Belinda S. W. Chang

Retinal counterion switch in the photoactivation of the G protein-coupled receptor rhodopsin

The biological function of Glu-181 in the photoactivation process of rhodopsin is explored through spectroscopic studies of site-specific mutants. Preresonance Raman vibrational spectra of the unphotolyzed E181Q mutant are nearly identical to spectra of the native pigment, supporting the view that Glu-181 is uncharged (protonated) in the dark state. The pH dependence of the absorption of the metarhodopsin I (Meta I)-like photoproduct of E181Q is investigated, revealing a dramatic shift of its Schiff base pKa compared with the native pigment. This result is most consistent with the assignment of Glu-181 as the primary counterion of the retinylidene protonated Schiff base in the Meta I state, implying that there is a counterion switch from Glu-113 in the dark state to Glu-181 in Meta I. We propose a model where the counterion switch occurs by transferring a proton from Glu-181 to Glu-113 through an H-bond network formed primarily with residues on extracellular loop II (EII). The resulting reorganization of EII is then coupled to movements of helix III through a conserved disulfide bond (Cys110–Cys187); this process may be a general element of G protein-coupled receptor activation.

The Burmese python genome reveals the molecular basis for extreme adaptation in snakes

Significance The molecular basis of morphological and physiological adaptations in snakes is largely unknown. Here, we study these phenotypes using the genome of the Burmese python (Python molurus bivittatus), a model for extreme phenotypic plasticity and metabolic adaptation. We discovered massive rapid changes in gene expression that coordinate major changes in organ size and function after feeding. Many significantly responsive genes are associated with metabolism, development, and mammalian diseases. A striking number of genes experienced positive selection in ancestral snakes. Such genes were related to metabolism, development, lungs, eyes, heart, kidney, and skeletal structure—all highly modified features in snakes. Snake phenotypic novelty seems to be driven by the system-wide coordination of protein adaptation, gene expression, and changes in genome structure. Snakes possess many extreme morphological and physiological adaptations. Identification of the molecular basis of these traits can provide novel understanding for vertebrate biology and medicine. Here, we study snake biology using the genome sequence of the Burmese python (Python molurus bivittatus), a model of extreme physiological and metabolic adaptation. We compare the python and king cobra genomes along with genomic samples from other snakes and perform transcriptome analysis to gain insights into the extreme phenotypes of the python. We discovered rapid and massive transcriptional responses in multiple organ systems that occur on feeding and coordinate major changes in organ size and function. Intriguingly, the homologs of these genes in humans are associated with metabolism, development, and pathology. We also found that many snake metabolic genes have undergone positive selection, which together with the rapid evolution of mitochondrial proteins, provides evidence for extensive adaptive redesign of snake metabolic pathways. Additional evidence for molecular adaptation and gene family expansions and contractions is associated with major physiological and phenotypic adaptations in snakes; genes involved are related to cell cycle, development, lungs, eyes, heart, intestine, and skeletal structure, including GRB2-associated binding protein 1, SSH, WNT16, and bone morphogenetic protein 7. Finally, changes in repetitive DNA content, guanine-cytosine isochore structure, and nucleotide substitution rates indicate major shifts in the structure and evolution of snake genomes compared with other amniotes. Phenotypic and physiological novelty in snakes seems to be driven by system-wide coordination of protein adaptation, gene expression, and changes in the structure of the genome.

Honeybee Blue- and Ultraviolet-Sensitive Opsins: Cloning, Heterologous Expression in Drosophila, and Physiological Characterization

The honeybee (Apis mellifera) visual system contains three classes of retinal photoreceptor cells that are maximally sensitive to light at 440 nm (blue), 350 nm (ultraviolet), and 540 nm (green). We performed a PCR-based screen to identify the genes encoding the Apis blue- and ultraviolet (UV)-sensitive opsins. We obtained cDNAs that encode proteins having a high degree of sequence and structural similarity to other invertebrate and vertebrate visual pigments. The Apis blue opsin cDNA encodes a protein of 377 amino acids that is most closely related to other invertebrate visual pigments that are thought to be blue-sensitive. The UV opsin cDNA encodes a protein of 371 amino acids that is most closely related to the UV-sensitive Drosophila Rh3 and Rh4 opsins. To test whether these novel Apis opsin genes encode functional visual pigments and to determine their spectral properties, we expressed them in the R1–6 photoreceptor cells of blindninaE mutant Drosophila, which lack the major opsin of the fly compound eye. We found that the expression of either the Apis blue- or UV-sensitive opsin in transgenic flies rescued the visual defect of ninaEmutants, indicating that both genes encode functional visual pigments. Spectral sensitivity measurements of these flies demonstrated that the blue and UV visual pigments are maximally sensitive to light at 439 and 353 nm, respectively. These maxima are in excellent agreement with those determined previously by single-cell recordings fromApis photoreceptor cells and provide definitive evidence that the genes described here encode visual pigments having blue and UV sensitivity.

