Christine Siligan

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

Mobility of single-file water molecules determined by H-bonds. 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.

The bacterial translocon SecYEG opens upon ribosome binding

Background: How SecYEG opens for co-translational translocation is unknown. Results: Ribosome binding to the SecY complex induces ion channel activity. Conclusion: SecYEG responds to ligand binding by a conformational transition. Significance: Dislocation of the plug prepares entry of the nascent chain. 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.

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.

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.

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.

Ion conductivity of the bacterial translocation channel SecYEG engaged in translocation

Background: How SecYEG excludes ions during protein translocation is not known. Results: SecYEG gating is voltage-sensitive. Conclusion: Voltage minimizes the ion leak that is induced by stalling a translocation intermediate. Significance: Preservation of the proton motif force requires voltage-driven conformational changes. 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.

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.

Voltage Sensitivity of the Bacterial Protein Translocation Channel

The heterotrimeric protein translocation channel SecYEG enables (i) soluble proteins to cross the inner membrane and (ii) hydrophobic proteins to enter the membrane interior. It contains an aqueous pore that, in its resting state, is sealed by a ring of six hydrophobic residues and a half helix, termed the plug [1]. Signal sequence binding or ribosome binding both dislocate the plug and break the ring, thereby opening the channel to ions [2]. The membrane barrier to ions is preserved, since physiological values of the transmembrane potential close the channel by a yet unknown mechanism [3]. Here we demonstrate that this voltage sensitivity does not depend on the ligand. To be precise, the open time decreases together with a decrease in voltage for SecYEG channels that are bound to (i) signal peptides, (ii) translocation intermediates (proOmpA) and the motor protein SecA, (iii) a ribosome-nascent chain (FtsQ) complex or (iv) empty ribosomes. The observations were made with planar lipid bilayers that contained the purified and reconstituted SecYEG complex. They indicate that the voltage sensor must be part of the SecYEG channel. In our search for the sensor, we mutated charged residues, deleted the plug and performed various cross-link experiments, the outcome of which will be discussed.1. Saparov, S.M., Erlandson, K., Cannon, K., Schaletzky, J., Schulman, S., Rapoport, T.A., Pohl, P. (2007). Determining the Conductance of the SecY Protein Translocation Channel for Small Molecules. Mol. Cell.2. Knyazev, D.G., Lents, A., Krause, E., Ollinger, N., Siligan, C., Papinski, D., Winter, L., Horner, A., Pohl, P. (2013). The Bacterial Translocon SecYEG Opens upon Ribosome Binding. J. Biol. Chem.3. Knyazev, D.G., Winter, L., Bauer, B.W., Siligan, C., Pohl, P. (2014). Ion Conductivity of the Bacterial Translocation Channel SecYEG Engaged in Translocation. J. Biol. Chem.