which when integrated with different investigative approaches shed more and more light on nature and functioning of photoreceptive pigments.

In the following we will briefly describe the characteristics of rhodopsin-like proteins and flavoproteins and compare spectroscopic and biochemical techniques that have been used to define the light-absorbing properties of photoreceptive proteins either in vivo (single cell or cell population) or in vitro (extracted material). To provide a clear perspective on the efficacy of different techniques as tools for the study of photoreceptive structures, a discussion of the pros and cons, the advantages and limitations of each technique are included.

Rhodopsin-Like Proteins
Rhodopsins are photoreceptor proteins, universally used from archeabacteria to humans, consisting of a proteic part, the opsin, organized in seven transmembrane a-helices, and a light absorbing group, the retinal (i.e., the chromophore). The retinal is located inside a pocket of the opsin, approximately in its center.

Why these proteins are so special? First, retinal–opsin complex has an intense absorption band whose maximum can be shifted into the visible region of the spectrum, over the entire range from 380 to 640 nm. Second, light isomerizes the retinal inside the protein very efficiently and rapidly. This isomerization, that is, the event initiating the vision reaction cascade, can be triggered almost exclusively by light; in the dark it occurs only about once in a thousand years. Third, remarkable structural changes (movements of single a-helix) are produced by isomerization of retinal. Light is converted into atomic motion of sufficient magnitude to trigger a signal reliably and reproducibly.

Fourth, the photocycle (the photoreceptive protein upon light excitation undergoes a series of conformational changes which can be driven back to the original conformational state) is very fast, hence the intracellular photoreceptive machinery is immediately reset for a new response. Fifth, retinal is derived from β-carotene, a precursor with a widespread biological distribution.

About 1000 rhodopsin-like protein genes have been so far detected in the microbial world. Genes were detected in Anabaena (aka Nostoc) sp. (Cyanophyta), Guillarda theta (Cryptophyta), Pyrocystis lunula (Dynophyta), and Chlamydomonas sp. (Chlorophyta). Biochemical and spectroscopical evidences for a retinal-based photoreceptor were reported in one lineage of prokaryotes, that is, in Leptolyngbya sp., (Cyanophyta), and at least in four lineages of eukaryotic algae, that is, Ochromonas sp. and Silvetia, sp. (Heterokonthophyta); Euglena sp. (Euglenophyta); Gymnodinium sp. (Dinophyta), and Dunaliella sp., Spermatozopsis sp., and Volvox sp. (Chlorophyta).

Flavoproteins
Flavoproteins, or yellow enzymes, are a diverse group of more than 70 oxidoreductases found in animal, plants, and microorganisms, which have a flavin as a prosthetic group covalently attached to the protein. The proposition that these proteins could function as a near-UV-visible-light detector dates back more than 40 years. Despite the ubiquity and ancient origins of flavoproteins, their role in acquiring information from the radiation environment still remains a complex area of study. Apart from difficulties in their identification, much of the reason for the lack of understanding lies in their diversity of function. Typical absorption spectra of flavoproteins show a dominant protein peak at 280 nm, and maior peaks at 380 and 460 nm. The overall similarity of many blue-light action spectra with flavoprotein absorption spectra is one of the main reasons for the belief that flavoproteins can function as blue-light photoreceptors. So far, the only biochemical identification of flavin-based photoreceptor has been carried out in Euglena gracilis. Despite this paucity of evidence, the hypothesis of a flavin-based photoreceptor in algae has withstood time and still remains an accepted working hypothesis.

Action Spectroscopy
Action spectroscopy still represents the classic way to investigate photopigments. By means of action spectroscopy the photosensitivity of a cell at different wavelengths can be measured, thus providing information on the nature of the pigments involved in photoreception. It is still a common belief that this approach represents the only feasible way to study the photosensory pigments of a large number of species. However, direct measure of an action spectrum is much more difficult than that of an absorption spectrum. Moreover, action spectra may not be directly correlated with the absorption peaks of the pigments involved in photoreception, as light scattering can cause several errors. When many pigments with similar absorption characteristics in the same visible range are present, action spectroscopy often fails to discriminate between them. Even when there is only one predominant pigment, it is not always possible to identify it. To obtain more reliable results, threshold action spectra should be preferred to eliminate adaptation phenomena and screening modulation, limiting the utilization of action spectroscopy to the study of changes or increases in the photosensitivity of a mutant cell, after the exogenous addition of a presumptive photoreceptor pigment which the cell lacks. It may be hasty to indicate the nature of a photoreceptor only on the basis of data obtained from action spectroscopy. This is especially true in the case of photoreceptors such as rhodopsins, which have retinal as the chromophoric group. Retinal absorption can be fine-tuned by amino acid charges of the retinal pocket, which allows the entire spectrum between 380 and 640 nm to be covered. Moreover, the presence or formation of photo-intermediates may shift the absorption maxima, and make the interpretation of the action spectrum difficult.


