How important are transmembrane helices of bitopic membrane proteins?

The topology of a bitopic membrane protein consists of a single transmembrane helix connecting two extra-membranous domains. As opposed to helices from polytopic proteins, the transmembrane helices of bitopic proteins were initially considered as merely hydrophobic anchors, while more recent studies have begun to shed light on their role in the protein's function. Herein the overall importance of transmembrane helices from bitopic membrane proteins was analyzed using a relative conservation analysis. Interestingly, the transmembrane domains of bitopic proteins are on average, significantly more conserved than the remainder of the protein, even when taking into account their smaller amino acid repertoire. Analysis of highly conserved transmembrane domains did not reveal any unifying consensus, pointing to a great diversity in their conservation patterns. However, Fourier power spectrum analysis was able to show that regardless of the conservation motif, in most sequences a significant conservation moment was observed, in that one side of the helix was conserved while the other was not. Taken together, it may be possible to conclude that a significant proportion of transmembrane helices from bitopic membrane proteins participate in specific interactions, in a variety of modes in the plane of the lipid bilayer.     

How important are transmembrane helices of bitopic membrane proteins?

The topology of a bitopic membrane protein consists of a single transmembrane helix connecting two extra-membranous domains. As opposed to helices from polytopic proteins, the transmembrane helices of bitopic proteins were initially considered as merely hydrophobic anchors, while more recent studies have begun to shed light on their role in the protein's function. Herein the overall importance of transmembrane helices from bitopic membrane proteins was analyzed using a relative conservation analysis. Interestingly, the transmembrane domains of bitopic proteins are on average, significantly more conserved than the remainder of the protein, even when taking into account their smaller amino acid repertoire. Analysis of highly conserved transmembrane domains did not reveal any unifying consensus, pointing to a great diversity in their conservation patterns. However, Fourier power spectrum analysis was able to show that regardless of the conservation motif, in most sequences a significant conservation moment was observed, in that one side of the helix was conserved while the other was not. Taken together, it may be possible to conclude that a significant proportion of transmembrane helices from bitopic membrane proteins participate in specific interactions, in a variety of modes in the plane of the lipid bilayer.     

How do membrane proteins sense water stress?

Maintenance of cell turgor is a prerequisite for almost any form of life as it provides a mechanical force for the expansion of the cell envelope. As changes in extracellular osmolality will have similar physicochemical effects on cells from all biological kingdoms, the responses to osmotic stress may be alike in all organisms. The primary response of bacteria to osmotic upshifts involves the activation of transporters, to effect the rapid accumulation of osmoprotectants, and sensor kinases, to increase the transport and/or biosynthetic capacity for these solutes. Upon osmotic downshift, the excess of cytoplasmic solutes is released via mechanosensitive channel proteins. A number of breakthroughs in the last one or two years have led to tremendous advances in our understanding of the molecular mechanisms of osmosensing in bacteria. The possible mechanisms of osmosensing, and the actual evidence for a particular mechanism, are presented for well studied, osmoregulated transport systems, sensor kinases and mechanosensitive channel proteins. The emerging picture is that intracellular ionic solutes (or ionic strength) serve as a signal for the activation of the upshift-activated transporters and sensor kinases. For at least one system, there is strong evidence that the signal is transduced to the protein complex via alterations in the protein-lipid interactions rather than direct sensing of ion concentration or ionic strength by the proteins. The osmotic downshift-activated mechanosensitive channels, on the other hand, sense tension in the membrane but other factors such as hydration state of the protein may affect the equilibrium between open and closed states of the proteins.     

How do Rab proteins function in membrane traffic?

The Rabs are a group of GTP-binding proteins implicated for some time in the targeting of different transport vesicles within the cell, but it has been unclear how they function, or how they relate to a second group of targeting proteins, the SNAREs. Recent work, discussed in this review, has used biophysical, biochemical and genetic approaches to begin to answer these questions for Rab3, Rab5 and the yeast protein Sec4p. However, the results from these three Rabs lead to a surprising conclusion: the different Rabs seem to function via highly diverse target proteins.     

