YidC from Escherichia coli Forms an Ion-Conducting Pore upon Activation by Ribosomes.

The universally conserved protein YidC aids in the insertion and folding of transmembrane polypeptides. Supposedly, a charged arginine faces its hydrophobic lipid core, facilitating polypeptide sliding along YidC's surface. How the membrane barrier to other molecules may be maintained is unclear. Here, we show that the purified and reconstituted E. coli YidC forms an ion-conducting transmembrane pore upon ribosome or ribosome-nascent chain complex (RNC) binding. In contrast to monomeric YidC structures, an AlphaFold parallel YidC dimer model harbors a pore. Experimental evidence for a dimeric assembly comes from our BN-PAGE analysis of native vesicles, fluorescence correlation spectroscopy studies, single-molecule fluorescence photobleaching observations, and crosslinking experiments. In the dimeric model, the conserved arginine and other residues interacting with nascent chains point into the putative pore. This result suggests the possibility of a YidC-assisted insertion mode alternative to the insertase mechanism.

Entropic barrier of water permeation through single-file channels.

Facilitated water permeation through narrow biological channels is fundamental for all forms of life. Despite its significance in health and disease as well as for biotechnological applications, the energetics of water permeation are still elusive. Gibbs free energy of activation is composed of an enthalpic and an entropic component. Whereas the enthalpic contribution is readily accessible via temperature dependent water permeability measurements, estimation of the entropic contribution requires information on the temperature dependence of the rate of water permeation. Here, we estimate, by means of accurate activation energy measurements of water permeation through Aquaporin-1 and by determining the accurate single channel permeability, the entropic barrier of water permeation through a narrow biological channel. Thereby the calculated value for [Formula: see text] = 2.01 ± 0.82 J/(mol·K) links the activation energy of 3.75 ± 0.16 kcal/mol with its efficient water conduction rate of ~1010 water molecules/second. This is a first step in understanding the energetic contributions in various biological and artificial channels exhibiting vastly different pore geometries.

Trapped Pore Waters in the Open Proton Channel HV 1.

The voltage-gated proton channel, HV 1, 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, the water mobility for the putative case of an uninterrupted wire is estimated. The predicted single-channel water permeability 2.3 × 10-12 cm3 s-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 predicted value. Osmotic deflation of reconstituted lipid vesicles reveals negligible water permeability of the HV 1 wild-type channel and the 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 by reducing the capture radius. At least one intervening amino acid contributes to H+ conductance while interrupting the water wire across the membrane.

Multiple pore lining residues modulate water permeability of GlpF.

The water permeability of aquaporins (AQPs) varies by more than an order of magnitude even though the pore structure, geometry, as well as the channel lining residues are highly conserved. However, channel gating by pH, divalent ions or phosphorylation was only shown for a minority of AQPs. Structural and in silico indications of water flux modulation by flexible side chains of channel lining residues have not been experimentally confirmed yet. Hence, the aquaporin "open state" is still considered to be a continuously open pore with water molecules permeating in a single-file fashion. Using protein mutations outside the selectivity filter in the aqua(glycerol)facilitator GlpF of Escherichia coli we, to the best of our knowledge, for the first time, modulate the position of the highly conserved Arg in the selectivity filter. This in turn enhances or reduces the unitary water permeability of GlpF as shown in silico by molecular dynamics (MD) simulations and in vitro with purified and reconstituted GlpF. This finding suggests that AQP water permeability can indeed be regulated by lipid bilayer asymmetry and the transmembrane potential. Strikingly, our long-term MD simulations reveal that not only the conserved Arg in the selectivity filter, but the position and dynamics of multiple other pore lining residues modulate water passage through GlpF. This finding is expected to trigger a wealth of future investigations on permeability and regulation of AQPs among others with the aim to tune water permeability for biotechnological applications.

The Hidden Intricacies of Aquaporins: Remarkable Details in a Common Structural Scaffold.

