Do evolutionary changes in cytochrome c structure reflect functional adaptations?

Following the demonstration that the rate of evolutionary change in the amino acid sequences of cytochromes c of eukaryotic species was not constant either for a single line of phylogenetic descent during different evolutionary intervals or for separate lines of descent, the concept that neutral mutations account for the vast majority of the evolutionary variations could no longer be accepted. Previous studies had shown that all eukaryotic cytochromes c tested appeared to be functionally indistinguishable in their reaction with mitochondrial respiratory chain components. However, an examination of the kinetics at low ionic strength led to the discovery of a high affinity reaction of cytochrome c with cytochrome c oxidase that revealed large differences in activity between the cytochromes of the horse, baker's yeast and the protist Euglena. Observed Km values for this reaction of 10(-7) to 10(-8) M appear to represent actual dissociation constants, as demonstrated by direct binding studies of cytochrome c with purified cytochrome c oxidase. The high affinity reaction is sensitive to ionic strength and inhibited by ADP and ATP in the range of physiological concentrations, ATP being three times as effective as ADP. The possibility is discussed that this effect of ATP on cytochrome c binding to its oxidase could provide the basis of a mechanism for mitochondrial respiratory control. The demonstration of differences between cytochrome c of various species in this kinetic system opens the way to a systematic study of the possible evolutionary adaptations of cytochromes c to their oxidases.     

Evolutionary genetics: what is driving male mutation?

In mammals, most new mutations occur in males. But a study of the evolution of a human X to Y chromosomal translocation has revealed a sex bias much lower than previous estimates. Patterns of substitution suggest that differential methylation between male and female germ lines is a key determinant of the mutation rate.     

From protein structure to biochemical function?

Here we describe various methods currently under development aimed at identifying a proteins function from its three-dimensional structure. We are combining a number of these methods to create a pipeline of applications, called ProFunc, which will take a given 3D structure, run all the applications on it and compile and summarise the results obtained. The aim is to provide a best guess as to the proteins function from the evidence provided by the different methods. Here we present three examples, using structures solved by the Midwest Center for Structural Genomics consortium, illustrating the strengths and weaknesses of current approaches.     

Evolutionary adaptations by fish to ecotonal complexity in spatially variable landscapes — a perspective

In most types of freshwater ecosystems fish diversity depends greatly on land/inland water ecotones. So, to maintain biodiversity of fish communities in inland waters, management and restoration of aquatic terrestrial ecotones will be an important tool. However, to provide a scientific background for such conservation activities, it will be desirable to test the importance of different types of ecotones in structuring and maintaining the genetic diversity of fish populations. The relevance of population genetics data to ecotone studies can only be understood in an ecological context as evolution is a function of environment. We suggest that as ecotone complexity increases opportunities for survival of individuals, improving trophic conditions and spatial habitat heterogeneity, so the population size and variation increase with increased genetic diversity and vulnerability to environment changes decreases.     

Do voltage-gated Kv1.1 and inward rectifier Kir2.1 potassium channels form heteromultimers?

Possible heteromultimer formation between Kv- and Kir-type K⁺ channels was investigated, in connection with the known functional diversity of K⁺ channels in vivo. Voltage-clamp experiments were performed on Xenopus oocytes, either injected with concatenated Kir2.1-Kv1.1 mRNA, or co-injected with Kv1.1 and Kir2.1 mRNA. K⁺ currents could be approximated by the algebraic sum of the 2 K⁺ current types alone. The tandem construct did not show functional expression, although it could be detected by Western blotting. We conclude that Kv1.1 and Kir2.1 α-subunit proteins fail to assemble and do not contribute functional diversity to K⁺ channels.     

