Experiments on cloned ktrB in the pBAD18 vector showed that V. alginolyticus KtrB alone was still active in E. coli. It mediated Na ؉ -independent, slow, high affinity, and mutation-specific K ؉ uptake as well as K ؉ -independent Na ؉ uptake. These data demonstrate that KtrB contains a selectivity filter for K ؉ ions and that all four conserved p-loop glycine residues are part of this filter. They also indicate that the role of KtrA lies in conferring velocity and ion coupling to the Ktr complex.
Membrane proteins are prime drug targets as they control the transit of information, ions, and solutes across membranes. Here, we present a membrane-on-nanopore platform to analyze nonelectrogenic channels and transporters that are typically not accessible by electrophysiological methods in a multiplexed manner. The silicon chip contains 250 000 femtoliter cavities, closed by a silicon dioxide top layer with defined nanopores. Lipid vesicles containing membrane proteins of interest are spread onto the nanopore-chip surface. Transport events of ligand-gated channels were recorded at single-molecule resolution by high-parallel fluorescence decoding.
The alkali cation potassium is the main osmolyte in the cytoplasm of prokaryotes (1). It binds close to the active center of the ribosome (2) and participates actively in pH homeostasis (3) and osmoadaptation (4, 5) by transport across the cell membrane. Hence, prokaryotes regulate their K ϩ contents and adapt them rapidly in response to changes in the environment. For this purpose, they possess a number of K ϩ channels, pumps, and transporters (1, 6). Several of the K ϩ -uptake systems contain a K ϩ -translocating subunit belonging to the superfamily of K ϩ transporters (7, 8) (termed SKT proteins (9)). These proteins may have evolved from simple K ϩ channels of the M 1 PM 2 type, like KcsA (10, 11) or Kir (12), by multiple gene duplications and gene fusions (7). Whereas the channels form homotetramers from four identical M 1 PM 2 subunits, SKT proteins consist of four covalently linked M 1 PM 2 motifs connected by cytoplasmic loops. The four p-loops (P) are thought to fold back from the external medium to the middle of the membrane, where they form a part of the permeation pathway for K ϩ through the channel center (7-9, 13). Within each p-loop, most SKT proteins contain one conserved glycine residue, which is part of their K ϩ selectivity filter (9, 14 -17). With single conserved glycine residues in SKT proteins, this filter appears to have a simpler structure than in K ϩ channels, in which the filter is formed by the well conserved p-loop sequence TVGYG from each subunit (18).The SKT-protein KtrB forms the K ϩ -translocating subunit of the Na ϩ -dependent K ϩ -uptake system KtrAB from bacteria (9, 14, 19 -22). KtrA, the other subunit from KtrAB, is located at the cytoplasmic side of the membrane and is a member of the RCK/KTN protein family (1, 23). KtrA may regulate K ϩ transport by binding ATP (24,25). It confers velocity, Na ϩ dependence, and K ϩ selectivity to the complex (9). KtrB alone transports K ϩ slowly in a process that is independent of Na ϩ . In addition, it transports Na ϩ with relatively low affinity (K m value of ϳ3 mM Na ϩ (9)). The exact structure of KtrB is unknown, but it has been modeled based on the structure of KcsA (11,13). Most of the KtrB structure was similar to that of KcsA. However, in particular, the C termini from the membrane spans M 2C and M 2D deviated from that of KcsA-M 2 . This may reflect the difference in function between the channel KcsA and the transporter KtrB (13). Subsequent cross-linking studies showed that the external half of KtrB is very similar to that of the KcsA tetramer, whereas its cytoplasmic half deviates. In addition, KtrB may form dimers (26). In their modeling studies, Durell and Guy (13) focused on membrane span M 2C . They divided it into three regions, from M 2C1 to M 2C3 (see Fig. 1A). M 2C1 and M 2C3 can form hydrophobic ␣-helices. However, M 2C2 contains many conserved small and polar residues (Ala, Gly, and Ser, Thr, Lys, respectively; see Fig. 1B). It may form a random coil or -turn structure (13). According to the first Durell and Guy model ...
After a survey of the special role, which the amino acid proline plays in the chemistry of life, the cell-penetrating properties of polycationic proline-containing peptides are discussed, and the widely unknown discovery by the Giralt group (J. Am. Chem. Soc. 2002, 124, 8876) is acknowledged, according to which fluorescein-labeled tetradecaproline is slowly taken up by rat kidney cells (NRK-49F). Here, we describe details of our previously mentioned (Chem. Biodiversity 2004, 1, 1111) observation that a hexa-β(3)-Pro derivative penetrates fibroblast cells, and we present the results of an extensive investigation of oligo-L- and oligo-D-α-prolines, as well as of oligo-β(2)h- and oligo-β(3)h-prolines without and with fluorescence labels (1-8; Fig. 1). Permeation through protein-free phospholipid bilayers is detected with the nanoFAST biochip technology (Figs. 2-4). This methodology is applied for the first time for quantitative determination of translocation rates of cell-penetrating peptides (CPPs) across lipid bilayers. Cell penetration is observed with mouse (3T3) and human foreskin fibroblasts (HFF; Figs. 5 and 6-8, resp.). The stabilities of oligoprolines in heparin-stabilized human plasma increase with decreasing chain lengths (Figs. 9-11). Time- and solvent-dependent CD spectra of most of the oligoprolines (Figs. 13 and 14) show changes that may be interpreted as arising from aggregation, and broadening of the NMR signals with time confirms this assumption.
Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed. The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of protein-harboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates. High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results. The chip technology is currently based on SiO2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.
Membrane protein transport on the single protein level still evades detailed analysis, if the substrate translocated is non-electrogenic. Considerable efforts have been made in this field, but techniques enabling automated high-throughput transport analysis in combination with solvent-free lipid bilayer techniques required for the analysis of membrane transporters are rare. This class of transporters however is crucial in cell homeostasis and therefore a key target in drug development and methodologies to gain new insights desperately needed.The here presented manuscript describes the establishment and handling of a novel biochip for the analysis of membrane protein mediated transport processes at single transporter resolution. The biochip is composed of microcavities enclosed by nanopores that is highly parallel in its design and can be produced in industrial grade and quantity. Protein-harboring liposomes can directly be applied to the chip surface forming self-assembled pore-spanning lipid bilayers using SSM-techniques (solid supported lipid membranes). Pore-spanning parts of the membrane are freestanding, providing the interface for substrate translocation into or out of the cavity space, which can be followed by multi-spectral fluorescent readout in real-time. The establishment of standard operating procedures (SOPs) allows the straightforward establishment of proteinharboring lipid bilayers on the chip surface of virtually every membrane protein that can be reconstituted functionally. The sole prerequisite is the establishment of a fluorescent read-out system for non-electrogenic transport substrates.High-content screening applications are accomplishable by the use of automated inverted fluorescent microscopes recording multiple chips in parallel. Large data sets can be analyzed using the freely available custom-designed analysis software. Three-color multi spectral fluorescent read-out furthermore allows for unbiased data discrimination into different event classes, eliminating false positive results.The chip technology is currently based on SiO 2 surfaces, but further functionalization using gold-coated chip surfaces is also possible.
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