Homology versus analogy: possible evolutionary relationship of immunoglobulins, cupredoxins, and Cu,Zn-superoxide dismutase

The 'immunoglobulin-like' fold is one of most common structural motifs observed in proteins. This topology is found in more than 80 superfamilies of proteins, including Cu,Zn-superoxide dismutase (SOD) and cupredoxin. Evolutionary relationships have not been identified, but may exist. The challenge remains, therefore, of resolving the issue of whether the diverse distribution of the fold is accounted for by divergent evolution of function or convergent evolution of structure following multiple independent origins of function. Since the early studies that revealed conformational similarity of immunoglobulins and other proteins, the number of primary structures available for comparison has dramatically increased and new computational approaches for analysis of sequences have been developed. It now appears that a hypothesis of a common evolutionary origin for cupredoxins, Cu,Zn-SOD, and immunoglobulins may be credible. The distinction between protein homology and protein analogy is fundamental. The immunoglobulin-like fold may represent a robust system within which to examine again the issue of protein homology versus analogy.     

The evolution fo rhodopsins and neurotransmitter receptors

Summary Rhodopsins share a limited number of amino acid identities with a variety of other integral membrane proteins. Most of these proteins have seven putative transmembrane segments and are likely to play a role in transmembrane signaling. We have undertaken a systematic series of comparisons of primary and secondary structure in order to clarify the functional and evolutionary significance of these sequence similarities. On the basis of consistently high similarity scores, we find that the most internally consistent definition of the rhodopsis gene family would ionclude vertebrate rhodospins, - and -adrenergic receptors, M1 and M2 muscarinic acetylcholine receptors, substance K receptors and insect rhodopsins, while excluding bacterirhodopsin, themas human oncogene, vertebrate and insect nicotinic acetylcholine receptors, and the yeast STE2 and STE3 peptide receptors. The rhodopsin gene family is highly diverged at the primary sequence level but has maintained a conserved secondary structure, including a previosuly unidentified hierarchy of transmembrane segment hydrophobicity. We have deevelope new computer alogithms for progressive multiple sequence alignment and the analysis of local conservation of protein domains, and we have used these algorithms to examined the phylogeny of the rhodopsin gene family and the changing domains of sequence conservation. The results show striking diffiierences and similarities in the conserved domains in each of the three main branches of the rhodopsin gene family, and indicte that color vision arose independently in the lines of descent leading to modern humans and fruit flies.     

Protein-assisted pericyclic reactions: an alternate hypothesis for the action of quantal receptors

The rules for allowable pericyclic reactions indicate that the photoisomerizations of retinals in rhodopsins can be formally analogous to thermally promoted Diels-Alder condensations of monoenes with retinols. With little change in the seven-transmembrane helical environment these latter reactions could mimic the retinal isomerization while providing highly sensitive chemical reception. In this way archaic progenitors of G-protein-coupled chemical quantal receptors such as those for pheromones might have been evolutionarily plagiarized from the photon quantal receptor, rhodopsin, or vice versa. We investigated whether the known structure of bacteriorhodopsin exhibited any similarity in its active site with those of the two known antibody catalysts of Diels-Alder reactions and that of the photoactive yellow protein. A remarkable three-dimensional motif of aromatic side chains emerged in all four proteins despite the drastic differences in backbone structure. Molecular orbital calculations supported the possibility of transient pericyclic reactions as part of the isomerization-signal transduction mechanisms in both bacteriorhodopsin and the photoactive yellow protein. It appears that reactions in all four of the proteins investigated may be biological analogs of the organic chemists' chiral auxiliary-aided Diels-Alder reactions. Thus the light receptor and the chemical receptor subfamilies of the heptahelical receptor family may have been unified at one time by underlying pericyclic chemistry.     

Infrared spectroscopic study of photoreceptor membrane and purple membrane. Protein secondary structure and hydrogen deuterium exchange