Evidence against functional interaction between aquaporin-4 water channels and Kir4.1 potassium channels in retinal Müller cells

Indirect evidence suggests that the Müller/glial cell water channel aquaporin-4 (AQP4) modulates K(+) channel function of the closely associated Kir4.1 protein. We used patch clamp to compare Kir4.1 K(+) channel function in freshly isolated Müller cells from retinas of wild-type (+/+) and AQP4 knock-out (-/-) mice. Immunocytochemistry showed a comparable Kir4.1 protein expression pattern in Müller cells from +/+ and -/- retinas, with greatest expression at their end feet. Osmotic water permeability was >4-fold reduced in -/- than in +/+ Müller cells. Resting membrane potential did not differ significantly in +/+ versus -/- Müller cells (-64 +/- 1 versus -64 +/- 1 mV, S.E., n = 24). Whole-cell K(+) currents recorded with a micropipette inserted into the cell soma were Ba(2+)-sensitive and showed no significant differences in magnitude in +/+ versus -/- Müller cells (1.3 +/- 0.1 versus 1.2 +/- 0.1 nA at -160 mV) or in inwardly rectifying current-voltage relationships. Spatially resolved K(+) currents generated by pulsed K(+) injections along Müller cell bodies were also comparable in +/+ versus -/- Müller cells. Single-channel cell-attached patch clamp showed comparable unitary conductance, current-voltage data, and open probability in +/+ versus -/- Müller cells. Thus, contrary to the generally accepted view, our results provide direct evidence against functionally significant AQP4 modulation of Müller cell Kir4.1 K(+) channel function.     

The emerging structure of the Extended Evolutionary Synthesis: where does Evo-Devo fit in?

The Extended Evolutionary Synthesis (EES) debate is gaining ground in contemporary evolutionary biology. In parallel, a number of philosophical standpoints have emerged in an attempt to clarify what exactly is represented by the EES. For Massimo Pigliucci, we are in the wake of the newest instantiation of a persisting Kuhnian paradigm; in contrast, Telmo Pievani has contended that the transition to an EES could be best represented as a progressive reformation of a prior Lakatosian scientific research program, with the extension of its Neo-Darwinian core and the addition of a brand-new protective belt of assumptions and auxiliary hypotheses. Here, we argue that those philosophical vantage points are not the only ways to interpret what current proposals to ‘extend’ the Modern Synthesis-derived ‘standard evolutionary theory’ (SET) entail in terms of theoretical change in evolutionary biology. We specifically propose the image of the emergent EES as a vast network of models and interweaved representations that, instantiated in diverse practices, are connected and related in multiple ways. Under that assumption, the EES could be articulated around a paraconsistent network of evolutionary theories (including some elements of the SET), as well as models, practices and representation systems of contemporary evolutionary biology, with edges and nodes that change their position and centrality as a consequence of the co-construction and stabilization of facts and historical discussions revolving around the epistemic goals of this area of the life sciences. We then critically examine the purported structure of the EES—published by Laland and collaborators in 2015—in light of our own network-based proposal. Finally, we consider which epistemic units of Evo-Devo are present or still missing from the EES, in preparation for further analyses of the topic of explanatory integration in this conceptual framework.     

Evolutionary changes in protein composition — evidence for an optimal strategy

The information contained in the composition of different proteins of the same family is analyzed. It is found that within each family the gain in information per amino acid replacement is constant. This finding is interpreted to imply that evolutionary changes in proteins follow an "optimal" path in the sense that they maximize the number of potentially functional sequences that can be generated by T accepted point mutations from a given protein, subject to restrictions due to biological function.     

Thermal denaturation of Bungarus fasciatus acetylcholinesterase: Is aggregation a driving force in protein unfolding?

A monomeric form of acetylcholinesterase from the venom of Bungarus fasciatus is converted to a partially unfolded molten globule species by thermal inactivation, and subsequently aggregates rapidly. To separate the kinetics of unfolding from those of aggregation, single molecules of the monomeric enzyme were encapsulated in reverse micelles of Brij 30 in 2,2,4-trimethylpentane, or in large unilamellar vesicles of egg lecithin/cholesterol at various protein/micelle (vesicle) ratios. The first-order rate constant for thermal inactivation at 45 degrees C, of single molecules entrapped within the reverse micelles (0.031 min(-1)), was higher than in aqueous solution (0.007 min(-1)) or in the presence of normal micelles (0.020 min(-1)). This clearly shows that aggregation does not provide the driving force for thermal inactivation of BfAChE. Within the large unilamellar vesicles, at average protein/vesicle ratios of 1:1 and 10:1, the first-order rate constants for thermal inactivation of the encapsulated monomeric acetylcholinesterase, at 53 degrees C, were 0.317 and 0.342 min(-1), respectively. A crosslinking technique, utilizing the photosensitive probe, hypericin, showed that thermal denaturation produces a distribution of species ranging from dimers through to large aggregates. Consequently, at a protein/vesicle ratio of 10:1, aggregation can occur upon thermal denaturation. Thus, these experiments also demonstrate that aggregation does not drive the thermal unfolding of Bungarus fasciatus acetylcholinesterase. Our experimental approach also permitted monitoring of recovery of enzymic activity after thermal denaturation in the absence of a competing aggregation process. Whereas no detectable recovery of enzymic activity could be observed in aqueous solution, up to 23% activity could be obtained for enzyme sequestered in the reverse micelles.     

