Mechanosensitive (MS) channels play a major role in protecting bacterial cells against hypo-osmotic shock. To understand their function, it is important to identify the conserved motifs using sequence analysis methods. In this study, the sequence conservation was investigated by an in silico analysis to generate sequence logos. We have identified new conserved motifs in the domains TM1, TM2 and the cytoplasmic helix from 231 homologs of MS channel of large conductance (MscL). In addition, we have identified new motifs for the TM3 and the cytoplasmic carboxy-terminal domain from 309 homologs of MS channel of small conductance (MscS). We found that the conservation in MscL homologs is high for TM1 and TM2 in the three domains of life. The conservation in MscS homologs is high only for TM3 in Bacteria and Archaea.

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We studied the properties of bilayers formed by ether-and ester-containing phospholipids, whose hydrocarbon chains can be either linear or branched, using sn-1,2 dipalmitoyl, dihexadecyl, diphytanoyl, and diphytanyl phosphatidylcholines (DPPC, DHPC, DPhoPC, and DPhPC, respectively) either pure or in binary mixtures. Differential scanning calorimetry and confocal fluorescence microscopy of giant unilamellar vesicles concurred in showing that equimolar mixtures of linear and branched lipids gave rise to gel/fluid phase coexistence at room temperature. Mixtures containing DHPC evolved in time (0.5 h) from initial reticulated domains to extended solid ones when an equilibrium was achieved. The nanomechanical properties of supported planar bilayers formed by each of the four lipids studied by atomic force microscopy revealed average breakdown forces Fb decreasing in the order DHPC ≥ DPPC > DPhoPC >> DPhPC. Moreover, except for DPPC, two different Fb values were found for each lipid. Atomic force microscopy imaging of DHPC was peculiar in showing two coexisting phases of different heights, probably corresponding to an interdigitated gel phase that gradually transformed, over a period of 0.5 h, into a regular tilted gel phase. Permeability to nonelectrolytes showed that linear-chain phospholipids allowed a higher rate of solute + water diffusion than branched-chain phospholipids, yet the former supported a smaller extent of swelling of the corresponding vesicles. Ether or ester bonds appeared to have only a minor effect on permeability.

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We systematically predict the internal flexibility of the S3 segment, one of the most mobile elements in the voltage-sensor domain. By analyzing the primary amino acid sequences of V-sensor containing proteins, including Hv1, TPC channels and the voltage-sensing phosphatases, we established correlations between the local flexibility and modes of activation for different members of the VGIC superfamily. Taking advantage of the structural information available, we also assessed structural aspects to understand the role played by the flexibility of S3 during the gating of the pore. We found that S3 flexibility is mainly determined by two specific regions: (1) a short NxxD motif in the N-half portion of the helix (S3a), and (2) a short sequence at the beginning of the so-called paddle motif where the segment has a kink that, in some cases, divide S3 into two distinct helices (S3a and S3b). A good correlation between the flexibility of S3 and the reported sensitivity to temperature and mechanical stretch was found. Thus, if the channel exhibits high sensitivity to heat or membrane stretch, local S3 flexibility is low. On the other hand, high flexibility of S3 is preferentially associated to channels showing poor heat and mechanical sensitivities. In contrast, we did not find any apparent correlation between S3 flexibility and voltage or ligand dependence. Overall, our results provide valuable insights into the dynamics of channel-gating and its modulation.

Understanding the lateral organization of biological membranes plays a key role on the road to fully appreciate the physiological functions of this fundamental barrier between the inside and outside regions of a cell. Ternary lipid bilayers composed of a high and a low melting temperature lipid and cholesterol represent a model system that mimics some of the important thermodynamical features of much more complex lipid mixtures such as those found in mammal membranes. The phase diagram of these ternary mixtures can be studied exploiting fluorescence microscopy in giant unilamellar vesicles, and it is typically expected to give rise, for specific combinations of composition and temperature, to regions of two-phase coexistence and a region with three-phase coexistence, namely, the liquid-ordered, liquid-disordered, and solid phases. Whereas the observation of two-phase coexistence is routinely possible using fluorescence microscopy, the three-phase region is more elusive to study. In this article, we show that particular lipid mixtures containing diphytanoyl-phosphatidylcholine and cholesterol plus different types of sphingomyelin (SM) are prone to produce bilayer regions with more than two levels of fluorescence intensity. We found that these intensity levels occur at low temperature and are linked to the copresence of long and asymmetric chains in SMs and diphytanoyl-phosphatidylcholine in the lipid mixtures. We discuss the possible interpretations for this observation in terms of bilayer phase organization in the presence of sphingolipids. Additionally, we also show that in some cases, liposomes in the three-phase coexistence state exhibit extreme sensitivity to lateral tension. We hypothesize that the appearance of the different phases is related to the asymmetric structure of SMs and to interdigitation effects.

Taxonomic distribution, repeats and functions of the S1 domain-containing proteins as members of the OB-fold family Evgeniia I. Deryusheva, Andrey V. Machulin, Olga M. Selivanova and Oxana V. Galzitskaya

Amphiphilic molecules which have a biological effect on specific membrane proteins, could also affect lipid bilayer properties possibly resulting in a modulation of the overall membrane behavior. In light of this consideration, it is important to study the possible effects of amphiphilic molecule of pharmacological interest on model systems which recapitulate some of the main properties of the biological plasma membranes. In this work we studied the effect of a neurosteroid, Allopregnanolone (3α,5α-tetrahydroprogesterone or Allo), on a model bilayer composed by the ternary lipid mixture DOPC/bSM/chol. We chose ternary mixtures which present, at room temperature, a phase coexistence of liquid ordered (Lo) and liquid disordered (Ld) domains and which reside near to a critical point. We found that Allo, which is able to strongly partition in the lipid bilayer, induces a marked increase in the bilayer area and modifies the relative proportion of the two phases favoring the Ld phase. We also found that the neurosteroid shifts the miscibility temperature to higher values in a way similarly to what happens when the cholesterol concentration is decreased. Interestingly, an isoform of Allo, isoAllopregnanolone (3β,5α-tetrahydroprogesterone or isoAllo), known to inhibit the effects of Allo on GABAA receptors, has an opposite effect on the bilayer properties.