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In concert with irreversible non-equilibrium peptide translation by the ribosome, the nascent polypeptide chain may integrate into the membrane or translocate to the other side of the membrane, facilitated by the conserved protein translocation channel SecYEG in bacteria and Sec61 in eukaryotes. Assuming equilibrium for the decision processes yielded the biological hydrophobicity scale, reflecting free-energy differences ΔG between the pore interior and membrane. Yet kinetic effects and molecular dynamic simulations suggested that a nascent polypeptide could not sample the two separate environments a sufficient number of times for partitioning in equilibrium. Here we tested the hypothesis employing purified and reconstituted SecYEG harboring a stalled ribosome nascent chain (RNC). The SecYEG-RNC complex was open in a de-energized membrane, allowing ion flow. Application of a membrane potential closed the channel if nascent chain hydrophobicity permitted membrane integration. Taking the ratio of steady-state to initial ion conductances as a measure of nascent chain hydrophobicity, we found ΔG for KvAP’s voltage sensor (4thhelix harboring four arginines) and FtsQ’s transmembrane helix to be equal to 0.3 and –2.1 kcal/mol, respectively. Thus, ΔG observed in our minimalistic system agrees very well with the position-dependent amino acid contribution of the biological hydrophobicity scale. Characteristic sampling times of ~2 s appear sufficient to reach a steady state for a ~20 amino acid-long segment invalidating the hypothesis of insufficient sampling.

The chase toward endowing chemical compounds with machine-like functions mimicking those of biological molecular machineries has yielded a variety of artificial molecular motors (AMMs). Pharmaceutical applications of photoexcited monomolecular unidirectionally-rotating AMMs have been envisioned in view of their ability to permeabilize biological membranes. Nonetheless, the mechanical properties of lipid membranes render the proposed drilling activity of AMMs doubtful. Here, we show that singlet oxygen released by a photoexcited "molecular drill" oxidized unsaturated lipids composing giant unilamellar vesicles. In contrast, giant liposomes built of saturated lipids were inert to AMM photoactuation. The AMM did not mechanically destroy gramicidin A ion channels in planar bilayer lipid membranes but instead photoinactivated them. Sodium azide, a singlet oxygen quencher, reduced both AMM-mediated light-induced dye release from unsaturated large unilamellar vesicles and protected gramicidin A from photoinactivation. Upon additional consideration of the underlying bilayer mechanics, we conclude that AMMs' envisioned therapeutic and pharmaceutical applications rely on their photodynamic activity rather than their nanomechanical drilling abilities.

The voltage-gated proton channel, HV1, is crucial for innate immune responses. According to alternative hypotheses, protons either hop on top of an uninterrupted water wire or bypass titratable amino acids, interrupting the water wire halfway across the membrane. To distinguish between both hypotheses, we estimate the water mobility for the putative case of an uninterrupted wire. The predicted single-channel water permeability 3×10−12cm3s−1 reflects the permeability-governing number of hydrogen bonds between water molecules in single-file configuration and pore residues. However, the measured unitary water permeability does not confirm the prediction, i.e., it is negligible. Osmotic deflation of reconstituted lipid vesicles reveals trapped water inside the HV1 wild-type channel and D174A mutant open at 0 mV. The conductance of 1400 H+ s−1 per wild-type channel agrees with the calculated diffusion limit for a ~2 Å capture radius for protons. Removal of a charged amino acid (D174) at the pore mouth decreases H+ conductance, conceivably by reducing the capture radius. At least one intervening amino acid contributes to H+ conductance while blocking water flow.

