Effects of the M153R mutation in pHluorin on the protein stability of its fusion products.

<p>(<b>A</b>) Immunoblotting, using polyclonal anti-FliG (left panel) or anti-MotB antibody (right panel), of whole cell proteins prepared from SJW1103 (WT), YVM1002 (pHluorin-FliG, indicated as pH-FliG), YVM1004 (pHluorin(M153R)-FliG, indicated as pH(M153R)-FliG), YVM1001 (pHluorin-MotB, indicated as pH-MotB) and YVM1003 (pHluorin(M153R)-MotB, indicated as pH(M153R)-MotB). The positions of molecular mass markers (kDa) are shown on the left. (<b>B</b>) Fluorescence images (EPI) and bright field images (BF) of YVM1002 and YVM1004. The cells were grown overnight in LB at 30°C and observed by fluorescence microscopy.</p

pH dependence of fluorescence excitation spectra and Ratio_410/470 of pHluorin(M153R)-FliG-His₆.

<p>(<b>A</b>) Fluorescence excitation spectra. The different colored lines refer to different pH values. red, pH 5.5; orange, pH 6.0; light green, pH 6.5; green, pH 7.0; cyan, pH 7.5; grey, pH 8.0; black, pH 8.5. (<b>B</b>) Ratio_410/470. The fluorescence excitation spectra of the purified protein were recorded on a fluorescence spectrophotometer. The measurements were done at 23°C.</p

Effect of the M153R mutation on Ratio_410/470.

<p>(<b>A</b>) Fluorescent intensities of SJW1103/pYC001 (pHluorin) and SJW1103/pYVM001 (pHluorin(M153R)) cells grown in T-broth at 30°C. Emission spectra with 395 nm excitation were measured by a fluorescence spectrophotometer. The measurements were done at 23°C. Inset: Immunoblotting, using polyclonal anti-GFP antibody, of whole cell proteins. (<b>B</b>) Fluorescent intensities of purified pHluorin and pHluorin(M153R).</p

Strains and Plasmids used in this study.

<p>*In this pHluorin-MotB-His₈ fusion construct, N-terminal 28 residues of MotB (Met1- Lys28) are attached to the N-terminus of pHluorin as described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019598#pone.0019598-Leake1" target="_blank">[9]</a>.</p

Primers used for construction of <i>pHluorin-fliG</i> and <i>pHluorin-motB</i> strains.

<p>Primers used for construction of <i>pHluorin-fliG</i> and <i>pHluorin-motB</i> strains.</p

Pressure dependence of Poisson’s ratio of glassy Baltic amber studied by Brillouin scattering spectroscopy

Amber is a typical natural glass with very long aging time. Elastic properties of amber at high pressures have been studied using Brillouin scattering and a diamond anvil cell. Both longitudinal and transverse acoustic modes have been observed up to 12 GPa. The pressure dependences of longitudinal, shear, Young's, and bulk moduli, compressibility, and Poisson's ratio were determined. The longitudinal, shear, Young's, bulk moduli show the remarkable increase, and compressibility shows a marked decrease with increasing pressure. However, it is found that the pressure dependence of Poisson's ratio is very small. The mechanism of this small pressure dependence was discussed. The Cauchy type relation between longitudinal and shear moduli was examined. Its coefficients show the deviation from the Cauchy relation owing to the coexistence of different intermolecular interactions in amber

pH dependence of fluorescence excitation spectra and Ratio_410/470 of pHIuorin and pHluorin(M153R).

<p>(<b>A</b>) Fluorescence excitation spectra. The different colored lines refer to different pH values. red, pH 5.5; orange, pH 6.0; light green, pH 6.5; green, pH 7.0; cyan, pH 7.5; grey, pH 8.0; black, pH 8.5. (<b>B</b>) Ratio_410/470. The fluorescence excitation spectra of purified proteins were recorded on a fluorescence spectrophotometer. The measurements were done at 23°C.</p

Interaction between Na⁺ Ion and Carboxylates of the PomA−PomB Stator Unit Studied by ATR-FTIR Spectroscopy

Bacterial flagellar motors are molecular machines powered by the electrochemical potential gradient of specific ions across the membrane. The PomA−PomB stator complex of Vibrio alginolyticus couples Na+ influx to torque generation in this supramolecular motor, but little is known about how Na+ associates with the PomA−PomB complex in the energy conversion process. Here, by means of attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, we directly observed binding of Na+ to carboxylates in the PomA−PomB complex, including the functionally essential residue Asp24. The Na+ affinity of Asp24 is estimated to be ∼85 mM, close to the apparent Km value from the swimming motility of the cells (78 mM). At least two other carboxylates are shown to be capable of interacting with Na+, but with somewhat lower affinities. We conclude that Asp24 and at least two other carboxylates constitute Na+ interaction sites in the PomA−PomB complex. This work reveals features of the Na+ pathway in the PomA−PomB Na+ channel by using vibrational spectroscopy

Presentation_1_Stator Dynamics Depending on Sodium Concentration in Sodium-Driven Bacterial Flagellar Motors.pdf

Bacterial flagellar motor (BFM) is a large membrane-spanning molecular rotary machine for swimming motility. Torque is generated by the interaction between the rotor and multiple stator units powered by ion-motive force (IMF). The number of bound stator units is dynamically changed in response to the external load and the IMF. However, the detailed dynamics of stator unit exchange process remains unclear. Here, we directly measured the speed changes of sodium-driven chimeric BFMs under fast perfusion of different sodium concentration conditions using computer-controlled, high-throughput microfluidic devices. We found the sodium-driven chimeric BFMs maintained constant speed over a wide range of sodium concentrations by adjusting stator units in compensation to the sodium-motive force (SMF) changes. The BFM has the maximum number of stator units and is most stable at 5 mM sodium concentration rather than higher sodium concentration. Upon rapid exchange from high to low sodium concentration, the number of functional stator units shows a rapidly excessive reduction and then resurrection that is different from predictions of simple absorption model. This may imply the existence of a metastable hidden state of the stator unit during the sudden loss of sodium ions.</p

Patient characteristics of the AMS 3–4 cohort.

Patient characteristics of the AMS 3–4 cohort.</p