Membrane-on-a-Chip: Microstructured Silicon/Silicon-Dioxide Chips for High-Throughput Screening of Membrane Transport and Viral Membrane Fusion

Screening of transport processes across biological membranes is hindered by the challenge to establish fragile supported lipid bilayers and the difficulty to determine at which side of the membrane reactants reside. Here, we present a method for the generation of suspended lipid bilayers with physiological relevant lipid compositions on microstructured Si/SiO₂ chips that allow for high-throughput screening of both membrane transport and viral membrane fusion. Simultaneous observation of hundreds of single-membrane channels yields statistical information revealing population heterogeneities of the pore assembly and conductance of the bacterial toxin α-hemolysin (αHL). The influence of lipid composition and ionic strength on αHL pore formation was investigated at the single-channel level, resolving features of the pore-assembly pathway. Pore formation is inhibited by a specific antibody, demonstrating the applicability of the platform for drug screening of bacterial toxins and cell-penetrating agents. Furthermore, fusion of H3N2 influenza viruses with suspended lipid bilayers can be observed directly using a specialized chip architecture. The presented micropore arrays are compatible with fluorescence readout from below using an air objective, thus allowing high-throughput screening of membrane transport in multiwell formats in analogy to plate readers

Overexpression, purification, and fluorescent labeling of <i>M</i>. <i>tuberculosis</i> SecA1 and SecA2.

<p>A) Overexpression of <i>M</i>. <i>tuberculosis</i> SecA1 (lane 2) and SecA2 (lane 4) in <i>E</i>. <i>coli</i> BL21(λDE3) with a molecular mass of 105 and 85 kDa, respectively. Molecular masses of protein standard are indicated on the left. The empty vector, pACYCDuet-1 and pET15b, are shown as controls (lane 1 and 3). B) Coomassie-stained SDS-PAGE of purified <i>M</i>. <i>tuberculosis</i> SecA1 (lane 1) and SecA2 (lane 2). C) Visualization of fluorescently labeled SecA1 and SecA2 in SDS-PAGE by fluorescence imaging. SecA1 and SecA2 were labeled with the fluorescent probe Cy5 (lane 1 and 2) or AF488 (lane 3 and 4).</p

MST analysis of SecA1 and SecA2 homodimerization.

<p>A) Scheme of the MST experiments and B) diffusion traces observed in MST. The mobility of molecules in a temperature gradient is followed by the fluorescence intensity in a central spot. When the IR-Laser is turned on, the initial fluorescence (1) drops due to the thermophoretic movement of fluorescently labeled proteins out of the heated spot (2). When the IR-Laser is turned off, back-diffusion of the fluorescently labeled proteins is observed which is driven by mass diffusion and depends on the hydration shell of the proteins (3). Dimers diffuse slower than monomers. SecA1 (C) and SecA2 (D) dimerization measured by MST. Unlabeled protein (1 nM to 10 μM) was titrated into a fixed concentration of labeled protein (25 nM). The thermophoretic signal is plotted as a function of the protein concentration resulting in a dimerization curve. The curves were fitted using the Hill-equation and apparent K_d values were determined. Error bars represent the standard error of 3 measurements. The apparent K_d for SecA1 and SecA2 dimerization at low salt concentrations were 65 ± 2.5 nM and 161 ± 6.2 nM, respectively. The measurement of samples at high salt concentrations showed no binding.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Pulldown analysis on SecA1 and SecA2.

<p>A) Coomassie-stained SDS-PAGE of cell lysate of wild type (lane1), co-expressed His₆-SecA1 and Strep-SecA2 (lane 2), His₆-SecA1 only (lane 3), and Strep-SecA2 only (lane 4). Molecular masses of protein standard are indicated on the left. B) Western blot analysis of cell lysate of wild type (lane1), co-expressed His₆-SecA1 and Strep-SecA2 (lane 2), His₆-SecA1 only (lane 3), and Strep-SecA2 only (lane 4) against α-Strep antibody. C) Coomassie-stained SDS-PAGE of purified His₆-SecA1 (lane 5–8). D) Western blot analysis of purified His₆-SecA1 against α-Strep antibody (lane 5–8). Western blot data showed that SecA2 was co-purified with SecA1 (lane 6).</p