Lipid domains less than 200 nm in size may form a scaffold, enabling the concerted function of plasma membrane proteins. The size-regulating mechanism is under debate. We tested the hypotheses that large values of spontaneous monolayer curvature are incompatible with micrometer-sized domains. Here, we used the transition of photoswitchable lipids from their cylindrical conformation to a conical conformation to increase the negative curvature of a bilayer-forming lipid mixture. In contrast to the hypothesis, pre-existing micrometer-sized domains did not dissipate in our planar bilayers, as indicated by fluorescence images and domain mobility measurements. Elasticity theory supports the observation by predicting the zero free energy gain for splitting large domains into smaller ones. It also indicates an alternative size-determining mechanism: The cone-shaped photolipids reduce the line tension associated with lipid deformations at the phase boundary and thus slow down the kinetics of domain fusion. The competing influence of two approaching domains on the deformation of the intervening lipids is responsible for the kinetic fusion trap. Our experiments indicate that the resulting local energy barrier may restrict the domain size in a dynamic system.

It is my pleasure to write a few words to introduce myself to the readers of Biophysical Reviews as part of the “Meet the Councilor Series.” Currently, I am serving the second period as IUPAB councilor after having been elected first in 2017. Initially, I studied Biophysics in Moscow (Russia) and later Medicine in Halle (Germany). My scientific carrier took me from the Medical School of the Martin Luther University of Halle-Wittenberg, via the Leibniz Institute for Molecular Pharmacology (Berlin) and the Institute for Biology at the Humboldt University (Berlin) to the Physics Department of the Johannes Kepler University in Linz (Austria). My key research interests lie in the molecular mechanisms of transport phenomena occurring at the lipid membrane, including (i) spontaneous and facilitated transport of water and other small molecules across membranes in reconstituted systems, (ii) proton migration along the membrane surface, (iii) protein translocation, and (iv) bilayer mechanics. Training of undergraduate, graduate, and postdoctoral researchers from diverse academic disciplines has been—and shall remain—a consistent part of my work.

Various nanoscopic channels of roughly equal diameter and length facilitate single-file diffusion at vastly different rates. The underlying variance of the energetic barriers to transport is poorly understood. First, water partitioning into channels so narrow that individual molecules cannot overtake each other incurs an energetic penalty. Corresponding estimates vary widely depending on how the sacrifice of two out of four hydrogen bonds is accounted for. Second, entropy differences between luminal and bulk water may arise: additional degrees of freedom caused by dangling OH-bonds increase entropy. At the same time, long-range dipolar water interactions decrease entropy. Here, we dissect different contributions to Gibbs free energy of activation, ΔG‡, for single-file water transport through narrow channels by analyzing experimental results from water permeability measurements on both bare lipid bilayers and biological water channels that (i) consider unstirred layer effects and (ii) adequately count the channels in reconstitution experiments. First, the functional relationship between water permeabilities and Arrhenius activation energies indicates negligible differences between the entropies of intraluminal water and bulk water. Second, we calculate ΔG‡ from unitary water channel permeabilities using transition state theory. Plotting ΔG‡ as a function of the number of H-bond donating or accepting pore-lining residues results in a 0.1 kcal/mol contribution per residue. The resulting upper limit for partial water dehydration amounts to 2 kcal/mol. In the framework of biomimicry, our analysis provides valuable insights for the design of synthetic water channels. It thus may aid in the urgent endeavor towards combating global water scarcity.

Membrane proteins can be regulated by alterations in material properties intrinsic to the hosting lipid bilayer. Here, we investigated whether the reversible photoisomerization of bilayer-embedded diacylglycerols (OptoDArG) with two azobenzene-containing acyl chains may trigger such regulatory events. We observed an augmented open probability of the mechanosensitive model channel gramicidin A (gA) upon photoisomerizing OptoDArG's acyl chains from trans to cis: integral planar bilayer conductance brought forth by hundreds of simultaneously conducting gA dimers increased by typically >50% - in good agreement with the observed increase in single-channel lifetime. Further, (i) increments in the electrical capacitance of planar lipid bilayers and protrusion length of aspirated giant unilamellar vesicles into suction pipettes, as well as (ii) changes of small-angle X-ray scattering of multilamellar vesicles indicated that spontaneous curvature, hydrophobic thickness, and bending elasticity decreased upon switching from trans- to cis-OptoDArG. Our bilayer elasticity model for gA supports the causal relationship between changes in gA activity and bilayer material properties upon photoisomerization. Thus, we conclude that photolipids are deployable for converting bilayers of potentially diverse origins into light-gated actuators for mechanosensitive proteins.

