Nicole Ollinger

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.

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.

Transport of Small Molecules across the Bacterial Translocation Channel SecYEG

Many proteins are translocated across the endoplasmic reticulum (ER) membrane or the bacterial plasma membrane through a conserved channel, formed by a heterotrimeric protein complex (called the Sec61p complex in eukaryotes and the SecYEG complex in bacteria and archaea). Cotranslational or postranslational translocation requires ribosome or SecA binding, respectively. The resting channel is impermeable to ions and water (Saparov et al., 2007). Here we have tested whether the channel remains a barrier to small molecules after a conformational transition occurred due to ligand binding. Therefore, we reconstituted the purified and fluorescently labeled translocation channel SecYEG into planar lipid bilayers. Positioning of the membrane on top of a laser scanning microscope with single molecule sensitivity allowed monitoring the reconstitution efficiency. Both the motor molecule SecA and ribosomes induced ion channel activity. The probability of the channel opening was derived from the number of open channels and the total number of reconstituted channels.Acknowledgments: The project was funded by the Austrian Science Fund.ReferenceS. M. Saparov, Karl Erlandson, Kurt Cannon, Julia Schaletzky, Sol Schulman, Tom A. Rapoport, and P. Pohl. Determining the Conductance of the SecY Protein Translocation Channel for Small Molecules. Mol.Cell 26 (4):501-509, 2007.