Infrared spectroscopy in the interval from 1800 to 1300 cm-1 has been used to investigate the secondary structure and the hydrogen/deuterium exchange behavior of bacteriorhodopsin and bovine rhodopsin in their respective native membranes. The amide I' and amide II' regions from spectra of membrane suspensions in D2O were decomposed into constituent bands by use of a curve-fitting procedure. The amide I' bands could be fit with a minimum of three theoretical components having peak positions at 1664, 1638, and 1625 cm-1 for bacteriorhodopsin and 1657, 1639, and 1625 cm-1 for rhodopsin. For both of these membrane proteins, the amide I' spectrum suggests that alpha-helix is the predominant form of peptide chain secondary structure, but that a substantial amount of beta-sheet conformation is present as well. The shape of the amide I' band was pH-sensitive for photoreceptor membranes, but not for purple membrane, indicating that membrane-bound rhodopsin undergoes a conformation change at acidic pH. Peptide hydrogen exchange of bacteriorhodopsin and rhodopsin was monitored by observing the change in the ratio of integrated absorbance (Aamide II'/Aamide I') during the interval from 1.5 to 25 h after membranes were introduced into buffered D2O. The fraction of peptide groups in a very slowly exchanging secondary structure was estimated to be 0.71 for bacteriorhodopsin at pD 7. The corresponding fraction in vertebrate rhodopsin was estimated to be less than or equal to 0.60. These findings are discussed in relationship to previous studies of hydrogen exchange behavior and to structural models for both proteins.     

Adaptive evolution of G-protein coupled receptor genes

The phylogeny and patterns of nucleotide substitutions in the visual pigment genes, adrenergic receptor genes, muscarinic receptor genes, and in the human mas oncogene were studied by comparing their DNA sequences. The evolutionary tree obtained shows that the visual pigment genes and mas oncogene form one cluster and that the receptor genes form another. In the evolution of rhodopsin genes, synonymous substitutions outnumber nonsynonymous substitutions. This is consistent with the neutral theory of molecular evolution. However, the early evolutionary stages of alpha- and beta-adrenergic and muscarinic receptors are notable for significantly more nonsynonymous substitutions than synonymous substitutions, suggesting the acquisition of novel functional adaptations. Variable rates of nonsynonymous changes in different domains of these proteins reveal DNA segments that might have been important in their functional adaptations.      

A galaxy of folds

Many protein classification systems capture homologous relationships by grouping domains into families and superfamilies on the basis of sequence similarity. Superfamilies with similar 3D structures are further grouped into folds. In the absence of discernable sequence similarity, these structural similarities were long thought to have originated independently, by convergent evolution. However, the growth of databases and advances in sequence comparison methods have led to the discovery of many distant evolutionary relationships that transcend the boundaries of superfamilies and folds. To investigate the contributions of convergent versus divergent evolution in the origin of protein folds, we clustered representative domains of known structure by their sequence similarity, treating them as point masses in a virtual 2D space which attract or repel each other depending on their pairwise sequence similarities. As expected, families in the same superfamily form tight clusters. But often, superfamilies of the same fold are linked with each other, suggesting that the entire fold evolved from an ancient prototype. Strikingly, some links connect superfamilies with different folds. They arise from modular peptide fragments of between 20 and 40 residues that co‐occur in the connected folds in disparate structural contexts. These may be descendants of an ancestral pool of peptide modules that evolved as cofactors in the RNA world and from which the first folded proteins arose by amplification and recombination. Our galaxy of folds summarizes, in a single image, most known and many yet undescribed homologous relationships between protein superfamilies, providing new insights into the evolution of protein domains.     

Proteomic traces of speciation

Recent work has shown that the network of structural similarity between protein domains exhibits a power-law distribution of edges per node. The scale-free nature of this graph, termed the protein domain universe graph or PDUG, may be reproduced via a divergent model of structural evolution. The performance of this model, however, does not preclude the existence of a successful convergent model. To further resolve the issue of protein structural evolution, we explore the predictions of both convergent and divergent models directly. We show that when nodes from the PDUG are partitioned into subgraphs on the basis of their occurrence in the proteomes of particular organisms, these subgraphs exhibit a scale-free nature as well. We explore a simple convergent model of structural evolution and find that the implications of this model are inconsistent with features of these organismal subgraphs. Importantly, we find that biased convergent models are inconsistent with our data. We find that when speciation mechanisms are added to a simple divergent model, subgraphs similar to the organismal subgraphs are produced, demonstrating that dynamic models can easily explain the distributions of structural similarity that exist within proteomes. We show that speciation events must be included in a divergent model of structural evolution to account for the non-random overlap of structural proteomes. These findings have implications for the long-standing debate over convergent and divergent models of protein structural evolution, and for the study of the evolution of organisms as a whole.     

Matrix-assisted laser desorption mass spectrometry of rhodopsin and bacteriorhodopsin.