Lipid rafts serve as anchoring platforms for membrane proteins. Thus far they escaped direct observation by light microscopy due to their small size. Here we used differently colored dyes as reporters for the registration of both ordered and disordered lipids from the two leaves of a freestanding bilayer. Photoswitchable lipids dissolved or reformed the domains. Measurements of domain mobility indicated the presence of 120 nm wide ordered and 40 nm wide disordered domains. These sizes are in line with the predicted roles of line tension and membrane undulation as driving forces for alignment.

This is a perspective article entitled “Frontiers in computational biophysics: understanding conformational dynamics of complex lipid mixtures relevant to biology” which is following a CECAM meeting with the same name. Fil: Friedman, Ran. Linnæus University; Argentina Fil: Khalid, Syma. University of Southampton; Reino Unido Fil: Aponte Santamaría, Camilo. Ruprecht-Karls-Universität Heidelberg; Alemania. Universidad de los Andes; Colombia Fil: Arutyunova, Elena. University of Alberta; Canadá Fil: Becker, Marlon. Westfälische Wilhelms Universität; Alemania Fil: Boyd, Kevin J.. University of Connecticut; Estados Unidos Fil: Christensen, Mikkel. University Aarhus; Dinamarca Fil: Coimbra, João T. S.. Universidad de Porto; Portugal Fil: Concilio, Simona. Universita di Salerno; Italia Fil: Daday, Csaba. Heidelberg Institute for Theoretical Studies; Alemania Fil: Eerden, Floris J. van. University of Groningen; Países Bajos Fil: Fernandes, Pedro A.. Universidad de Porto; Portugal Fil: Gräter, Frauke. Heidelberg University; Alemania. Heidelberg Institute for Theoretical Studies; Alemania Fil: Hakobyan, Davit. Westfälische Wilhelms Universität; Alemania Fil: Heuer, Andreas. Westfälische Wilhelms Universität; Alemania Fil: Karathanou, Konstantina. Freie Universität Berlin; Alemania Fil: Keller, Fabian. Westfälische Wilhelms Universität; Alemania Fil: Lemieux, M. Joanne. University of Alberta; Canadá Fil: Marrink, Siewert J.. University of Groningen; Países Bajos Fil: May, Eric R.. University of Connecticut; Estados Unidos Fil: Mazumdar, Antara. University of Groningen; Países Bajos Fil: Naftalin, Richard. Colegio Universitario de Londres; Reino Unido Fil: Pickholz, Mónica Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Física de Buenos Aires. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Física de Buenos Aires; Argentina Fil: Piotto, Stefano. Universita di Salerno; Italia Fil: Pohl, Peter. Johannes Kepler University; Austria Fil: Quinn, Peter. Colegio Universitario de Londres; Reino Unido Fil: Ramos, Maria J.. Universidad de Porto; Portugal Fil: Schiøtt, Birgit. University Aarhus; Dinamarca Fil: Sengupta, Durba. National Chemical Laboratory India; India Fil: Sessa, Lucia. Universita di Salerno; Italia Fil: Vanni, Stefano. University Of Fribourg; Fil: Zeppelin, Talia. University Aarhus; Dinamarca Fil: Zoni, Valeria. University of Fribourg; Suiza Fil: Bondar, Ana-Nicoleta. Freie Universität Berlin; Alemania Fil: Domene, Carmen. University of Oxford; Reino Unido. University of Bath; Reino Unido