Evolution turned aquaporins (AQPs) into the most efficient facilitators of passive water flow through cell membranes at no expense of solute discrimination. In spite of a plethora of solved AQP structures, many structural details remain hidden. Here, by combining extensive sequence- and structural-based analysis of a unique set of 20 non-redundant high-resolution structures and molecular dynamics simulations of four representatives, key aspects of AQP stability, gating, selectivity, pore geometry, and oligomerization, with a potential impact on channel functionality, are identified. The general view of AQPs possessing a continuous open water pore is challenged and it is depicted that AQPs' selectivity is not exclusively shaped by pore-lining residues but also by the relative arrangement of transmembrane helices. Moreover, this analysis reveals that hydrophobic interactions constitute the main determinant of protein thermal stability. Finally, a numbering scheme of the conserved AQP scaffold is established, facilitating direct comparison of, for example, disease-causing mutations and prediction of potential structural consequences. Additionally, the results pave the way for the design of optimized AQP water channels to be utilized in biotechnological applications.

Scattering versus fluorescence self-quenching: more than a question of faith for the quantification of water flux in large unilamellar vesicles?

The endeavors to understand the determinants of water permeation through membrane channels, the effect of the lipid or polymer membrane on channel function, the development of specific water flow inhibitors, the design of artificial water channels and aquaporins for the use in industrial water filtration applications all rely on accurate ways to quantify water permeabilities (P f). A commonly used method is to reconstitute membrane channels into large unilamellar vesicles (LUVs) and to subject these vesicles to an osmotic gradient in a stopped-flow device. Fast recordings of either scattered light intensity or fluorescence self-quenching signals are taken as a readout for vesicle volume change, which in turn can be recalculated to accurate P f values. By means of computational and experimental data, we discuss the pros and cons of using scattering versus self-quenching experiments or subjecting vesicles to hypo- or hyperosmotic conditions. In addition, we explicate for the first time the influence of the LUVs size distribution, channel distribution between vesicles and remaining detergent after protein reconstitution on P f values. We point out that results such as the single channel water permeability (p f) depend on the membrane matrix or on the direction of the applied osmotic gradient may be direct results of the measurement and analysis procedure.

Micropipette Aspiration-Based Assessment of Single Channel Water Permeability.

Measurements of the unitary hydraulic conductivity of membrane channels, pf , may be hampered by difficulties in producing sufficient quantities of purified and reconstituted proteins. Low yield expression, the purely empiric choice of detergents, as well as protein aggregation and misfolding during reconstitution may result in an average of less than one reconstituted channel per large unilamellar vesicle. This limits their applicability for pf measurements, independent of whether light scattering or fluorescence quenching of encapsulated dyes is monitored. Here the micropipette aspiration technique is adopted because its superb sensitivity allows resolving pf values for one order of magnitude smaller protein densities in sphingomyelin and cholesterol rich giant unilamellar vesicles (GUVs). Protein density is derived from intensity fluctuations that fluorescently labeled channels in the aspirated GUV induce by diffusing through the diffraction limited spot. A perfusion system minimizes unstirred layers in the immediate membrane vicinity as demonstrated by the distribution of both encapsulated and extravesicular aqueous dyes. pf amounted to 2.4 ± 0.1 × 10-13 cm³ s-1 for aquaporin-1 that served as a test case. The new assay paves the way for directly monitoring the effect that interaction of aquaporins with other proteins or inhibitors may have on pf on a single sample.

Voltage Sensing in Bacterial Protein Translocation.