How do BCL-2 proteins induce mitochondrial outer membrane permeabilization?

The mitochondrial pathway of apoptosis proceeds when molecules sequestered between the outer and inner mitochondrial membranes are released to the cytosol by mitochondrial outer membrane permeabilization (MOMP). This process is controlled by the BCL-2 family, which is composed of both pro- and anti-apoptotic proteins. Although there is no disagreement that BCL-2 proteins regulate apoptosis, the mechanism leading to MOMP remains controversial. Current debate focuses on what interactions within the family are crucial to initiate MOMP. Specifically, do the BH3-only proteins directly engage BAX and/or BAK activation or do these proteins solely promote apoptosis by neutralization of anti-apoptotic BCL-2 proteins? We describe these models and contend that BH3-only proteins must perform both functions to efficiently engage MOMP and apoptosis.     

Do biomarkers of exposure and effect correlate with internal exposure to PAHs in swine?

Humans are commonly exposed to polycyclic aromatic hydrocarbons (PAHs), a family of compounds present as mixtures in the environment. This study exposed swine to PAH mixtures in single and subacute dose regimens and collected liver and ileum tissue to measure cytochrome P450 mRNA expression and enzyme activity as biomarkers of exposure and DNA adducts and oxidized proteins as biomarkers of effect. Micronucleated reticulocytes were measured as systemic biomarkers of effect. Duration of exposure did not influence biomarkers of exposure, though exposure duration produced significant increases in DNA adducts and oxidative stress. Micronucleated reticulocyte numbers were not affected by exposure length.     

How do bees learn and recognize visual patterns?

The basic task of perceptual systems is the recognition and localization of objects. The central nervous system has to solve these problems on the basis of the excitation patterns of sensory nerves, in spite of the fact that these provide only ambiguous information about objects. Two processing principles seem to be fundamental for an efficient formation of object representations: the extraction of characteristic features and the ability to assess similarities between different objects. This article reviews investigations in which different training paradigms were applied in order to explore the honeybee's capacities to learn and recognize visual patterns. One aim of these experiments is to assess whether insects use similar processing mechanisms as vertebrates, for instance human beings. By comparing the computational performance of perceptual systems in animals with different evolutionary history we can hope to learn more about the operation of basic rules in nervous systems. “The messages which the brain receives have not the least similarity with the stimuli. They consist in pulses of given intensities and frequencies, characteristic for the transmitting nerve-fiber, which ends at a definite place of the cortex. ... From this information it produces the image of the world by a process which can metaphorically be called a consummate piece of combinatorial mathematics: it sorts out of the maze of indifferent and varying signals, invariant shapes and relations which form the world of ordinary experience.” Max Born (1949) Received: 4 October 1997 / Accepted in revised form: 26 August 1998     

How reliably do amino acid composition comparisons predict sequence similarities between proteins?

A method for comparing amino acid compositions of proteins (Cornish-Bowden, 1977) has been extended to allow proteins of unequal lengths to be compared. The method has been tested by applying it to proteins of known sequence. It tends to exaggerate the amount of difference between unrelated proteins. It is therefore a reliable guide to possible sequence similarities, in that it does not suggest that sequences are similar when they are not, though it sometimes fails to detect genuine similarities. When applied to related proteins the method gives results in good agreement with those predicted. A phylogenetic tree for 37 snake venom toxins has been constructed from their compositions and is similar in most important respects to one constructed from the corresponding sequences.     

How do microbiota associated with an invasive seaweed vary across scales?

Communities are shaped by scale dependent processes. To study the diversity and variation of microbial communities across scales, the invasive and widespread seaweed Agarophyton vermiculophyllum presents a unique opportunity. We characterized pro‐ and eukaryotic communities associated with this holobiont across its known distribution range, which stretches over the northern hemisphere. Our data reveal that community composition and diversity in the holobiont vary at local but also larger geographic scales. While processes acting at the local scale (i.e., within population) are the main structuring drivers of associated microbial communities, changes in community composition also depend on processes acting at larger geographic scales. Interestingly, the largest analysed scale (i.e., native and non‐native ranges) explained variation in the prevalence of predicted functional groups, which could suggest a functional shift in microbiota occurred over the course of the invasion process. While high variability in microbiota at the local scale supports A. vermiculophyllum to be a generalist host, we also identified a number of core taxa. These geographically independent holobiont members imply that cointroduction of specific microbiota may have additionally promoted the invasion process.     