Absorption and Fluorescence Microspectroscopy
These techniques do not disturb the integrity of the organism or subcellular components, and allow the examination of an uninjured system with its physiological functions intact. The spectroscopic overshadowing of one pigment by another is avoided because each pigment is packaged in a different structure. Thus, cellular structure can be easily correlated with pigment type by direct observation. It is possible to make exact quantitative determinations of various reactions at the time of their occurrence in the sample, the progressive changes in these reactions, and their relationships to different conditions in the external medium. Because of the fundamental connection between optical parameters and properties of molecular structures, microspectroscopy allows assessments of minute changes in the state of the molecules of various substances in the organism, the degree of their aggregation, and the interconversions of various forms of pigments and other important biochemical compounds with characteristic spectra. In many cases the lability and reversibility of such changes make microspectroscopy the only possible method of investigation. There is virtually no light scattering problem associated with microspectroscopic measurements, even if the analyzed structure has a dimension of 1 µm. Obviously, the absorption spectrum cannot provide adequate information about the photochemical action of photons as a function of their fundamental energy; however, the identification of the chromophores in the photoreceptive structures does provide information about possible mechanisms of energy transfer. The measurements are very difficult when small changes in absorption have to be measured in the presence of a strong total signal (luminous background), as photon noise is proportional to the square root of the intensity of the incident light. Then, fluorescence spectroscopy is recommended: it can achieve more reliable results compared with absorption spectroscopy, because the background emission is much reduced. In this case the sensitivity of detection is not limited by the signal-to-noise ratio, but rather by the presence, virtually unavoidable, of fluorescent contaminants.

Biochemical and Spectroscopic Study of Extracted Visual Pigments
Extraction of visual pigments (chromophore or protein), either by means of detergents such as digitonin and TRITON or by organic solvents, could be the best method for providing large quantities of photoreceptive pigments in an accessible in vitro form for subsequent detailed biochemical analysis. Such samples allow a very accurate determination of spectroscopic parameters. Spectroscopy of solubilized pigment may be complicated, however, by the simultaneous extraction of several other pigments in the cell, which cause distortion of absolute spectra and necessitate special procedures of purification. These problems can be solved by separating the different pigments after extraction, for example, by high performance liquid chromatography (HPLC) and final identification with gas chromatography – mass spectrography (GC–MS) for chromophores or affinity chromatography for proteins. As pigment extraction permanently removes the identifying link to a particular cell structure and may change the spectroscopic properties of a receptor because native interactions are disrupted, these detrimental factors mandate a careful evaluation of the results obtained by this method.

Electrophysiology
Light excitation of the photoreceptor generates a cascade of electrical events. Electrophysiology was the elective method in the study of vertebrate photoreceptors. However, this technique has been applied with less success in the algae because it is very difficult to locate the photoreceptors in the cell body and, when this is possible, to produce a good sealing between the glass pipette and the cell membrane.

Flash-induced transient depolarizing potentials using intracellular glass microelectrode were first identified in Acetabularia crenulata. However, the first detailed analysis of photocurrents were possible by employing a suction pipette technique (patch clamp technique) in Haematococcus pluvialis and in the wall-free mutants of the unicellular green alga Chlamydomonas sp. In these experiments whole cells were gently sucked into fire-polished pipettes, forming seals with resistances up to 250 MW, allowing cell attached recordings from a relatively large membrane area, though higher resistance seals were not achieved. Recently, Negel et al. (2002) demonstrated by means of electrophysiology that the rhodopsin-like protein of Chlamydomonas, expressed in Xenopus laevis oocytes in the presence of all-trans retinal produces a light gated conductance that shows characteristics of a channel selectively permeable for protons.

Molecular Biology Investigations
DNA hybridization is useful in attempting to determine phylogenetic interrelationship between species. The rationale is that similarities between DNA structures correlate to interrelatedness. It is used to detect and isolate specific sequences and to measure the extent of homology between nucleic acids. It represents an alternative to the study of visual pigments at the protein level, as the genes encoding these proteins can be identified, their sequences determined, and the comparative genetic information assessed. Genomic Southern blot hybridization is used to probe the genomes of a variety of species in a manner analogous to that reported for other protein families. The potential for using bovine rhodopsin opsin complementary DNA (cDNA) probe to identify homologous genes in other species was demonstrated by Martin et al. (1986). These authors identified coding regions of bovine opsin that are homologous with visual pigment genes of vertebrate, invertebrate, and phototactic unicellular species. Successful application of this method requires closely homologous genes, and in general additional criteria, such as protein sequence information, is desirable for eliminating false positives on Southern blots. A molecular biology approach has been used also by Sineshchekov et al. (2002) in Chlamydomonas. These authors identified gene fragments with homology to the archaeal rhodopsin apoprotein genes in the expressed sequence-tag data bank of Chlamydomonas reinhardtii. Two quite similar genes were identified having almost all the residues of bacteriorhodopsin in the retinal binding site. The authors suspected that these genes were related to the putative retinal-based pigments already suggested for Chlamydomonas.

However, to show that the pigments are a part of the genuine signaling system, ideally one would like to delete each gene by using homologous recombination, but it is not easy to do such gene knockouts in any algal species. The problem can be overcome partially by using RNA interference (RNAi) technology to preferentially suppress the synthesis of the pigments to convincingly show that the pigment is a genuine segment of the algal phototactic response.

Understanding the molecular mechanism used by algal cells to “see the light,” as we have tried to explain, is a very difficult task. At least a century has been wasted without any success. It is discouraging to think that even if the algae are not as intelligent as men are, they have “understood” very well how to orientate themselves in their light environment, and do it very efficiently. Maybe the compass mechanism they use is too simple for our complex brain.