The bacterial channel SecYEG efficiently translocates both hydrophobic and hydrophilic proteins across the plasma membrane. Translocating polypeptide chains may dislodge the plug, a half helix that blocks the permeation of small molecules, from its position in the middle of the aqueous translocation channel. Instead of the plug, six isoleucines in the middle of the membrane supposedly seal the channel, by forming a gasket around the translocating polypeptide. However, this hypothesis does not explain how the tightness of the gasket may depend on membrane potential. Here, we demonstrate voltage-dependent closings of the purified and reconstituted channel in the presence of ligands, suggesting that voltage sensitivity may be conferred by motor protein SecA, ribosomes, signal peptides, and/or translocating peptides. Yet, the presence of a voltage sensor intrinsic to SecYEG was indicated by voltage driven closure of pores that were forced-open either by crosslinking the plug to SecE or by plug deletion. We tested the involvement of SecY's half-helix 2b (TM2b) in voltage sensing, since clearly identifiable gating charges are missing. The mutation L80D accelerated voltage driven closings by reversing TM2b's dipolar orientation. In contrast, the L80K mutation decelerated voltage induced closings by increasing TM2b's dipole moment. The observations suggest that TM2b is part of a larger voltage sensor. By partly aligning the combined dipole of this sensor with the orientation of the membrane-spanning electric field, voltage may drive channel closure.

Author Correction: Quantification of Water Flux in Vesicular Systems.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Interaction of the motor protein SecA and the bacterial protein translocation channel SecYEG in the absence of ATP.

Translocation of many secretory proteins through the bacterial plasma membrane is facilitated by a complex of the SecYEG channel with the motor protein SecA. The ATP-free complex is unstable in detergent, raising the question how SecA may perform several rounds of ATP hydrolysis without being released from the membrane embedded SecYEG. Here we show that dual recognition of (i) SecYEG and (ii) vicinal acidic lipids confers an apparent nanomolar affinity. High-speed atomic force microscopy visualizes the complexes between monomeric SecA and SecYEG as being stable for tens of seconds. These long-lasting events and complementary shorter ones both give rise to single ion channel openings of equal duration. Furthermore, luminescence resonance energy transfer reveals two conformations of the SecYEG-SecA complex that differ in the protrusion depth of SecA's two-helix finger into SecYEG's aqueous channel. Such movement of the finger is in line with the power stroke mechanism of protein translocation.

Positively charged residues at the channel mouth boost single-file water flow.

Water molecules lose two of their four bulk neighbours when entering single-file channels. This process may be sensitive to the presence of positive and negative charges at the channel mouth, since the costs for dehydrating cations and anions differ by a large margin. However, it is not known whether entrance charges affect the single channel water permeability (pf). So far, pf is only known to be governed by H-bond formation between permeating water molecules and wall-lining residues. Here we compare the pf values of five different aquaporin species (AQP1, AQPZ, AQP4 wild type, and two phosphorylation mimicking AQP4 mutants) that offer the same number of hydrogen bond donating and receiving residues in their single-file region but display different entrance charges. The pf measurements were performed with reconstituted lipid vesicles. We assessed (i) the osmotically induced vesicle deflation from the light scattering intensity in a stopped-flow device and (ii) the aquaporin abundance by fluorescence correlation spectroscopy. Substitution of serine at positions 111 and 180 in AQP4 for aspartic acid showed only a marginal effect on pf, suggesting that negative entrance charges are of minor importance. In contrast, the total number of positively charged amino acid side chains at entrances and exits correlates with pf: a total of three, four and seven charges of AQP4, AQPZ, and AQP1 translate into pf values of 1.1, 1.8, and 3.2 × 10-13 cm3 s-1, respectively. Thus, positive interfacial charges boost the pf value of AQP1 to three times the value of AQP4. Nevertheless, the number of hydrogen bond donating and receiving residues in the single-file region remains the major determinant of pf. Their effect on pf may be a hundredfold larger than that of interfacial charges.

Quantification of Water Flux in Vesicular Systems.

