Single-Cell Census of Mechanosensitive Channels in Living Bacteria

Bacteria are subjected to a host of different environmental stresses. One such insult occurs when cells encounter changes in the osmolarity of the surrounding media resulting in an osmotic shock. In recent years, a great deal has been learned about mechanosensitive (MS) channels which are thought to provide osmoprotection in these circumstances by opening emergency release valves in response to membrane tension. However, even the most elementary physiological parameters such as the number of MS channels per cell, how MS channel expression levels influence the physiological response of the cells, and how this mean number of channels varies from cell to cell remain unanswered. In this paper, we make a detailed quantitative study of the expression of the mechanosensitive channel of large conductance (MscL) in different media and at various stages in the growth history of bacterial cultures. Using both quantitative fluorescence microscopy and quantitative Western blots our study complements earlier electrophysiology-based estimates and results in the following key insights: i) the mean number of channels per cell is much higher than previously estimated, ii) measurement of the single-cell distributions of such channels reveals marked variability from cell to cell and iii) the mean number of channels varies under different environmental conditions. The regulation of MscL expression displays rich behaviors that depend strongly on culturing conditions and stress factors, which may give clues to the physiological role of MscL. The number of stress-induced MscL channels and the associated variability have far reaching implications for the in vivo response of the channels and for modeling of this response. As shown by numerous biophysical models, both the number of such channels and their variability can impact many physiological processes including osmoprotection, channel gating probability, and channel clustering

Applications of REDOR for distance measurements in biological solids

Knowledge of structural details at an atomic level is important in understanding the function and molecular properties of biomolecules. In situations where crystallography or solution state NMR methods are not applicable, solid state NMR can provide valuable insight without the limitations imposed by the availability of crystals, solubility or molecular weight. In particular dedicated solid state NMR methods enable the determination of selective internuclear distances at high accuracy. A popular and robust technique is rotational echo double resonance (REDOR), which allows the measurement of distances of heteronuclear spin pairs. This magic angle spinning method uses radiofrequency pulses to prevent the dipole-dipole interaction from being averaged by the sample spinning, leading to a reduction of the NMR signal which depends on the internuclear distance. Biological systems constitute a major area of application of the REDOR experiment, and are the focus of this review. First, the theoretical background, developments and experimental considerations of REDOR are discussed, regarding in particular applications on biological samples. Then, an overview of the use of REDOR in a wide range of biological applications, such as ligand binding sites of enzymes, fibrilar proteins, membrane active peptides, transmembrane helices, biomaterials like the peptidoglycan of bacterial cell walls or nucleic acids, is presented. © 2007 Elsevier Ltd. All rights reserved

2H[19F] REDOR for distance measurements in biological solids using a double resonance spectrometer.

A new approach for distance measurements in biological solids employing 2H[19F] rotational echo double resonance was developed and validated on 2H,19F-D-alanine and an imidazopyridine based inhibitor of the gastric H+/K+-ATPase. The 2H-19F double resonance experiments presented here were performed without 1H decoupling using a double resonance NMR spectrometer. In this way, it was possible to benefit from the relatively longer distance range of fluorine without the need of specialized fluorine equipment. A distance of 2.5 +/- 0.3 A was measured in the alanine derivative, indicating a gauche conformation of the two labels. In the case of the imidazopyridine compound a lower distance limit of 5.2 A was determined and is in agreement with an extended conformation of the inhibitor. Several REDOR variants were compared, and their advantages and limitations discussed. Composite fluorine dephasing pulses were found to enhance the frequency bandwidth significantly, and to reduce the dependence of the performance of the experiment on the exact choice of the transmitter frequency

Identifying anisotropic constraints in multiply labeled bacteriorhodopsin by 15N MAOSS NMR: a general approach to structural studies of membrane proteins.

Structural models of membrane proteins can be refined with sets of multiple orientation constraints derived from structural NMR studies of specifically labeled amino acids. The magic angle oriented sample spinning (MAOSS) NMR approach was used to determine a set of orientational constraints in bacteriorhodopsin (bR) in the purple membrane (PM). This method combines the benefits of magic angle spinning (MAS), i.e., improved sensitivity and resolution, with the ability to measure the orientation of anisotropic interactions, which provide important structural information. The nine methionine residues in bacteriorhodopsin were isotopically (15)N labeled and spectra simplified by deuterium exchange before cross-polarization magic angle spinning (CPMAS) experiments. The orientation of the principal axes of the (15)N chemical shift anisotropy (CSA) tensors was determined with respect to the membrane normal for five of six residual resonances by analysis of relative spinning sideband intensities. The applicability of this approach to large proteins embedded in a membrane environment is discussed in light of these results

Membrane protein structure determination using solid-state NMR.

Solid-state NMR is emerging as a method for resolving structural information for large biomolecular complexes, such as membrane-embedded proteins. In principle, there is no molecular weight limit to the use of the approach, although the complexity and volume of data is still outside complete assignment and structural determinations for any large (Mr > approx 30,000) complex unless specific methods to reduce the information content to a manageable amount are employed. Such methods include specific residue-type labeling, labeling of putative segments of a protein, or examination of complexes made up of smaller, manageable units, such as oligomeric ion channels. Labeling possibilities are usually limited to recombinant or synthesized proteins, and labeling strategies often follow models from a bioinformatics approach. In all cases, and in common with most membrane studies, sample preparation is vital, and this activity alone can take considerable effort before NMR can be applied--peptide or protein production (synthesis or expression) followed by reconstitution into bilayers and resolution of suitable sample geometry is still technically challenging. As experience is gained in the field, this development time should decrease. Here, the practical aspects of the use of solid-state NMR for membrane protein structural determinations are presented, as well as how the methodology can be applied. Some successes to date are discussed, with an indication of how the area might develop

Folding and Self-Assembly of the Pore-Forming Unit Tat-A of the Bacterial Twin-Arginine Translocase

The bacterial Twin-arginine translocation pathway is able to transport fully folded proteins across membranes. In B. subtilis it consists only of two components: TatCd, which serves as a receptor for the signal peptide, and the pore forming unit TatAd, which occurs in high stoichiometric excess. According to circular dichroism TatA contains a transmembrane segment, an amphiphilic helix, and an unstructured C-terminus [1]. Its detailed moelcular structure was resolved by solid-state NMR spectroscopy in oriented bilayers [2]. A striking pattern on the monomeric protein surface allowed us to assemble several units into protomers and into an open oligomeric pore. The stability of these complexes was supported by all-atom MD simulations and using structure-based modeling [3]. The observed interactions suggest that a novel motif for folding and self-assembly motif is present in this membrane-bound transport system, which allows reversible pore formation. Our comprehensive three-dimensional model thus reconciles for the first time TatA transport with a pore size of variable diameter, which can open and close by an energetically feasible mechanism. [1] Müller, S.D., A.A. De Angelis, T.H. Walther, S.L. Grage, C. Lange, S.J. Opella & A.S. Ulrich (2007) Biochim. Biophys. Acta 1768: 3071-3079 [2] Walther, T.H., S.L. Grage, N. Roth & A.S. Ulrich (2010) J. Am. Chem. Soc., in press [3] Grage, S.L., T.H. Walther, M. Wolf, A. Vargiu, M.J. Klein, P. Ruggerone, W. Wenzel, A.S. Ulrich (2010) submitte