Recreating ancestral proteins.

Tracing the history of molecular changes using phylogenetic methods can provide powerful insights into how and why molecules work the way they do. It is now possible to recreate inferred ancestral proteins in the laboratory and study the function of these molecules. This provides a unique opportunity to study the paths and the mechanisms of functional change during molecular evolution. What insights have already emerged from such phylogenetic studies of protein evolution and function, what are the impediments to progress and what are the prospects for the future?

An improved likelihood ratio test for detecting site-specific functional divergence among clades of protein-coding genes

Maximum likelihood codon substitution models have proven useful for studying when and how protein function evolves, but they have recently been criticized on a number of fronts. The strengths and weaknesses of such methods must therefore be identified and improved upon. Here, using simulations, we show that the Clade model C versus M1a test for functional divergence among clades is prone to false positives under simple evolutionary conditions. We then propose a new null model (M2a_rel) that better accounts for among-site variation in selective constraint. We show that the revised test has an improved false-positive rate and good power. Applying this test to previously analyzed data sets of primate ribonucleases and mammalian rhodopsins reveals that some conclusions may have been misled by the original method. The improved test should prove useful for identifying patterns of divergence in selective constraint among paralogous gene families and among orthologs from ecologically divergent species.

Long-wavelength sensitive visual pigments of the guppy (Poecilia reticulata): six opsins expressed in a single individual

The diversity of visual systems in fish has long been of interest for evolutionary biologists and neurophysiologists, and has recently begun to attract the attention of molecular evolutionary geneticists. Several recent studies on the copy number and genomic organization of visual pigment proteins, the opsins, have revealed an increased opsin diversity in fish relative to most vertebrates, brought about through recent instances of opsin duplication and divergence. However, for the subfamily of opsin genes that mediate vision at the long-wavelength end of the spectrum, the LWS opsins, it appears that most fishes possess only one or two loci, a value comparable to most other vertebrates. Here, we characterize the LWS opsins from cDNA of an individual guppy, Poecilia reticulata, a fish that is known exhibit variation in its long-wavelength sensitive visual system, mate preferences and colour patterns. We identified six LWS opsins expressed within a single individual. Phylogenetic analysis revealed that these opsins descend from duplication events both pre-dating and following the divergence of the guppy lineage from that of the bluefin killifish, Lucania goodei, the closest species for which comparable data exists. Numerous amino acid substitutions exist among these different LWS opsins, many at sites known to be important for visual pigment function, including spectral sensitivity and G-protein activation. Likelihood analyses using codon-based models of evolution reveal significant changes in selective constraint along two of the guppy LWS opsin lineages. The guppy displays an unusually high number of LWS opsins compared to other fish, and to vertebrates in general. Observing both substitutions at functionally important sites and the persistence of lineages across species boundaries suggests that these opsins might have functionally different roles, especially with regard to G-protein activation. The reasons why are currently unknown, but may relate to aspects of the guppys behavioural ecology, in which both male colour patterns and the female mate preferences for these colour patterns experience strong, highly variable selection pressures.

Cloning of the gene encoding honeybee long-wavelength rhodopsin: A new class of insect visual pigments

Rhodopsins (Rh), G-protein-coupled receptors with seven transmembrane (TM) helices, form the first step in visual transduction in most organisms. Although many long-wavelength (LW) vertebrate opsin sequences are known, less information is available for invertebrate LW sequences. By a combination of RT-PCR and cDNA library screening, we have cloned and sequenced the honeybee LW Rh gene. The deduced protein is composed of 378 amino acids (aa), appears to have seven TM regions, and contains many of the structures and key aa thought to be important for Rh function. Phylogenetic analysis of this sequence in relation to other invertebrate Rh reveals it to be a member of a new group of insect LW Rh.