Building on Skinner’s theory of verbal behavior, research over the last few decades confirmed verbal speaker operants, added the role of the listener, added the identification of speaker and listener interaction between and within individuals, and identified verbal behavior developmental cusps. Meanwhile, comparative biology focused on how and why language evolved in Homo sapiens. Findings about differences in behavior that neurotypical children demonstrated in their verbal development, and even more so in research that identified and established missing verbal behavior cusps, suggested changes analogous to metamorphosis. These striking changes in stimulus control found in the onset of cusps from the preverbal to the fully verbal child led us to an expansion of the concept of metamorphosis from morphology to the domain of behavior. The major findings of this comparative perspective are presented here as they have led us from experimental analyses of verbal development to metamorphosis as complex verbal behavior transformation and finally to a novel hypothesis about the evolution of language based on the concepts and research described here. To our knowledge, this is the first formulation of verbal development as behavioral metamorphosis in the context of evolutionary developmental biology.

Significance Lipid bilayer membranes are responsible for compartmentalization, signaling, transport, and flow of charge in living cells. Membranes self-assemble in aqueous solutions. Without a hydrating environment, membranes cannot exist. It is therefore surprising to note that the hydrating water is neglected in most membrane-related studies. We imaged membrane-bound oriented water by means of label-free second harmonic microscopy. We tracked, on millisecond time scales, membrane domain diffusion of condensed charged lipid domains, domain structure, and the spatial distribution of charge. Real-time electrostatic membrane potential maps were constructed using nonlinear optical theory. The spatiotemporal fluctuation in the membrane potential is surprisingly large and reveals the importance of charge fluctuations on membranes., Biological membranes are highly dynamic and complex lipid bilayers, responsible for the fate of living cells. To achieve this function, the hydrating environment is crucial. However, membrane imaging typically neglects water, focusing on the insertion of probes, resonant responses of lipids, or the hydrophobic core. Owing to a recent improvement of second-harmonic (SH) imaging throughput by three orders of magnitude, we show here that we can use SH microscopy to follow membrane hydration of freestanding lipid bilayers on millisecond time scales. Instead of using the UV/VIS resonant response of specific membrane-inserted fluorophores to record static SH images over time scales of >1,000 s, we SH imaged symmetric and asymmetric lipid membranes, while varying the ionic strength and pH of the adjacent solutions. We show that the nonresonant SH response of water molecules aligned by charge−dipole interactions with charged lipids can be used as a label-free probe of membrane structure and dynamics. Lipid domain diffusion is imaged label-free by means of the hydration of charged domains. The orientational ordering of water is used to construct electrostatic membrane potential maps. The average membrane potential depends quadratically on an applied external bias, which is modeled by nonlinear optical theory. Spatiotemporal fluctuations on the order of 100-mV changes in the membrane potential are seen. These changes imply that membranes are very dynamic, not only in their structure but also in their membrane potential landscape. This may have important consequences for membrane function, mechanical stability, and protein/pore distributions.

This review focusses on the energetics of protein translocation via the Sec translocation machinery. First we complement structural data about SecYEG’s conformational rearrangements by insight obtained from functional assays. These include measurements of SecYEG permeability that allow assessment of channel gating by ligand binding and membrane voltage. Second we will discuss the power stroke and Brownian ratcheting models of substrate translocation and the role that the two models assign to the putative driving forces: (i) ATP (SecA) and GTP (ribosome) hydrolysis, (ii) interaction with accessory proteins, (iii) membrane partitioning and folding, (iv) proton motive force (PMF), and (v) entropic contributions. Our analysis underlines how important energized membranes are for unravelling the translocation mechanism in future experiments.

