Resorcin[4]arene cavitands with four quinoxaline bridges are a family of macrocycles that adopt, at elevated temperature, a contracted, vase‐type conformation, capable of guest inclusion, whereas at low temperature they switch to an expanded, kite‐type conformation with a large flat surface. The present investigations lay the foundation for the use of such dynamic cavitands as miniaturized mechanical grippers for supramolecular construction at the single‐molecule level. New vase–kite switching modes, stimulated by pH changes or stoichiometric metal‐ion complexation, have been discovered and monitored by 1H NMR and optical absorption spectroscopy. The solid‐state geometries of the two states have been revealed by X‐ray crystallography, and the kinetics and thermodynamics of the switching processes in solution as well as their solvent dependency has been investigated in great detail. Monolayers of the cavitand in the vase form have been studied by scanning tunneling microscopy at molecular resolution; conformational switching is also observed in Langmuir monolayers at the air/water interface. Synthetic protocols have been developed for preparation of partially and asymmetrically bridged resorcin[4]arene cavitands, which are also shown to undergo conformational switching. These synthetic advances pave the way to new, dynamic molecular receptors for steroids, tetrathiofulvalene‐bridged grippers with the potential to undergo electrochemically induced conformational switching, and systems with greatly extended, rigid cavity walls functionalized at the termini by dipyrrometheneboron difluoride dyes. The latter cavitands are shown by fluorescence resonance energy transfer to undergo geometrically precisely defined motions between a contracted (≈ 7 Å linear extension) and a strongly expanded (≈ 7 nm linear extension) state.

Gram-negative bacteria acquire iron with TonB-dependent uptake systems. The TonB-ExbBD inner membrane complex is hypothesized to transfer energy to outer membrane (OM) iron transporters. Fluorescence microscopic characterization of green fluorescent protein (GFP)-TonB hybrid proteins revealed an unexpected, restricted localization of TonB in the cell envelope. Fluorescence polarization measurements demonstrated motion of TonB in living cells, which likely was rotation. By determining the anisotropy of GFP-TonB in the absence and presence of inhibitors, we saw the dependence of its motion on electrochemical force and on the actions of ExbBD. We observed higher anisotropy for GFP-TonB in energy-depleted cells and lower values in bacteria lacking ExbBD. However, the metabolic inhibitors did not change the anisotropy of GFP-TonB in ΔexbBD cells. These findings demonstrate that TonB undergoes energized motion in the bacterial cell envelope and that ExbBD couples this activity to the electrochemical gradient. The results portray TonB as an energized entity in a regular array underlying the OM bilayer, which promotes metal uptake through OM transporters by a rotational mechanism.bioenergetics | membrane transport | FepA | iron transport F rom its importance in aerobic metabolism, iron is essential to most pro-and eukaryotes and therefore is a determinant of bacterial disease. Its sequestration by transferrin, lactoferrin, ferritin, heme compounds, and lipocalins defends animal cells, fluids, and tissues by "nutritional immunity" (1). However, efficient pathogens overcome this barrier and capture Fe 3+ either by producing siderophores (2) or by directly removing the metal from eukaryotic proteins (3). The trilaminar cell envelope of Gram-negative bacteria, composed of inner membrane (IM), outer membrane (OM), and the periplasm between them, contains protein components that confer the uptake of metabolic solutes, including sugars, amino acids, nucleotides, vitamins, and metals such as iron (4, 5). Enigmatic OM active transporters acquire metal complexes (ferric siderophores, heme, vitamin B 12 ) from the environment (6). The OM protein ferric enterobactin permease A (FepA), for example, internalizes the siderophore ferric enterobactin (FeEnt) (7). It is typical of many homologous metal transporters in commensal and pathogenic organisms. These uptake reactions also require TonB (8), a cell envelope protein that long ago was proposed to transduce energy (9-12). However, many questions exist about TonB's mediation of iron uptake, including its physical mechanism and its relationship to bioenergetics. Proton motive force (PMF) may drive OM active transport (6-8), but the mode of energy transmission to the OM and TonB's potential role in it are unknown. We addressed these topics by characterizing the localization of TonB in the cell membranes, by monitoring its physical motion, and by determining the dependence of its movements on metabolic energy and the additional IM proteins ExbBD.Iron chelates bind to their OM transporter...