Is phylogeny driving tendon length in lizards?

Tulli, M.J., Herrel, A., Vanhooydonck, B. and Abdala, V. 2012. Is phylogeny driving tendon length in lizards?—Acta Zoologica (Stockholm) 93 : 319–329. Tendons transmit tensile forces generated by muscles and are a crucial part of the musculoskeletal system in vertebrates. Because tendons and tendon cells respond to altered mechanical load by increasing collagen synthesis, we hypothesized that a correlation between tendon morphology and the loading regime imposed by locomotor style or habitat use exists. This makes tendons an interesting model for studying the relationship between morphology and environment. In this study, we compare the general morphology of the palmar flexor plate, the length of the digital tendons, and the length of the flexor carpi ulnaris tendon in species of lizards that exploit a variety of structural habitats. The results from statistical analyses show that phylogenetic relatedness has a major impact on our ability to detect differences between habitat groups, and no differences in tendon length could be detected between iguanian species occupying different habitats when taking into account the relatedness between species. Our data for lizards diverge from the general mammalian paradigm where variation in tendon is often associated with habitat use or locomotor style.     

Phylogenetic conservation of protein–lipid motifs in pentameric ligand-gated ion channels

Using the crosstalk between the nicotinic acetylcholine receptor (nAChR) and its lipid microenvironment as a paradigm, this short overview analyzes the occurrence of structural motifs which appear not only to be conserved within the nAChR family and contemporary eukaryotic members of the pentameric ligand-gated ion channel (pLGIC) superfamily, but also extend to prokaryotic homologues found in bacteria. The evolutionarily conserved design is manifested in: 1) the concentric three-ring architecture of the transmembrane region, 2) the occurrence in this region of distinct lipid consensus motifs in prokaryotic and eukaryotic pLGIC and 3) the key participation of the outer TM4 ring in conveying the influence of the lipid membrane environment to the middle TM1–TM3 ring and this, in turn, to the inner TM2 channel-lining ring, which determines the ion selectivity of the channel. The preservation of these constant structural–functional features throughout such a long phylogenetic span likely points to the successful gain-of-function conferred by their early acquisition. This article is part of a Special Issue entitled: Lipid–protein interactions.     

Does secondary structure determine tertiary structure in proteins?

Is highly approximate knowledge of a protein's backbone structure sufficient to successfully identify its family, superfamily, and tertiary fold? To explore this question, backbone dihedral angles were extracted from the known three‐dimensional structure of 2,439 proteins and mapped into 36 labeled, 60° × 60° bins, called mesostates. Using this coarse‐grained mapping, protein conformation can be approximated by a linear sequence of mesostates. These linear strings can then be aligned and assessed by conventional sequence‐comparison methods. We report that the mesostate sequence is sufficient to recognize a protein's family, superfamily, and fold with good fidelity. Proteins 2005. © 2005 Wiley‐Liss, Inc.     

Angular disorder in actin: is it consistent with general principles of protein structure?

Harold Erickson has recently provided a useful analysis of helical structures having one class versus two classes of intersubunit bonds. His analysis is based upon an assumption that the subunits themselves are essentially unchanged upon bond formation (polymerization). He shows that such a structure having two classes of bonds (i.e. one in which each subunit interacts with four of its neighbors rather than two) can explain some of the features of actin. While he acknowledges that for actin there could be a conformational change and that, in principle, it could explain such features, he argues that the allowed magnitude of such a conformational change is inadequate. Since kinetics and thermodynamics cannot distinguish between the energy derived from the formation of a bond from that due to a conformational change, the question of whether the features of F-actin are derived from a conformational change or a system of two classes of bonds or both must be answered with high-resolution structural information. Recent studies by K. C. Holmes and others suggest that the second possibility might be closest to the truth. The heart of our disagreement is not whether Erickson's thermodynamic analysis is correct, given rigid subunits, but whether all protein polymers are characterized by rigid subunits with rigid intersubunit contacts. Erickson maintains that the observation of an angular disorder of 12 degrees per subunit within the actin filament conflicts with his formalism of rigid subunit interfaces and must therefore result from the erroneous interpretation of measurements. He presents an alternative model to explain the observations. His model, however, does not account for the observations and we will argue that, ultimately, like the resolution of the matter of the number of classes of bonds and the extent of their contact, the amount of angular disorder will require higher-resolution structural studies.     