Water at interfaces governs many processes on the molecular scale from electrochemical and enzymatic reactions to protein folding. Here we focus on water transport through proteinaceous pores that are so narrow that the water molecules cannot overtake each other in the pore. After a short introduction into the single-file transport theory, we analyze experiments in which the unitary water permeability, p f, of water channel proteins (aquaporins, AQPs), potassium channels (KcsA), and antibiotics (gramicidin-A derivatives) has been obtained. A short outline of the underlying methods (scanning electrochemical microscopy, fluorescence correlation spectroscopy, measurements of vesicle light scattering) is also provided. We conclude that p f increases exponentially with a decreasing number N H of hydrogen bond donating or accepting residues in the channel wall. The variance in N H is responsible for a more than hundredfold change in p f. The dehydration penalty at the channel mouth has a smaller effect on p f. The intricate link between p f and the Gibbs activation energy barrier, ∆Gt‡ for water flow suggests that conformational transitions of water channels act as a third determinant of p f.

The mechanism of action of photosensitizers used in the photodynamic tumor therapy is studied by using bilayer lipid membranes (BLM) which adequately simulate the plasma membranes of cells. The formation of singlet oxygen upon photoexcitation of photosensitizer (tetrasulfonated alumophthalocyanine) is revealed by the photosensitizer-induced decomposition of styryl dyes di-4-ANEPPS or RH-421 the adsorption of which on BLM gives rise to changes in the dipole potential at the membrane interface. This potential decreases upon illumination of the membrane covered with adsorbed phthalocyanine and dye molecules as a result of the dye oxidation by singlet oxygen and is recovered upon interruption of illumination as a result of adsorption of new dye molecules from the solution. The changes in the potential are observed both when the dye molecules are adsorbed on the same (cis) membrane side as the photosensitizer molecules and also when they are adsorbed on the opposite (trans) membrane side. When the dye molecules are adsorbed on the trans side, the kinetics of potential variations in the light and its subsequent recovery in the dark depends on the membrane size. This dependence is explained within the framework of a model which assumes that dye molecules can get into the lipid bilayer not only from the aqueous solution but also as a result of lateral diffusion from the bilayer-surrounding circular reservoir containing a lipid solution in decane. The quantitative description of experimental data in terms of this model allows the coefficient of lateral diffusion of di-4- ANEPPS molecules in the lipid bilayer to be assessed as 10–7–10–6 cm2/s. The oxidation rate of dye molecules on the cis side is shown to be lower, which is explained by inhibition of the formation of singlet oxygen by dye molecules.

Spontaneous solute and solvent permeation through membranes is of vital importance to human life, be it gas exchange in red blood cells, metabolite excretion, drug/toxin uptake, or water homeostasis. Knowledge of the underlying molecular mechanisms is the sine qua non of every functional assignment to membrane transporters. The basis of our current solubility diffusion model was laid by Meyer and Overton. It correlates the solubility of a substance in an organic phase with its membrane permeability. Since then, a wide range of studies challenging this rule have appeared. Commonly, the discrepancies have their origin in ill-used measurement approaches, as we demonstrate on the example of membrane CO2 transport. On the basis of the insight that scanning electrochemical microscopy offered into solute concentration distributions in immediate membrane vicinity of planar membranes, we analyzed the interplay between chemical reactions and diffusion for solvent transport, weak acid permeation, and enzymatic reactions adjacent to membranes. We conclude that buffer reactions must also be considered in spectroscopic investigations of weak acid transport in vesicular suspensions. The evaluation of energetic contributions to membrane translocation of charged species demonstrates the compatibility of the resulting membrane current with the solubility diffusion model. A local partition coefficient that depends on membrane penetration depth governs spontaneous membrane translocation of both charged and uncharged molecules. It is determined not only by the solubility in an organic phase but also by other factors like cholesterol concentration and intrinsic electric membrane potentials.

