Motivation: The enormous amount of protein sequence data uncovered by genome research has increased the demand for computer software that can automate the recognition of new proteins. We discuss the relative merits of various automated methods for recognizing G-protein coupled receptors (GPCRs), a superfamily of cell membrane proteins. GPCRs are found in a wide range of organisms and are central to a cellular signalling network that regulates many basic physiological processes. They are the focus of a signicant amount of current pharmaceutical research because they play a key role in many diseases. However, their tertiary structures remain largely unsolved. The methods described in this paper use only primary sequence information to make their predictions. We compare a simple nearest neighbor approach (BLAST), methods based on multiple alignments generated by a statistical prole hidden Markov model, and methods, including support vector machines, that transform protein sequences into xed-length feature vectors. Results: The last is the most computationally expensive method, but our experiments show that, for those interested in annotation-quality classication, the results are worth the eort. In two-fold cross-validation experiments testing recognition of GPCR subfamilies that bind a specic ligand (such as a histamine molecule), the errors per sequence at the minimum error point (MEP) were 13.7% for multi-class SVMs, 17.1% for our SVMtree method of hierarchical multi-class SVM classication, 25.5% for BLAST, 30% for prole HMMs, and 49% for classication based on nearest neighbor feature vector (kernNN). The percentage of true positives recognized before the rst false positive was 65% for both SVM methods, 13% for BLAST, 5% for prole HMMs and 4% ...

this paper. Here, we are interested in the packing of peptide chains, therefore all ligands and water molecules are removed before computation. Voids and pockets in the TM regions are identied and measured with a probe radius of 1.4 A using the CAST method

Motivation: Integral membrane proteins play important roles in living cells. Although these proteins are estimated to constitute around 25 % of proteins at a genomic scale, the Protein Data Bank (PDB) contains only a few hundred membrane proteins due to the dif-ficulties with experimental techniques. The presence of transmembrane proteins in the structure data bank, however, is quite invisible, as the annotation of these entries is rather poor. Even if a protein is identified as a transmembrane one, the possible location of the lipid bilayer is not indicated in the PDB because these proteins are crystallized without their natural lipid bilayer, and currently no method is publicly available to detect the pos-sible membrane plane using the atomic coordinates of membrane proteins. Results: Here we present a new geometrical approach to distinguish between trans-membrane and globular proteins using structural information only and to locate the most likely position of the lipid bilayer. An automated algorithm (TMDET) is given to deter-mine the membrane planes relative to the position of atomic coordinates, together with a discrimination function which is able to separate transmembrane and globular proteins

Spectral tuning of visual pigments is typically accomplished through changes in opsin amino acid sequence. Within a given opsin class, changes at a few key sites control wavelength specificity. To investigate known differences in the visual pigment spectral sensitivity of the Lake Malawi cichlids, Metriaclima zebra (368, 488, and 533 nm) and Dimidiochromis compressiceps (447, 536, and 569 nm), we sequenced cone opsin genes from these species as well as Labeotropheus fuelleborni and Oreochromis niloticus. These cichlids have five distinct classes of cone opsin genes, including two unique SWS-2 genes. Comparisons of the inferred amino acid sequences from the five cone opsin genes of M. zebra, D. compressiceps, and L. fuelleborni show the sequences to be nearly identical. Therefore, evolution of key opsin sites cannot explain the differences in visual pigment sensitivities. Real-time PCR demonstrates that different cichlid species express different subsets of the available cone opsin genes. Metriaclima zebra and L. fuelleborni express a complement of genes which give them UV-shifted visual pigments, while D. compressiceps expresses a different set to produce a red-shifted visual system. Thus, variations in cichlid spectral sensitivity have arisen through evolution of gene regulation, rather than through changes in opsin amino acid sequence.

A simple extension of the EEF1 energy function to heterogeneous membrane-aqueous media is proposed. The extension consists of (a) development of solvation parameters for a nonpolar phase using experimental data for the transfer of amino acid side-chains from water to cyclohexane, (b) introduction of a heterogeneous membraneaqueous system by making the reference solvation free energy of each atom dependent on the vertical coordinate, (c) a modification of the distancedependent dielectric model to account for reduced screening of electrostatic interactions in the membrane, and (d) an adjustment of the EEF1 aqueous model in light of recent calculations of the potential of mean force between amino acid side-chains in water. The electrostatic model is adjusted to match experimental observations for polyalanine, polyleucine, and the glycophorin A dimer. The resulting energy function (IMM1) reproduces the preference of Trp and Tyr for the membrane interface, gives reasonable energies of insertion into or adsorption onto a membrane, and allows stable 1-ns MD simulations of the glycophorin A dimer. We find that the lowest-energy orientation of melittin in bilayers varies, depending on the thickness of the hydrocarbon layer. Proteins 2003;52:176--192.