How do chemical denaturants affect the mechanical folding and unfolding of proteins?

We present the first single-molecule atomic force microscopy study on the effect of chemical denaturants on the mechanical folding/unfolding kinetics of a small protein GB1 (the B1 immunoglobulin-binding domain of protein G from Streptococcus). Upon increasing the concentration of the chemical denaturant guanidinium chloride (GdmCl), we observed a systematic decrease in the mechanical stability of GB1, indicating the softening effect of the chemical denaturant on the mechanical stability of proteins. This mechanical softening effect originates from the reduced free-energy barrier between the folded state and the unfolding transition state, which decreases linearly as a function of the denaturant concentration. Chemical denaturants, however, do not alter the mechanical unfolding pathway or shift the position of the transition state for mechanical unfolding. We also found that the folding rate constant of GB1 is slowed down by GdmCl in mechanical folding experiments. By combining the mechanical folding/unfolding kinetics of GB1 in GdmCl solution, we developed the “mechanical chevron plot” as a general tool to understand how chemical denaturants influence the mechanical folding/unfolding kinetics and free-energy diagram in a quantitative fashion. This study demonstrates great potential in combining chemical denaturation with single-molecule atomic force microscopy techniques to reveal invaluable information on the energy landscape underlying protein folding/unfolding reactions.     

Relationship between structure and amino acid sequence of strongly twisted and coiled β-hairpins in globular proteins

β-Hairpins are widespread in proteins, and it is possible to find them both within β-sheets and separately. In this work, a comparative analysis of amino acid sequences of β-strands within strongly twisted β-hairpins from different structural protein subclasses has been conducted. Strongly twisted and coiled β-hairpin generates in the space a right double helix out of β-strands that are connected by a loop region (connections). The frequencies of amino acid residues on the internal (concave) and external (convex) surfaces of strongly twisted β-hairpins have been determined (220 β-hairpins from nonhomologous proteins were studied). The concave surface of these β-hairpins is mainly generated by hydrophobic residues, while the convex surface by hydrophilic residues; accordingly, the alternation of hydrophobic internal and hydrophilic external residues is observed in their amino acid sequences. Amino acid residues of glycine and alanine (especially in places of the largest twisting of the strands) were anomalously frequently found in internal positions of strongly twisted and coiled β-hairpins. It was established that internal positions never contain the proline residues, while external positions in the twisting region contain them in a relatively large amount. It was demonstrated that at least one amino acid residue in α_L- or ε-conformation is required for generation of relatively short (up to 7 amino acid residues) connection. As a rule, these positions are occupied by glycines. Thus, not only the alternation of hydrophobic and hydrophilic amino acid residues, but also the presence of one or two glycine residues in the connection region and the excess of glycines and alanines in the places of the largest strand twisting on the concave surface, as well as the presence of prolines on the convex surface, are required to generate a strongly twisted and coiled β-hairpin.     

How do Bax and Bak lead to permeabilization of the outer mitochondrial membrane?

Bcl-2 family members, like the structurally similar translocation domain of diphtheria toxin, can form ion-selective channels and larger-diameter pores in artificial lipid bilayers. Recent studies show how Bcl-2 family members change topology in membranes during apoptosis and that these different states may either promote or inhibit apoptosis. Binding of BH3-only proteins alters the subcellular localization and/or membrane topology and probably affects the channel formation of Bcl-2, Bcl-xL and Bcl-w. However, it remains unclear how the pore-forming activity functions in cells to regulate mitochondrial membrane permeabilization and cell death. Bcl-2 family members in flies and worms regulate apoptosis by mechanisms seemingly unrelated to membrane permeabilization, leaving a unifying model for the biochemical activity of this protein family unknown. Work linking Bcl-2 family members to mitochondrial morphogenesis in worms and mammals suggests some common functions of Bcl-2 family proteins may exist.     