Matrix-assisted laser desorption ionization (MALDI) mass spectrometry has been used to obtain accurate molecular weight information for the integral membrane proteins bacteriorhodopsin and bovine rhodopsin desorbed from solubilized membrane preparations. Mass differences in the molecular weights measured for bleached and unbleached bacteriorhodopsin and rhodopsin indicate the removal of the retinal chromophores upon bleaching. The MALDI technique was also successful for determination of the major cleavage products obtained upon treatment of membrane bound rhodopsin with endoproteinase Asp-N and thermolysin. Our results indicate that the MALDI method is a useful means of obtaining accurate molecular weight information on hydrophobic proteins isolated in their native membranes.     

Analysis of protein homology by assessing the (dis)similarity in protein loop regions

Two proteins are considered to have a similar fold if sufficiently many of their secondary structure elements are positioned similarly in space and are connected in the same order. Such a common structural scaffold may arise due to either divergent or convergent evolution. The intervening unaligned regions ("loops") between the superimposable helices and strands can exhibit a wide range of similarity and may offer clues to the structural evolution of folds. One might argue that more closely related proteins differ less in their nonconserved loop regions than distantly related proteins and, at the same time, the degree of variability in the loop regions in structurally similar but unrelated proteins is higher than in homologs. Here we introduce a new measure for structural (dis)similarity in loop regions that is based on the concept of the Hausdorff metric. This measure is used to gauge protein relatedness and is tested on a benchmark of homologous and analogous protein structures. It has been shown that the new measure can distinguish homologous from analogous proteins with the same or higher accuracy than the conventional measures that are based on comparing proteins in structurally aligned regions. We argue that this result can be attributed to the higher sensitivity of the Hausdorff (dis)similarity measure in detecting particularly evident dissimilarities in structures and draw some conclusions about evolutionary relatedness of proteins in the most populated protein folds.     

Electrospray-ionization mass spectrometry of intact intrinsic membrane proteins.

Membrane proteins drive and mediate many essential cellular processes making them a vital section of the proteome. However, the amphipathic nature of these molecules ensures their detailed structural analysis remains challenging. A versatile procedure for effective electrospray-ionization mass spectrometry (ESI-MS) of intact intrinsic membrane proteins purified using reverse-phase chromatography in aqueous formic acid/isopropanol is presented. The spectra of four examples, bacteriorhodopsin and its apoprotein from Halobacterium and the D1 and D2 reaction-center subunits from spinach thylakoids, achieve mass measurements that are within 0.01% of calculated theoretical values. All of the spectra reveal lesser quantities of other molecular species that can usually be equated with covalently modified subpopulations of these proteins. Our analysis of bovine rhodopsin, the first ESI-MS study of a G-protein coupled receptor, yielded a complex spectrum indicative of extensive molecular heterogeneity. The range of masses measured for the native molecule agrees well with the range calculated based upon variable glycosylation and reveals further heterogeneity arising from other covalent modifications. The technique described represents the most precise way to catalogue membrane proteins and their post-translational modifications. Resolution of the components of protein complexes provides insights into native protein/protein interactions. The apparent retention of structure by bacteriorhodopsin during the analysis raises the potential of obtaining tertiary structure information using more developed ESI-MS experiments.     

A microbial rhodopsin with a unique retinal composition shows both sensory rhodopsin II and bacteriorhodopsin-like properties

Rhodopsins possess retinal chromophore surrounded by seven transmembrane α-helices, are widespread in prokaryotes and in eukaryotes, and can be utilized as optogenetic tools. Although rhodopsins work as distinctly different photoreceptors in various organisms, they can be roughly divided according to their two basic functions, light-energy conversion and light-signal transduction. In microbes, light-driven proton transporters functioning as light-energy converters have been modified by evolution to produce sensory receptors that relay signals to transducer proteins to control motility. In this study, we cloned and characterized two newly identified microbial rhodopsins from Haloquadratum walsbyi. One of them has photochemical properties and a proton pumping activity similar to the well known proton pump bacteriorhodopsin (BR). The other, named middle rhodopsin (MR), is evolutionarily transitional between BR and the phototactic sensory rhodopsin II (SRII), having an SRII-like absorption maximum, a BR-like photocycle, and a unique retinal composition. The wild-type MR does not have a light-induced proton pumping activity. On the other hand, a mutant MR with two key hydrogen-bonding residues located at the interaction surface with the transducer protein HtrII shows robust phototaxis responses similar to SRII, indicating that MR is potentially capable of the signaling. These results demonstrate that color tuning and insertion of the critical threonine residue occurred early in the evolution of sensory rhodopsins. MR may be a missing link in the evolution from type 1 rhodopsins (microorganisms) to type 2 rhodopsins (animals), because it is the first microbial rhodopsin known to have 11-cis-retinal similar to type 2 rhodopsins.     