Water transport across lipid membranes is fundamental to all forms of life and plays a major role in health and disease. However, not only typical water facilitators like aquaporins facilitate water flux, but also transporters, ion channels or receptors represent potent water pathways. The efforts directed towards a mechanistic understanding of water conductivity determinants in transmembrane proteins, the development of water flow inhibitors, and the creation of biomimetic membranes with incorporated membrane proteins or artificial water channels depend on reliable and accurate ways of quantifying water permeabilities Pf. A conventional method is to subject vesicles to an osmotic gradient in a stopped-flow device: Fast recordings of scattered light intensity are converted into the time course of vesicle volume change. Even though an analytical solution accurately acquiring Pf from scattered light intensities exists, approximations potentially misjudging Pf by orders of magnitude are used. By means of computational and experimental data we point out that erroneous results such as that the single channel water permeability pf depends on the osmotic gradient are direct results of such approximations. Finally, we propose an empirical solution of which calculated permeability values closely match those calculated with the analytical solution in the relevant range of parameters.

YidC and SecYEG form a heterotetrameric protein translocation channel.

The heterotrimeric SecYEG complex cooperates with YidC to facilitate membrane protein insertion by an unknown mechanism. Here we show that YidC contacts the interior of the SecY channel resulting in a ligand-activated and voltage-dependent complex with distinct ion channel characteristics. The SecYEG pore diameter decreases from 8 Å to only 5 Å for the YidC-SecYEG pore, indicating a reduction in channel cross-section by YidC intercalation. In the presence of a substrate, YidC relocates to the rim of the pore as indicated by increased pore diameter and loss of YidC crosslinks to the channel interior. Changing the surface charge of the pore by incorporating YidC into the channel wall increases the anion selectivity, and the accompanying change in wall hydrophobicity is liable to alter the partition of helices from the pore into the membrane. This could explain how the exit of transmembrane domains from the SecY channel is facilitated by YidC.

Tuning membrane protein mobility by confinement into nanodomains.

High-speed atomic force microscopy (HS-AFM) can be used to visualize function-related conformational changes of single soluble proteins. Similar studies of single membrane proteins are, however, hampered by a lack of suitable flat, non-interacting membrane supports and by high protein mobility. Here we show that streptavidin crystals grown on mica-supported lipid bilayers can be used as porous supports for membranes containing biotinylated lipids. Using SecYEG (protein translocation channel) and GlpF (aquaglyceroporin), we demonstrate that the platform can be used to tune the lateral mobility of transmembrane proteins to any value within the dynamic range accessible to HS-AFM imaging through glutaraldehyde-cross-linking of the streptavidin. This allows HS-AFM to study the conformation or docking of spatially confined proteins, which we illustrate by imaging GlpF at sub-molecular resolution and by observing the motor protein SecA binding to SecYEG.

Curli mediate bacterial adhesion to fibronectin via tensile multiple bonds.

Many enteric bacteria including pathogenic Escherichia coli and Salmonella strains produce curli fibers that bind to host surfaces, leading to bacterial internalization into host cells. By using a nanomechanical force-sensing approach, we obtained real-time information about the distribution of molecular bonds involved in the adhesion of curliated bacteria to fibronectin. We found that curliated E. coli and fibronectin formed dense quantized and multiple specific bonds with high tensile strength, resulting in tight bacterial binding. Nanomechanical recognition measurements revealed that approximately 10 bonds were disrupted either sequentially or simultaneously under force load. Thus the curli formation of bacterial surfaces leads to multi-bond structural components of fibrous nature, which may explain the strong mechanical binding of curliated bacteria to host cells and unveil the functions of these proteins in bacterial internalization and invasion.

The Sodium Glucose Cotransporter SGLT1 Is an Extremely Efficient Facilitator of Passive Water Transport.