A new test facility is presented, enabling single-phase isothermal fluid mechanics experiments to support nuclear reactor safety studies. The applied measurement technique Magnetic Resonance Velocimetry (MRV) is based on the medical imaging modality Magnetic Resonance Imaging (MRI). The experiments in this laboratory aim at providing the mean velocity field in models of nuclear fuel assemblies using isothermal water. Additionally, turbulence statistics in terms of the Reynolds stress tensor can be acquired. Since MRV is a non-optical measurement technique, there are no requirements on optical properties, and the investigated models can be quickly built with additive manufacturing techniques. Even though MRV offers a relatively low temporal and spatial resolution compared to laser-optical techniques and has some limitations regarding the test section materials, e.g., no metals, the main gain is that MRV provides a full three-dimensional representation of the flow field in short times. Both the model preparation and the measurement time are significantly faster than with laser-optical measurement techniques. The full-field experimental data obtained with MRV is often referred to as 'CFD grade data' as it enables a full-field validation of numerical solutions obtained with Computational Fluid Dynamics (CFD). As a commissioning experiment, measurements inside a 5x5 fuel element model of a pressurized water reactor (PWR) are conducted and compared with CFD results. The measured mean velocity field inside the PWR replica comprised of 2.9 Million velocity vectors was acquired in 79 min. In addition, turbulence measurements in a periodic hill channel demonstrate how MRV can be used to obtain all components of the Reynolds stress tensor. Although the measurements in this proof-of-concept study suffer from some measurement errors, all major error contributions are well known, and validated methods exist to overcome these shortcomings. In conclusion, this study shows how the velocity field in a specific fuel assembly configuration can be obtained highly efficiently using a specialized MRI laboratory.

The study of verbal behavior focuses on communicative functions of the speakers/producers as they affect the behavior of listeners/observers. Effects on the listener reinforce the speaker and the listener/observer benefits (i.e., is reinforced) from the behavior of the speaker/producer. The interlocking of, and exchange of, the speaker and listener behavior between individuals and within one’s own skin constitute bidirectional operants. These bidirectional operants are instances of social interactions and measures of social behavior. Evidence suggests that the act of listening, among other observing responses, is initially developmentally independent from speaker behavior. How they become joined parallels the biological phenomenon of metamorphosis. The succession of changes has been empirically identified as a succession of verbal behavior development cusps, which are described in their sequence biologically as a manifestation of functional metamorphosis. The onset of a cusp constitutes first instances of behavior and accompanying stimulus control that allows infants and children to contact parts of the environment for the first time resulting in their learning things impossible to learn before or learning faster. Cusps for the intercept of the speaker and listener lead to bidirectional operants and provide explanations for how children incidentally learn the names of things, become increasingly social, and make subsequent complex behavior possible. Many of the cusps identified in our research resulted from the missing behavior and stimulus control of children with autism. Once cusps were established, these children learned things they could not learn before, learned faster, and learned by contacting parts of the social environment they could not contact before. These findings led to a theory of verbal behavior development that point to the selection of bidirectional operants as behavioral phenotypes during functional metamorphosis, which has enhanced the survival of Homo sapiens through emergent symbolic skills for more effective collaboration between two or more individuals.

Phase separation in biological membranes plays an important role in protein targeting and transmembrane signaling. Its occurrence in both membrane leaflets commonly gives rise to matching liquid or liquid-ordered domains in the opposing monolayers. The underlying mechanism of such co-localization is not fully understood. The decrease of the line tension around the thicker ordered domain constitutes an important driving force. Yet, robust domain coupling requires an additional energy source, which we have now identified as thermal undulations. Our theoretical analysis of elastic deformations in a lipid bilayer shows that stiffer lipid domains tend to distribute into areas with lower fluctuations of monolayer curvature. These areas naturally align in the opposing monolayers. Thus, coupling requires both membrane leafs to display a heterogeneity in splay rigidities. The heterogeneity may either originate from intrinsic lipid properties or be acquired by adsorption of peripheral molecules. Undulations and line tension act synergistically: the gain in energy due a minimized line tension is proportional to domain radius and thus primarily fuels the registration of smaller domains; whereas the energetic contribution of undulations increases with membrane area and thus primarily acts to coalesce larger domains.