Use of an in situ disulfide cross-linking strategy to map proximities between amino acid residues in transmembrane domains I and VII of the M3 muscarinic acetylcholine receptor

In this study, we employed an in situ disulfide cross-linking strategy to gain insight into the structure of the inactive and active state of the M(3) muscarinic acetylcholine receptor. Specifically, this study was designed to identify residues in TM I that are located in close to Cys532 (position 7.42), an endogenous cysteine residue present in the central portion of TM VII. Cysteine residues were substituted, one at a time, into 10 consecutive positions of TM I (Ala71-Val80) of a modified version of the M(3) muscarinic receptor that lacked most endogenous cysteine residues and contained a factor Xa cleavage site within the third intracellular loop. Following their expression in COS-7 cells, the 10 resulting cysteine mutant receptors were oxidized in their native membrane environment, either in the absence or in the presence of muscarinic ligands. Disulfide cross-link formation was monitored by examining changes in the electrophoretic mobility of oxidized and factor Xa-digested receptors on SDS gels. When molecular iodine was used as the oxidizing agent, the L77C receptor (position 1.42) was the only mutant receptor that displayed significant disulfide cross-linking, either in the absence or in the presence of muscarinic agonists or antagonists. On the other hand, when the Cu(II)-(1,10-phenanthroline)(3) complex served as the redox catalyst, muscarinic ligands inhibited disulfide cross-linking of the L77C receptor, probably because of impaired access of this relatively bulky oxidizing agent to the ligand binding crevice. The iodine cross-linking data suggest that M(3) muscarinic receptor activation is not associated with significant changes in the relative orientations of the outer and/or central segments of TM I and VII. In bovine rhodopsin, the residues present at the positions corresponding to Cys532 and Leu77 in the rat M(3) muscarinic receptor are not located directly adjacent to each other, raising the possibility that the relative orientations of TM I and VII are not identical among different class I GPCRs. Alternatively, dynamic protein backbone fluctuation may occur, enabling Cys532 to move within cross-linking distance of Leu77 (Cys77).     

Primary structure of sensory rhodopsin I, a prokaryotic photoreceptor.

The gene coding for sensory rhodopsin I (SR-I) has been identified in a restriction fragment of genomic DNA from the Halobacterium halobium strain L33. Of the 1014 nucleotides whose sequence was determined, 720 belong to the structural gene of SR-I. In the 5' non-coding region two putative promoter elements and a ribosomal binding site have been identified. The 3' flanking region bears a potential terminator structure. The SR-I protein moiety carries no signal peptide and is not processed at its N terminus. The C terminus, however, lacks the last aspartic acid residue encoded by the gene. Analysis of the primary structure of SR-I reveals no consistent homology with the eukaryotic photoreceptor rhodopsin, but 14% homology with the halobacterial ion pumps, bacteriorhodopsin (BR) and halorhodopsin (HR). Residues conserved in all three proteins are discussed with respect to their contribution to secondary structure, retinal binding and ion translocation. The aspartic acid residue which mediates in BR the reprotonation of the Schiff base (D96) is replaced in SR-I by a tyrosine (Y87). This amino acid replacement is proposed to be of crucial importance in the evolution of the slow-cycling photosensing pigment SR-I.     

Bacterioopsin, haloopsin, and sensory opsin I of the halobacterial isolate Halobacterium sp. strain SG1: three new members of a growing family.

The genes coding for bacterioopsin, haloopsin, and sensory opsin I of a halobacterial isolate from the Red Sea called Halobacterium sp. strain SG1 have been cloned and sequenced. The deduced protein sequences were aligned to the previously known halobacterial retinal proteins. The addition of these new sequences lowered the number of conserved residues to only 23 amino acids, or 8% of the alignment. Data base searches with two highly conserved peptides as well as with an alignment profile yielded no significant similarity to any other protein, so the halobacterial retinal proteins should be regarded as a distinct protein family. The protein alignment was used to make predictions about the structure of the retinal proteins as well as about the amino acids in contact with retinal proteins. These results were in excellent agreement with the structural model of bacteriorhodopsin of Halobacterium halobium as well as with mutant studies, indicating that (i) structure predictions based on the sequences of a membrane protein family can be quite accurate; (ii) halorhodopsin and sensory rhodopsin I have tertiary structures similar to that of bacteriorhodopsin; (iii) conserved amino acids do not take part in reactions specific for one group of proteins, e.g., proton translocation for bacteriorhodopsins, but have a crucial role in determining the conformation and reactions of the chromophore; and (iv) the general mode of action (light-induced chromophore and protein movements) is the same for all halobacterial retinal proteins, ion pumps as well as sensors.     