The small intestine is void of aquaporins adept at facilitating vectorial water transport, and yet it reabsorbs ∼8 liters of fluid daily. Implications of the sodium glucose cotransporter SGLT1 in either pumping water or passively channeling water contrast with its reported water transporting capacity, which lags behind that of aquaporin-1 by 3 orders of magnitude. Here we overexpressed SGLT1 in MDCK cell monolayers and reconstituted the purified transporter into proteoliposomes. We observed the rate of osmotic proteoliposome deflation by light scattering. Fluorescence correlation spectroscopy served to assess (i) SGLT1 abundance in both vesicles and plasma membranes and (ii) flow-mediated dilution of an aqueous dye adjacent to the cell monolayer. Calculation of the unitary water channel permeability, pf, yielded similar values for cell and proteoliposome experiments. Neither the absence of glucose or Na(+), nor the lack of membrane voltage in vesicles, nor the directionality of water flow grossly altered pf Such weak dependence on protein conformation indicates that a water-impermeable occluded state (glucose and Na(+) in their binding pockets) lasts for only a minor fraction of the transport cycle or, alternatively, that occlusion of the substrate does not render the transporter water-impermeable as was suggested by computational studies of the bacterial homologue vSGLT. Although the similarity between the pf values of SGLT1 and aquaporin-1 makes a transcellular pathway plausible, it renders water pumping physiologically negligible because the passive flux would be orders of magnitude larger.

The mobility of single-file water molecules is governed by the number of H-bonds they may form with channel-lining residues.

Channel geometry governs the unitary osmotic water channel permeability, pf, according to classical hydrodynamics. Yet, pf varies by several orders of magnitude for membrane channels with a constriction zone that is one water molecule in width and four to eight molecules in length. We show that both the pf of those channels and the diffusion coefficient of the single-file waters within them are determined by the number NH of residues in the channel wall that may form a hydrogen bond with the single-file waters. The logarithmic dependence of water diffusivity on NH is in line with the multiplicity of binding options at higher NH densities. We obtained high-precision pf values by (i) having measured the abundance of the reconstituted aquaporins in the vesicular membrane via fluorescence correlation spectroscopy and via high-speed atomic force microscopy, and (ii) having acquired the vesicular water efflux from scattered light intensities via our new adaptation of the Rayleigh-Gans-Debye equation.

High-speed AFM images of thermal motion provide stiffness map of interfacial membrane protein moieties.

The flexibilities of extracellular loops determine ligand binding and activation of membrane receptors. Arising from fluctuations in inter- and intraproteinaceous interactions, flexibility manifests in thermal motion. Here we demonstrate that quantitative flexibility values can be extracted from directly imaging the thermal motion of membrane protein moieties using high-speed atomic force microscopy (HS-AFM). Stiffness maps of the main periplasmic loops of single reconstituted water channels (AqpZ, GlpF) revealed the spatial and temporal organization of loop-stabilizing intraproteinaceous H-bonds and salt bridges.

Ion conductivity of the bacterial translocation channel SecYEG engaged in translocation.

While engaged in protein transport, the bacterial translocon SecYEG must maintain the membrane barrier to small ions. The preservation of the proton motif force was attributed to (i) cation exclusion, (ii) engulfment of the nascent chain by the hydrophobic pore ring, and (iii) a half-helix partly plugging the channel. In contrast, we show here that preservation of the proton motif force is due to a voltage-driven conformational change. Preprotein or signal peptide binding to the purified and reconstituted SecYEG results in large cation and anion conductivities only when the membrane potential is small. Physiological values of membrane potential close the activated channel. This voltage-dependent closure is not dependent on the presence of the plug domain and is not affected by mutation of 3 of the 6 constriction residues to glycines. Cellular ion homeostasis is not challenged by the small remaining leak conductance.

The bacterial translocon SecYEG opens upon ribosome binding.

In co-translational translocation, the ribosome funnel and the channel of the protein translocation complex SecYEG are aligned. For the nascent chain to enter the channel immediately after synthesis, a yet unidentified signal triggers displacement of the SecYEG sealing plug from the pore. Here, we show that ribosome binding to the resting SecYEG channel triggers this conformational transition. The purified and reconstituted SecYEG channel opens to form a large ion-conducting channel, which has the conductivity of the plug deletion mutant. The number of ion-conducting channels inserted into the planar bilayer per fusion event roughly equals the number of SecYEG channels counted by fluorescence correlation spectroscopy in a single proteoliposome. Thus, the open probability of the channel must be close to unity. To prevent the otherwise lethal proton leak, a closed post-translational conformation of the SecYEG complex bound to a ribosome must exist.