Understanding the structure and dynamics of water’s constituent ions, proton and hydroxide, has been a subject of numerous experimental and theoretical studies over the last century. Besides their obvious importance in acid-base chemistry, these ions play an important role in numerous applications ranging from enzyme catalysis to environmental chemistry. Despite a long history of research, many fundamental issues regarding their properties continue to be an active area of research. Here, we provide a review of the experimental and theoretical advances made in the last several decades in understanding the structure, dynamics, and transport of the proton and hydroxide ions in different aqueous environments, ranging from water clusters to the bulk liquid and its interfaces with hydrophobic surfaces. The propensity of these ions to accumulate at hydrophobic surfaces has been a subject of intense debate, and we highlight the open issues and challenges in this area. Biological applications reviewed include proton transport along the hydration layer of various membranes and through channel proteins, problems that are at the core of cellular bioenergetics.

Water is an essential participant in the stability, structure, dynamics, and function of proteins and other biomolecules. Thermodynamically, changes in the aqueous environment affect the stability of biomolecules. Structurally, water participates chemically in the catalytic function of proteins and nucleic acids and physically in the collapse of the protein chain during folding through hydrophobic collapse and mediates binding through the hydrogen bond in complex formation. Water is a partner that slaves the dynamics of proteins, and water interaction with proteins affect their dynamics. Here we provide a review of the experimental and computational advances over the past decade in understanding the role of water in the dynamics, structure, and function of proteins. We focus on the combination of X-ray and neutron crystallography, NMR, terahertz spectroscopy, mass spectroscopy, thermodynamics, and computer simulations to reveal how water assist proteins in their function. The recent advances in computer simulations and the enhanced sensitivity of experimental tools promise major advances in the understanding of protein dynamics, and water surely will be a protagonist.

Archaeal single-cell microorganisms are characterised by particularly strong resistance to harsh environments at extreme temperature ranges, pH values, high pressure and salinity. The recognition of molecules rendering the archaeal membranes stable in such conditions represents an important step towards understanding the molecular mechanisms, which underlie the cellular survival, in order to effectively exploit them in biomedicine and bionanotechnology. Here we report on the bending elasticity and phase behaviour of model lipid membranes containing tetraether archaeal lipids. The bending elasticity modulus of membranes composed of palmitoyl-oleoyl phosphatidylcholine and glycerol dialkyl glycerol tetraether extract from the archaeon Thermoplasma acidophilum’s plasma membrane is measured by analysis of the thermal shape fluctuations of nearly spherical giant unilamellar vesicles (tens of micrometers in size). The bending rigidity is reported to non-monotonically depend on the archaeal lipids’ concentration in the bilayer. At 50 wt% of tetraether lipids in the membrane we measure around 20% decrease of the bending modulus compared to the studied single-component phosphocholine bilayer. The membrane resistance to bending increases at archaeal lipids’ content higher than 70 wt%. These findings support the hypothesis about a probable looping conformation at very low amounts of bipolar lipids in the membrane and prevailing spanning tetraether molecules at higher concentrations. Fluorescence microscopy reveals structural phase coexistence at low temperatures (6 °C) for 75 wt% of bolalipids in the bilayer. Laurdan spectroscopy measurements of large unilamellar vesicles (hundreds of nanometers in size) provide evidences for increased lipid ordering at the glycerol level induced by the presence of up to 90 wt% of bolalipid in the bilayer. At this archaeal lipid content we measure increased bending rigidity of the membrane. The fatty acyl chain mobility probed by DPH fluorescence spectroscopy is significantly reduced in the presence of bolalipids. The lipid ordering decreases with increasing the temperature to an extent depending on the bolalipid content in the membrane. The presence of bipolar lipids in the bilayer affects the lipid packing more strongly at the glycerol level compared to the hydrophobic core of the membrane. By elucidating the effect of tetraether lipids on the structural and mechanical properties of the bilayer, the reported results are expected to help in advancing on liposome-based pharmaceuticals and biomedical applications as well as for developments in sensorics and nanotechnology.