The Evolution of Acetyl-CoA Synthase

Acetyl-coenzyme A synthases (ACS) are Ni–Fe–S containingenzymes found in archaea and bacteria. They are divisible into 4 classes. Class I ACSs catalyze the synthesis of acetyl-CoAfrom CO2 + 2e-, CoA, and a methyl group, and contain5 types of subunits (α, β, γ, δ, and ε). Class II enzymes catalyze essentially the reversereaction and have similar subunit composition. Class III ACSscatalyze the same reaction as Class I enzymes, but use pyruvateas a source of CO2 and 2e-, and are composed of 2 autonomous proteins, an α2β2 tetramerand a γδ heterodimer. Class IV enzymes catabolize CO to CO2 and are α-subunit monomers. Phylogeneticanalyses were performed on all five subunits. ACS α sequences divided into 2 major groups, including Class I/II sequences and Class III/IV-like sequences. Conserved residuesthat may function as ligands to the B- and C-clusters wereidentified. Other residues exclusively conserved in Class I/IIsequences may be ligands to additional metal centers in Class I and II enzymes. ACS β sequences also separated into twogroups, but they were less divergent than the αs, and the separation was not as distinct. Class III-like β sequences contained ∼300 residues at their N-termini absent in Class I/II sequences. Conserved residues identifiedin β sequences may function as ligands to active siteresidues used for acetyl-CoA synthesis. ACS γ-sequencesseparated into 3 groups (Classes I, II, and III), while δ-sequences separated into 2 groups (Class I/II and III). These groups are less divergent than those of α sequences. ACS ε-sequence topology showed greaterdivergence and less consistency vis-à-vis the other subunits, possibly reflecting reduced evolutionary constraintsdue to the absence of metal centers. The α subunit phylogeny may best reflect the functional diversity of ACS enzymes. Scenarios of how ACS and ACS-containing organisms mayhave evolved are discussed.

Ancestral reconstruction of the ligand-binding pocket of Family C G protein-coupled receptors

The metabotropic glutamate receptors (mGluRs) within the Family C subclass of G protein-coupled receptors are crucial modulators of synaptic transmission. However, their closest relatives include a diverse group of sensory receptors whose biological functions are not associated with neurotransmission, raising the question of the evolutionary origin of amino acid-binding Family C receptors. A common feature of most, if not all, functional Family C receptors is the presence of an amino acid-binding site localized within the large extracellular Venus flytrap domain. Here, we used maximum likelihood methods to infer the ancestral state of key residues in the amino acid-binding pocket of a primordial Family C receptor. These residues were reconstructed in the background of the fish 5.24 chemosensory receptor, a broad-spectrum amino acid-activated receptor. Unlike the WT 5.24 receptor, which was not activated by mGluR agonists and displayed low sensitivity toward l-glutamate, the reconstructed ancestral receptor possessed a pharmacological profile characterized by high affinity for both l-glutamate and selective Group I mGluR agonists. This pharmacological phenotype could be largely recapitulated by mutating only two residues in the 5.24 receptor-binding pocket. Our results suggest that this primordial Family C receptor may have arisen early in metazoan evolution and that it already was preadapted as a glutamate receptor for its later use at excitatory synapses in glutamate-mediated neurotransmission.

Spectral tuning in vertebrate short wavelength-sensitive 1 (SWS1) visual pigments: can wavelength sensitivity be inferred from sequence data?

The molecular mechanisms underlying the enormous diversity of visual pigment wavelength sensitivities found in nature have been the focus of many molecular evolutionary studies, with particular attention to the short wavelength-sensitive 1 (SWS1) visual pigments that mediate vision in the ultraviolet to violet range of the electromagnetic spectrum. Over a decade of study has revealed that the remarkable extension of SWS1 absorption maxima (λ max ) into the ultraviolet occurs through a deprotonation of the Schiff base linkage of the retinal chromophore, a mechanism unique to this visual pigment type. In studies of visual ecology, there has been mounting interest in inferring visual sensitivity at short wavelengths, given the importance of UV signaling in courtship displays and other behaviors. Since experimentally determining spectral sensitivities can be both challenging and time-consuming, alternative strategies such as estimating λ max based on amino acids at sites known to affect spectral tuning are becoming increasingly common. However, these estimates should be made with knowledge of the limitations inherent in these approaches. Here, we provide an overview of the current literature on SWS1 site-directed mutagenesis spectral tuning studies, and discuss methodological caveats specific to the SWS1-type pigments. We focus particular attention on contrasting avian and mammalian SWS1 spectral tuning mechanisms, which are the best studied among vertebrates. We find that avian SWS1 visual pigment spectral tuning mechanisms are fairly consistent, and therefore more predictable in terms of wavelength absorption maxima, whereas mammalian pigments are not well suited to predictions of λ max from sequence data alone.