We report the synthesis of modified Cram-type cavitands bearing one or two fluorescent labels for singlemolecule spectroscopic studies of vaseÀkite conformational switching (Scheme 3). Syntheses were performed by stepwise bridging of the four couples of neighboring H-bonded OH groups of resorcin[4]arene bowls (Schemes 2 and 3). The new substitution patterns enable the construction of a large variety of future functional architectures.1 H-NMR Investigations showed that the new partially and differentially bridged cavitands feature temperature-and pH-triggered vaseÀkite conformational isomerism similar to symmetrical cavitands with four identical quinoxaline bridges (Table). It was discovered that vaseÀkite switching of cavitands is strongly solventdependent.Cavitands, which consist of a resorcin[4]arene bowl bridged by four quinoxaline (1) or pyrazine moieties, were introduced by Cram and co-workers [1] as a family of macrocycles capable of adopting two preferred conformations with profound geometry and property differences (Scheme 1). The vase conformation, with a deep cavity for guest inclusion, is preferred at elevated temperatures (> 318 K), whereas the kite conformation, with a large flat surface, dominates at low temperatures (< 213 K). Conformational switching can also be reversibly induced at ambient temperature by pH changes, with the kite conformation being preferred at low pH [2]. The vaseÀkite isomerization is conveniently monitored by

Diffusion of two Escherichia coli outer membrane proteins-the cobalamin (vitamin B12) receptor (BtuB) and the OmpF porin, which are implicated in the cellular import pathways of colicins and phages-was measured in vivo. The lateral mobility of these proteins is relevant to the mechanism of formation of the translocon for cellular import of colicins such as the rRNase colicin E3. The diffusion coefficient (D) of BtuB, the primary colicin receptor, complexed to fluorescent antibody or colicin, is 0.05±0.01 μm2/s and 0.10±0.02 μm2/s, respectively, over a timescale of 25-150 ms. Mutagenesis of the BtuB TonB box, which eliminates or significantly weakens the interaction between BtuB and the TonB energy-transducing protein that is anchored in the cytoplasmic membrane, resulted in a fivefold larger value of D, 0.27±0.06 μm2/s for antibody-labeled BtuB, indicating a cytoskeletal-like interaction of TonB with BtuB. OmpF has a diffusion coefficient of 0.006±0.002 μm2/s, ∼10-fold smaller than that of BtuB, and is restricted within a domain of diameter 100 nm, showing it to be relatively immobile compared to BtuB. Thus, formation of the outer membrane translocon for cellular import of the nuclease colicins is a demonstrably dynamic process, because it depends on lateral diffusion of BtuB and collisional interaction with relatively immobile OmpF.

We report on an in vivo single-molecule study of the signaling kinetics of G protein-coupled receptors (GPCR) performed using the neurokinin 1 receptor (NK1R) as a representative member. The NK1R signaling cascade is triggered by the specific binding of a fluorescently labeled agonist, substance P (SP). The diffusion of single receptor-ligand complexes in plasma membrane of living HEK 293 cells is imaged using fast single-molecule wide-field fluorescence microscopy at 100 ms time resolution. Diffusion trajectories are obtained which show intra- and intertrace heterogeneity in the diffusion mode. To investigate universal patterns in the diffusion trajectories we take the ligand-binding event as the common starting point. This synchronization allows us to observe changes in the character of the ligand-receptor-complex diffusion. Specifically, we find that the diffusion of ligand-receptor complexes is slowed down significantly and becomes more constrained as a function of time during the first 1000 ms. The decelerated and more constrained diffusion is attributed to an increasing interaction of the GPCR with cellular structures after the ligand-receptor complex is formed.