Membrane-on-a-Chip: Microstructured Silicon/Silicon-Dioxide Chips for High-Throughput Screening of Membrane Transport and Viral Membrane Fusion

Screening of transport processes across biological membranes is hindered by the challenge to establish fragile supported lipid bilayers and the difficulty to determine at which side of the membrane reactants reside. Here, we present a method for the generation of suspended lipid bilayers with physiological relevant lipid compositions on microstructured Si/SiO₂ chips that allow for high-throughput screening of both membrane transport and viral membrane fusion. Simultaneous observation of hundreds of single-membrane channels yields statistical information revealing population heterogeneities of the pore assembly and conductance of the bacterial toxin α-hemolysin (αHL). The influence of lipid composition and ionic strength on αHL pore formation was investigated at the single-channel level, resolving features of the pore-assembly pathway. Pore formation is inhibited by a specific antibody, demonstrating the applicability of the platform for drug screening of bacterial toxins and cell-penetrating agents. Furthermore, fusion of H3N2 influenza viruses with suspended lipid bilayers can be observed directly using a specialized chip architecture. The presented micropore arrays are compatible with fluorescence readout from below using an air objective, thus allowing high-throughput screening of membrane transport in multiwell formats in analogy to plate readers

Interactions of SecA1 and SecA2.

<p>A) MST measurement of SecA1-Cy5 titrated with increasing concentrations of unlabeled SecA2 B) MST measurement of SecA2-Cy5 titrated with increasing concentrations of unlabeled SecA1. The curves were fitted using the Hill-equation and the apparent K_ds were determined. Error bars represent the standard error of 3 measurements. The formation of the SecA1/SecA2 heterodimer is observed with an apparent K_d of 378 ± 22.4 nM and 438 ± 32.8 nM. C) FCCS analysis on SecA1 and SecA2. SecA1-Cy5 and SecA2-AF488 were mixed at ratio of 1:1. The measurements were done in 10 s and 10 repetitions. Auto-correlations of SecA1-Cy5 (red curve), SecA2-AF488 (blue curve) and cross-correlations (black curve) were recorded. The amplitude of cross-correlation was compared to the amplitude of autocorrelation. Positive cross-correlations is observed indicating the heterodimerization of SecA1 and SecA2. D) A control experiment of samples at high salt concentrations showed no cross-correlations indicating that the proteins stay in monomeric form.</p

Model of protein secretion.

<p>A) The canonical secretion pathway, in which protein translocation process is mediated by protein-conducting pore SecYEG and the ATP-driven motor protein SecA. First, dimeric SecA binds to the SecYEG channel. The chaperone protein maintains the newly synthesized preproteins in their unfolded form and targets them to SecYEG bound SecA. SecA delivers the chemical energy by the cycles of ATP binding and hydrolysis to drive preproteins through SecYEG and across the cytoplasmic membrane. B) Proposed model of the mycobacterial SecA1/SecA2 pathway. SecA2 and SecA1 form a heterodimer and bind asymmetrically to the canonical SecYEG. The ATPase activity of either SecA2 or SecA1 or both then provides the translocation of the substrate through SecYEG and across the cytoplasmic membrane. SecA1 dimers or possibly SecA2 dimers may also work on their own. CM, cytoplasmic membrane. SPase, signal peptidase.</p

Crystallographic analysis of RBF binding to LmrR.

<p><b>A</b>) Chemical structure of RBF. <b>B</b>) Composite omit 2<i>F</i>_o-<i>F</i>_c electron density for RBF in the LmrR•RBF structure calculated at 2.35 Å resolution and contoured at 1σ. The two crystallographically independent binding conformations of RBF are shown in stick representation with the carbon atoms colored green or cyan (oxygen and nitrogen atoms are colored red and blue, respectively). These binding conformations differ by a ~180° rotation of the heterocyclic isoalloxazine core relative to the ribityl moiety. The other two binding modes of RBF (shown with dark green lines) are related to the first two by 2-fold crystallographic symmetry (the location of the crystallographic dyad is indicated with an arrow). C) Overall structure of the LmrR•RBF dimer shown in two orientations. D) Close-up view of the RBF binding site and ligand-interacting residues. Amino acid residues within a radius of 4.5 Å from a ligand are shown in stick representation and labeled.</p