17 research outputs found

    NMR and Computation Reveal a Pressure-Sensitive Folded Conformation of Trp-Cage

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    Beyond defining the structure and stability of folded states of proteins, primary amino acid sequences determine all of the features of their conformational landscapes. Characterizing how sequence modulates the population of protein excited states or folding pathways requires atomic level detailed structural and energetic information. Such insight is essential for improving protein design strategies, as well as for interpreting protein evolution. Here, high pressure NMR and molecular dynamics simulations were combined to probe the conformational landscape of a small model protein, the tryptophan cage variant, Tc5b. Pressure effects on protein conformation are based on volume differences between states, providing a subtle continuous variable for perturbing conformations. 2D proton TOCSY spectra of Tc5b were acquired as a function of pressure at different temperature, pH, and urea concentration. In contrast to urea and pH which lead to unfolding of Tc5b, pressure resulted in modulation of the structures that are populated within the folded state basin. The results of molecular dynamics simulations on Tc5b displayed remarkable agreement with the NMR data. Principal component analysis identified two structural subensembles in the folded state basin, one of which was strongly destabilized by pressure. The pressure-dependent structural perturbations observed by NMR coincided precisely with the changes in secondary structure associated with the shifting populations in the folded state basin observed in the simulations. These results highlight the deep structural insight afforded by pressure perturbation in conjunction with high resolution experimental and advanced computational tools

    High Pressure ZZ-Exchange NMR Reveals Key Features of Protein Folding Transition States

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    Understanding protein folding mechanisms and their sequence dependence requires the determination of residue-specific apparent kinetic rate constants for the folding and unfolding reactions. Conventional two-dimensional NMR, such as HSQC experiments, can provide residue-specific information for proteins. However, folding is generally too fast for such experiments. ZZ-exchange NMR spectroscopy allows determination of folding and unfolding rates on much faster time scales, yet even this regime is not fast enough for many protein folding reactions. The application of high hydrostatic pressure slows folding by orders of magnitude due to positive activation volumes for the folding reaction. We combined high pressure perturbation with ZZ-exchange spectroscopy on two autonomously folding protein domains derived from the ribosomal protein, L9. We obtained residue-specific apparent rates at 2500 bar for the N-terminal domain of L9 (NTL9), and rates at atmospheric pressure for a mutant of the C-terminal domain (CTL9) from pressure dependent ZZ-exchange measurements. Our results revealed that NTL9 folding is almost perfectly two-state, while small deviations from two-state behavior were observed for CTL9. Both domains exhibited large positive activation volumes for folding. The volumetric properties of these domains reveal that their transition states contain most of the internal solvent excluded voids that are found in the hydrophobic cores of the respective native states. These results demonstrate that by coupling it with high pressure, ZZ-exchange can be extended to investigate a large number of protein conformational transitions

    Evolutionarily Conserved Pattern of Interactions in a Protein Revealed by Local Thermal Expansion Properties

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    The way in which the network of intramolecular interactions determines the cooperative folding and conformational dynamics of a protein remains poorly understood. High-pressure NMR spectroscopy is uniquely suited to examine this problem because it combines the site-specific resolution of the NMR experiments with the local character of pressure perturbations. Here we report on the temperature dependence of the site-specific volumetric properties of various forms of staphylococcal nuclease (SNase), including three variants with engineered internal cavities, as measured with high-pressure NMR spectroscopy. The strong temperature dependence of pressure-induced unfolding arises from poorly understood differences in thermal expansion between the folded and unfolded states. A significant inverse correlation was observed between the global thermal expansion of the folded proteins and the number of strong intramolecular hydrogen bonds, as determined by the temperature coefficient of the backbone amide chemical shifts. Comparison of the identity of these strong H-bonds with the co-evolution of pairs of residues in the SNase protein family suggests that the architecture of the interactions detected in the NMR experiments could be linked to a functional aspect of the protein. Moreover, the temperature dependence of the residue-specific volume changes of unfolding yielded residue-specific differences in expansivity and revealed how mutations impact intramolecular interaction patterns. These results show that intramolecular interactions in the folded states of proteins impose constraints against thermal expansion and that, hence, knowledge of site-specific thermal expansivity offers insight into the patterns of strong intramolecular interactions and other local determinants of protein stability, cooperativity, and potentially also of function

    Effect of Internal Cavities on Folding Rates and Routes Revealed by Real-Time Pressure-Jump NMR Spectroscopy