The ancestors of the archosaurs, a major branch of the diapsid reptiles, originated more than 240 MYA near the dawn of the Triassic Period. We used maximum likelihood phylogenetic ancestral reconstruction methods and explored different models of evolution for inferring the amino acid sequence of a putative ancestral archosaur visual pigment. Three different types of maximum likelihood models were used: nucleotide-based, amino acid-based, and codon-based models. Where possible, within each type of model, likelihood ratio tests were used to determine which model best fit the data. Ancestral reconstructions of the ancestral archosaur node using the best-fitting models of each type were found to be in agreement, except for three amino acid residues at which one reconstruction differed from the other two. To determine if these ancestral pigments would be functionally active, the corresponding genes were chemically synthesized and then expressed in a mammalian cell line in tissue culture. The expressed artificial genes were all found to bind to 11-cis-retinal to yield stable photoactive pigments with max values of about 508 nm, which is slightly redshifted relative to that of extant vertebrate pigments. The ancestral archosaur pigments also activated the retinal G protein transducin, as measured in a fluorescence assay. Our results show that ancestral genes from ancient organisms can be reconstructed de novo and tested for function using a combination of phylogenetic and biochemical methods.

GnRH and its analogs are used extensively for the treatment of hormone-dependent diseases and assisted reproductive tech-niques. They also have potential as novel contraceptives in men and women. A thorough delineation of the molecular mecha-nisms involved in ligand binding, receptor activation, and in-tracellular signal transduction is kernel to understanding dis-ease processes and the development of specific interventions. Twenty-three structural variants of GnRH have been identified in protochordates and vertebrates. In many vertebrates, three GnRHs and three cognate receptors have been identified with distinct distributions and functions. In man, the hypothalamic GnRH regulates gonadotropin secretion through the pituitary GnRH type I receptor via activation of Gq. In-depth studies have identified amino acid residues in both the ligand and recep-tor involved in binding, receptor activation, and translation into

Visual systems of vertebrates exhibit a striking level of diversity, reflecting their adaptive responses to various color environments. The photosensitive molecules, visual pigments, can be synthesized in vitro and their absorption spectra can be determined. Comparing the amino acid sequences and absorption spectra of various visual pigments, we can identify amino acid changes that have modified the absorption spectra of visual pigments. These hypotheses can then be tested using the in vitro assay. This approach has been a powerful tool in elucidating not only the molecular bases of color vision, but the processes of adaptive evolution at the molecular level.q 2002 Elsevier Science B.V. All rights reserved.

G-protein-coupled receptors (GPCRs) constitute a large and diverse family of proteins whose primary function is to transduce extracellular stimuli into intracellular signals. They are among the largest and most diverse protein families in mammalian genomes. On the basis of homology with rhodopsin, they are predicted to contain seven membrane-spanning helices, an extracellular N-terminus and an intracellular C-terminus. This gives rise to their other names, the 7-TM receptors or the heptahelical receptors. GPCRs transduce extracellular stimuli to give intracellular signals through interaction of their intracellular domains with heterotrimeric G proteins, and the crystal structure of one member of this group, bovine rhodopsin, has recently been solved (Palczewski et al., 2000). The presence of GPCRs in the genomes of bacteria, yeast, plants, nematodes and other invertebrate groups argues in favor of a relatively early evolutionary origin of this group of molecules. The diversity of GPCRs is dictated both by the multiplicity of stimuli to which they respond, as well as by the variety of intracellular signalling pathways they activate. These include light, neurotransmitters, odorants, biogenic amines, lipids, proteins, amino acids, hormones, nucleotides, chemokines and, undoubtedly, many others. In addition, there are at least 18 different human Ga proteins to which GPCRs can be coupled (Hermans, 2003; Wong, 2003). These Ga proteins form heterotrimeric complexes with G b subunits, of which there are at least 5 types, and Gg subunits, of which there are at least 11 types (Hermans, 2003). Estimates of the number of GPCRs in the human genome vary widely. Based on their sequences, as well as on their known or suspected functions, there are estimated to be five or six major classes of GPCR. In a recent analysis of the

For over 30 years, photoreceptors have been an outstanding model system for elucidating basic principles in sensory transduction and G protein signaling. Recently, photoreceptors have become an equally attractive model for studying many facets of neuronal cell biology. The primary goal of this review is to illustrate this rapidly growing trend. We will highlight the areas of active research in photoreceptor biology that reveal how different specialized compartments of the cell cooperate in fulfilling its overall function: converting photon absorption into changes in neurotransmitter release. The same trend brings us closer to understanding how defects in photoreceptor signaling can lead to cell death and retinal degeneration.