Lipid–protein nanodiscs promote in vitro folding of transmembrane domains of multi-helical and multimeric membrane proteins

Production of helical integral membrane proteins (IMPs) in a folded state is a necessary prerequisite for their functional and structural studies. In many cases large-scale expression of IMPs in cell-based and cell-free systems results in misfolded proteins, which should be refolded in vitro. Here using examples of the bacteriorhodopsin ESR from Exiguobacterium sibiricum and full-length homotetrameric K⁺ channel KcsA from Streptomyces lividans we found that the efficient in vitro folding of the transmembrane domains of the polytopic and multimeric IMPs could be achieved during the protein encapsulation into the reconstructed high-density lipoprotein particles, also known as lipid–protein nanodiscs. In this case the self-assembly of the IMP/nanodisc complexes from a mixture containing apolipoprotein, lipids and the partially denatured protein solubilized in a harsh detergent induces the folding of the transmembrane domains. The obtained folding yields showed significant dependence on the properties of lipids used for nanodisc formation. The largest recovery of the spectroscopically active ESR (~ 60%) from the sodium dodecyl sulfate (SDS) was achieved in the nanodiscs containing anionic saturated lipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPG) and was approximately twice lower in the zwitterionic DMPC lipid. The reassembly of tetrameric KcsA from the acid-dissociated monomer solubilized in SDS was the most efficient (~ 80%) in the nanodiscs containing zwitterionic unsaturated lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC). The charged and saturated lipids provided lower tetramer quantities, and the lowest yield (< 20%) was observed in DMPC. The overall yield of the ESR and KcsA folding was mainly restricted by the efficiency of the protein encapsulation into the nanodiscs.     

How do drought and fire influence the patterns of resprouting in Australian deserts?

Rainfall is the key driver of woody cover and life-history attributes in arid grassy biomes where disturbance is mostly rare and of low intensity. However, relatively little is known about the causes of woody community assembly in arid systems that are subject to periodic intense fire disturbance. In the central Australian desert region, grassland and shrubland fire can occur following above average rainfall. Patterns of species regeneration response (resprouting vs. reseeding) are poorly documented in this region. We tested the effects of rainfall and fire on species’ resprouting response across the latitudinal rainfall–fire gradient using constrained ordination of 385 sites and general linear models. A resprouting response was significantly greater in grassland habitat as well as at the high end of the rainfall–fire gradient. The frequency of epicormic stem resprouting also increased along the rainfall–fire gradient. We attribute this pattern to the combined effects of frequent fire and rapid gap closure on seedlings of slow-growing, fire-killed woody species in higher rainfall grasslands. In addition, we also demonstrated that rapidly maturing fire-recruiting species are similarly favoured by high fire disturbance. In arid grassy ecosystems, unlike in mesic savanna, flammable grassland supports a mix of resprouting and recruitment functional types, and habitat membership cannot be predicted by resprouting capacity. Regions, such as central Australia, that are characterised by grassland–shrubland mosaics of high and low fuel biomass, respectively, pose specific challenges to fire ecology research that are possibly best dealt with by focussing modelling at the habitat scale.     

Protein domains correlate strongly with exons in multiple eukaryotic genomes--evidence of exon shuffling?

We conducted a multi-genome analysis correlating protein domain organization with the exon-intron structure of genes in nine eukaryotic genomes. We observed a significant correlation between the borders of exons and domains on a genomic scale for both invertebrates and vertebrates. In addition, we found that the more complex organisms displayed consistently stronger exon-domain correlation, with substantially more significant correlations detected in vertebrates compared with invertebrates. Our observations concur with the principles of exon shuffling theory, including the prediction of predominantly symmetric phase of introns flanking the borders of correlating exons. These results suggest that extensive exon shuffling events during evolution significantly contributed to the shaping of eukaryotic proteomes.