Contingency Table Browser ? prediction of early stage protein structure

The Early Stage (ES) intermediate represents the starting structure in protein folding simulations based on the Fuzzy Oil Drop (FOD) model. The accuracy of FOD predictions is greatly dependent on the accuracy of the chosen intermediate. A suitable intermediate can be constructed using the sequence-structure relationship information contained in the so-called contingency table − this table expresses the likelihood of encountering various structural motifs for each tetrapeptide fragment in the amino acid sequence. The limited accuracy with which such structures could previously be predicted provided the motivation for a more indepth study of the contingency table itself. The Contingency Table Browser is a tool which can visualize, search and analyze the table. Our work presents possible applications of Contingency Table Browser, among them − analysis of specific protein sequences from the point of view of their structural ambiguity.     

Does Intrinsic Disorder in Proteins Favor Their Interaction with Lipids?

Intrinsically disordered proteins (IDPs) are implicated in a range of human diseases, some of which are associated with the ability to bind to lipids. Although the presence of solvent‐exposed hydrophobic regions in IDPs should favor their interactions with low‐molecular‐weight hydrophobic/amphiphilic compounds, this hypothesis has not been systematically explored as of yet. In this study, the analysis of the DisProt database with regard to the presence of lipid‐binding IDPs (LBIDPs) reveals that they comprise, at least, 15% of DisProt entries. LBIDPs are classified into four groups by ligand type, functional categories, domain structure, and conformational state. 57% of LBIDPs are classified as ordered according to the CH‐CDF analysis, and 70% of LBIDPs possess lengths of disordered regions below 50%. To investigate the lipid‐binding properties of IDPs for which lipid binding is not reported, three proteins from different conformational groups are rationally selected. They all are shown to bind linoleic (LA) and oleic (OA) acids with capacities ranging from 9 to 34 LA/OA molecules per protein molecule. The association with LA/OA causes the formation of high‐molecular‐weight lipid–protein complexes. These findings suggest that lipid binding is common among IDPs, which can favor their involvement in lipid metabolism.     

Refinement of the protein backbone angle ψ in NMR structure calculations

Cross-correlated relaxation rates involving the C-H dipolar interaction and the carbonyl (C) chemical shift anisotropy (CSA) have been measured using two complementary 3D experiments. We show that the protein backbone angle can be directly refined against such cross-correlated relaxation rates (^{H C,C}) and the three-bond H/D isotope effect on the C chemical shifts (³C _(ND)). By simultaneously using both experimental parameters as restraints during NMR structure calculations, a unique value for the backbone angle is defined. We have applied the new refinement method to the -Spectrin SH3 domain (a -sheet protein) and to the Sgs1p HRDC domain (an -helical protein) and show that the quality of the NMR structures is substantially improved, judging from the atomic coordinate precision and the Ramachandran map. In addition, the -refined NMR structures of the SH3 domain deviate less from the 1.8 Å crystal structure, suggesting an improved accuracy. The proposed refinement method can be used to significantly improve the quality of NMR structures and will be applicable to larger proteins.     

Mechanosensitive channels in bacteria: signs of closure?

Bacterial mechanosensitive channels are activated by increases in tension in the lipid bilayer of the cytoplasmic membrane, where they transiently create large pores in a controlled manner. Mechanosensitive channel research has benefited from advances in electrophysiology, genomics and molecular genetics as well as from the application of biophysical techniques. Most recently, new analytical methods have been used to complement existing knowledge and generate insights into the molecular interactions that take place between mechanosensitive channel proteins and the surrounding membrane lipids. This article reviews the latest developments.