Fourier transform infrared evidence for a predominantly alpha-helical structure of the membrane bound channel forming COOH-terminal peptide of colicin E1.

The structure of the membrane bound state of the 178-residue thermolytic COOH-terminal channel forming peptide of colicin E1 was studied by polarized Fourier transform infrared (FTIR) spectroscopy. This fragment was reconstituted into DMPC liposomes at varying peptide/lipid ratios ranging from 1/25-1/500. The amide I band frequency of the protein indicated a dominant alpha-helical secondary structure with limited beta- and random structures. The amide I and II frequencies are at 1,656 and 1,546 cm-1, close to the frequency of the amide I and II bands of rhodopsin, bacteriorhodopsin and other alpha-helical proteins. Polarized FTIR of oriented membranes revealed that the alpha-helices have an average orientation less than the magic angle, 54.6 degrees, relative to the membrane normal. Almost all of the peptide groups in the membrane-bound channel protein undergo rapid hydrogen/deuterium (H/D) exchange. These results are contrasted to the alpha-helical membrane proteins, bacteriorhodopsin, and rhodopsin.     

A novel measure characterized by a polar energy surface approximation for recognition and classification of transmembrane protein structures

A new theoretical method has been developed for recognition and classification of membrane proteins. The method is based on computation of a polar energy surface that can reveal characteristic interaction patterns for individual helices even if crystal or NMR structure coordinates are not available. A protein with N transmembrane helices is described as a set of N vectors that are derived from a Fourier analysis of this polar energy surface computed for each helix. We then derive a polarity difference score (PDS) for any two proteins computed as the root mean square deviation between the respective vector coordinate sets. The score was found to correlate with the degree of structural similarity between the following three protein families for which tertiary structures have been determined: bacteriorhodopsin, rhodopsin, and the cytochrome c oxidase III subunit.     

Rhodopsin and phototransduction: a model system for G protein-linked receptors.

Rhodopsin is the photoreceptor protein in rod cells of the vertebrate retina and the first member of the class of G protein-coupled receptors for which the amino acid sequence was determined. Rhodopsin is available in greater quantities than any other receptor of its class and therefore has been studied biochemically and biophysically by methods difficult or impossible to apply to its fellow receptors. Such studies support a model in which rhodopsin consists of seven transmembrane helices that form a binding pocket for its ligand, 11-cis retinal. Insights into the structure and function of rhodopsin serve as a model for understanding the structure and function of other members of the receptor class. Rhodopsin undergoes a change in conformation upon photoexcitation and activates a G protein, transducin, and is phosphorylated by a receptor-specific kinase, rhodopsin kinase. The phosphorylated photoactivated rhodopsin is bound by arrestin, thereby terminating activity of the receptor in the signal transduction process. These auxiliary proteins that function with rhodopsin on rod cells serve as models for understanding how other members of the receptor family may function in conjunction with other G proteins, kinases, and arrestin-like proteins.     

The regular surface layer of Acetogenium kivui: some structural, developmental and evolutionary aspects

Abstract The structure of the regular surface layer of Acetogenium kivui has been investigated by electron microscopy in conjunction with image processing techniques. From the averaged unit cell structure a tentative model of the subunit organization is derived. The occurrence of mono- and polycrystalline S-layer sheets is interpreted in terms of transitions related to the growth cycle of cell. The question is raised as to whether the striking structural similarity of S-layers, even from evolutionary rather distant species, is due to divergent or convergent evolution.     

NMR studies on fully hydrated membrane proteins, with emphasis on bacteriorhodopsin as a typical and prototype membrane protein

The 3D structures or dynamic feature of fully hydrated membrane proteins are very important at ambient temperature, in relation to understanding their biological activities, although their data, especially from the flexible portions such as surface regions, are unavailable from X-ray diffraction or cryoelectron microscope at low temperature. In contrast, high-resolution solid-state NMR spectroscopy has proved to be a very convenient alternative means to be able to reveal their dynamic structures. To clarify this problem, we describe here how we are able to reveal such structures and dynamic features, based on intrinsic probes from high-resolution solid-state NMR studies on bacteriorhodopsin (bR) as a typical membrane protein in 2D crystal, regenerated preparation in lipid bilayer and detergents. It turned out that their dynamic features are substantially altered upon their environments where bR is present. We further review NMR applications to study structure and dynamics of a variety of membrane proteins, including sensory rhodopsin, rhodopsin, photoreaction centers, diacylglycerol kinases, etc.