Probing Conformational States of the Finger and Thumb Subdomains of HIV‑1 Reverse Transcriptase Using Double Electron–Electron Resonance Electron Paramagnetic Resonance Spectroscopy

The configurational space sampled by the finger and thumb subdomains of the p66 subunit of HIV-1 reverse transcriptase was investigated by Q-band double electron–electron resonance pulsed electron paramagnetic resonance spectroscopy, a method for determining long-range distances between pairs of nitroxide spin-labels introduced via surface-engineered cysteine residues. Four constructs were examined, each containing two spin-labels in the p66 subunit, one in the finger subdomain and the other in the thumb subdomain. In the unliganded state, open and closed configurations for the finger and thumb subdomains are observed with the distribution between these states modulated by the spin-labels and associated mutations, in contrast to crystallographic data in which the unliganded state crystallizes in the closed conformation. Upon addition of double-stranded DNA, all constructs adopt open conformations consistent with previous crystallographic data in which the position of the thumb and finger subdomains is determined by contacts with the bound oligonucleotide duplex (DNA or DNA/RNA). Likewise, binary complexes with five different non-nucleoside reverse transcriptase inhibitors are in open or partially open conformations, indicating that binding of the inhibitor to the palm subdomain indirectly restricts the conformational space sampled by the finger and thumb subdomains

Direct Evaluation of Protein–Lipid Contacts Reveals Protein Membrane Immersion and Isotropic Bicelle Structure

The solvation of membrane proteins by both lipids and water makes their membrane immersion difficult to predict and the choice of a membrane mimic challenging. To characterize protein–lipid contacts and bicelle membrane mimics, we examined protein–lipid cross-relaxation of integrin αIIb and β3­(A711P) transmembrane helices in isotropic phospholipid bicelles (<i>q</i> = 0.5 and 0.7). Long-chain bicelle lipids dominated contacts with central helix segments, whereas both short- and long-chain lipids contacted the terminal turns of each helix in corroboration of the mixed bicelle model. The saturation transfer profiles from long-chain lipids directly established helix midpoints in the lipid bilayer. Lipid headgroups and water molecules engaged the side chains of buried serine and threonine in competition with intrahelical hydrogen bonding, illustrating that polar side chains seek the most favorable electrostatic contacts

Monitoring Functions in Managed Microbial Systems by Cytometric Bar Coding

Cytometric monitoring of microbial community dynamics can be used to estimate stability of technical microbial processes like biogas production by analysis of segregated cell abundance changes. In this study, structure variations of a biogas community were cytometrically recorded over 9 months and found to be of diagnostic value for process details. The reactor regime was intentionally disturbed with regard to substrate overload or H₂S accumulation. A single-cell based approach called cytometric bar coding (CyBar) for fast identification of reactive subcommunities was used. Functionality of specific subcommunities was uncovered by processing CyBar data with abiotic reactor parameters using Spearman’s correlation coefficient. Twenty subcommunities showed a discrete and divergent behavior. For example, a 4-fold substrate overload increased the cell number of two acidogenic index subcommunities to 176 and 193% within three days. Supplementary analyses were done using DNA fingerprinting, cloning, and sequencing. Bioreactor perturbations were shown to create cell abundance changes in subcommunities rather than variations in their phylogenetic composition. The used workflow and macros are ready-to-use tools and allow on-site monitoring and interpretation of variation in microbial community functions within a few hours

Single-molecule imaging and PICS analysis (S1 Fig Data).

<p><i>A</i>: Signal of individual eYFP-GR molecules on an emCCD camera. <i>B</i>: The signal of an individual molecule is fitted to a Gaussian yielding the position, the width and the strength of the signal. <i>C</i>: Distance calculation between molecules in subsequent frames. <i>D</i>: Cumulative distribution function (cdf) of distances of molecules in subsequent frames correlated by diffusion.</p

MOESM1 of Calcium binding protects E-cadherin from cleavage by Helicobacter pylori HtrA

Additional file 1. Supplementary information

Depth-of-Focus Correction in Single-Molecule Data Allows Analysis of 3D Diffusion of the Glucocorticoid Receptor in the Nucleus

<div><p>Single-molecule imaging of proteins in a 2D environment like membranes has been frequently used to extract diffusive properties of multiple fractions of receptors. In a 3D environment the apparent fractions however change with observation time due to the movements of molecules out of the depth-of-field of the microscope. Here we developed a mathematical framework that allowed us to correct for the change in fraction size due to the limited detection volume in 3D single-molecule imaging. We applied our findings on the mobility of activated glucocorticoid receptors in the cell nucleus, and found a freely diffusing fraction of 0.49±0.02. Our analysis further showed that interchange between this mobile fraction and an immobile fraction does not occur on time scales shorter than 150 ms.</p></div

Calibration of the depth of field (DOF). eYFP was coated on a glass slide and the objective was moved by a piezo scanner (S1 Fig Data).

<p>The resulting peak-widths were fitted as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141080#pone.0141080.ref025" target="_blank">25</a>]. The data were subsequently fit to Eq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141080#pone.0141080.e001" target="_blank">1</a>) yielding the signal width at focus, <i>σ</i>₀ = 263 nm and the DOF = 750 nm. All data characterized by a width larger than √2 × 263 nm = 372 nm (dashed line) were discarded from further analysis.</p

Result of Eq (6) for DOF = 750 nm and four different time lags, t (S1 Fig Data).

<p>For a diffusion constant of D = 2 μm²/s the probability to reside inside the DOF after t = 10 ms is 0.79, whereas for D = 0.1 mm2/s the probability is 0.97.</p

PICS analysis of glucocorticoid receptor at different time lags (S1 Fig Data).

<p>In blue the uncorrected result. A decrease of the fast fraction is observed. In green the result corrected by Eq (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0141080#pone.0141080.e008" target="_blank">8</a>) taking into account the DOF. The fast fraction stays constant for time lags at least up to 150 ms. Dashed lines are linear fits to the data. Error-bars represent the standard deviation.</p

Water Sorption Cycle Measurements on Functionalized MIL-101Cr for Heat Transformation Application

The water loading capacity and water cycle stability (40 adsorption/desorption cycles) of four nitro- or amino-functionalized MIL-101Cr materials (<b>1</b>–<b>4</b>) is assessed for heat transformation applications. Amino- or nitro-functionalized (<b>1</b>, <b>3</b>) and partially amino- or nitro-functionalized MIL-101Cr (<b>2</b>, <b>4</b>) have been synthesized through time-controlled postsynthetic modification of MIL-101Cr. The partially functionalized materials (<b>2</b>, <b>4</b>) contain about 78 mol % amino- or nitro-functionalized terephthalate linker. Hydrophilic nitro or amino functionalities were introduced into MIL-101Cr in order to achieve water loading at lower <i>p</i>/<i>p</i>₀ values for possible use in thermally driven adsorption chillers or heat pumps. Among the four materials studied, fully aminated MIL-101Cr-NH₂, <b>1</b>, and partially aminated MIL-101Cr-pNH₂, <b>2</b>, showed the best water loadings (about 1.0 gH₂O/gMIL) as well as water stability over 40 adsorption–desorption cycles. After 40 cycles, the X-ray powder diffractogram and Brunauer–Emmett–Teller (BET) surface determination of amino-functionalized materials indicated structural integrity with Δ_BET = −6.3% after 40 cycles, while the nitro-functionalized MIL-101Cr exhibited a decrease in their BET surface of Δ_BET = −25% and −20% for <b>3</b> and <b>4</b>, respectively