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    The time required to fold proteins usually increases significantly under conditions of high pressure. Taking advantage of this general property of proteins, we combined P-jump experiments with NMR spectroscopy to examine in detail the folding reaction of staphylococcal nuclease (SNase) and of some of its cavity-containing variants. The nearly 100 observables that could be measured simultaneously collectively describe the kinetics of folding as a function of pressure and denaturant concentration with exquisite site-specific resolution. SNase variants with cavities in the central core of the protein exhibit a highly heterogeneous transition-state ensemble (TSE) with a smaller solvent-excluded void volume than the TSE of the parent SNase. This heterogeneous TSE experiences Hammond behavior, becoming more native-like (higher molar volume) with increasing denaturant concentration. In contrast, the TSE of the L125A variant, which has a cavity at the secondary core, is only slightly different from that of the parent SNase. Because pressure acts mainly to eliminate solvent-excluded voids, which are heterogeneously distributed throughout structures, it perturbs the protein more selectively than chemical denaturants, thereby facilitating the characterization of intermediates and the consequences of packing on folding mechanisms. Besides demonstrating how internal cavities can affect the routes and rates of folding of a protein, this study illustrates how the combination of P-jump and NMR spectroscopy can yield detailed mechanistic insight into protein folding reactions with exquisite site-specific temporal information

    Recruitment, Assembly, and Molecular Architecture of the SpoIIIE DNA Pump Revealed by Superresolution Microscopy

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    <div><p></p><p>ATP-fuelled molecular motors are responsible for rapid and specific transfer of double-stranded DNA during several fundamental processes, such as cell division, sporulation, bacterial conjugation, and viral DNA transport. A dramatic example of intercompartmental DNA transfer occurs during sporulation in <i>Bacillus subtilis</i>, in which two-thirds of a chromosome is transported across a division septum by the SpoIIIE ATPase. Here, we use photo-activated localization microscopy, structured illumination microscopy, and fluorescence fluctuation microscopy to investigate the mechanism of recruitment and assembly of the SpoIIIE pump and the molecular architecture of the DNA translocation complex. We find that SpoIIIE assembles into ∌45 nm complexes that are recruited to nascent sites of septation, and are subsequently escorted by the constriction machinery to the center of sporulation and division septa. SpoIIIE complexes contain 47±20 SpoIIIE molecules, a majority of which are assembled into hexamers. Finally, we show that directional DNA translocation leads to the establishment of a compartment-specific, asymmetric complex that exports DNA. Our data are inconsistent with the notion that SpoIIIE forms paired DNA conducting channels across fused membranes. Rather, our results support a model in which DNA translocation occurs through an aqueous DNA-conducting pore that could be structurally maintained by the divisional machinery, with SpoIIIE acting as a checkpoint preventing membrane fusion until completion of chromosome segregation. Our findings and proposed mechanism, and our unique combination of innovating methodologies, are relevant to the understanding of bacterial cell division, and may illuminate the mechanisms of other complex machineries involved in DNA conjugation and protein transport across membranes.</p></div

    (A) Distribution of the ADC of CD9, CD55, and CD46 treated or not treated with MÎČCD (∌50% of the membrane Chl was removed)

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    CD55 is a raft marker, and CD46 is excluded from rafts and TEAs. Mean values of ADC of all the molecules are available in . (B) Comparison of trajectories (thin white lines) in living PC3 cells before (left) or after (right) MÎČCD treatment. Bars, 7.5 ÎŒm.<p><b>Copyright information:</b></p><p>Taken from "Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web"</p><p></p><p>The Journal of Cell Biology 2008;182(4):765-776.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518714.</p><p></p

    (A) ADC distribution and mean value (±SD) of CD55 molecules labeled with Atto647N-conjugated mAb 12A12

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    D is the mean value of the ADC calculated from a linear fit of the MSD-τ plot, and the dashed line delineates two different populations corresponding to pure confined trajectories (lower ADC) or mixed and Brownian trajectories. (B) Histograms (open boxes) representing the percentage of each CD55 diffusion mode as compared with the total number of trajectories. The gray part corresponds to the proportion of trajectories associated with TEAs (identified with the ensemble membrane labeling) for each diffusion mode (B, Brownian; C, confined; M, mixed). Compare with . (C) Trajectories of a single CD55 molecule. The inset is a magnification of the transient confinement area delineated by the boxed area.<p><b>Copyright information:</b></p><p>Taken from "Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web"</p><p></p><p>The Journal of Cell Biology 2008;182(4):765-776.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518714.</p><p></p

    SpoIIIE localization at superresolution.

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    <p>(a) SpoIIIE is observed during all stages of the cell cycle. Individual cells were recognized and classified as described in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001557#pbio-1001557-g001" target="_blank">Figure 1f</a>. Pixel size was 110 nm. From each 55 ms image, we automatically determined the localization of each single molecule in the image by using MTT <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001557#pbio.1001557-Serge1" target="_blank">[57]</a>. Each of these localizations is called a single-molecule event. In our pointilist representation, each single-molecule event is represented by a single green dot, whereas membrane stain is shown in white (see SM10 in Methods S1 for more details). Cells without septum were classified as stage 1 (vegetative/pre-divisional, left panel). Cells having a symmetric division septum were classified as stage 2 (division, middle panel), whereas those showing an asymmetric septum (at 1/5<sup>th</sup> or 4/5<sup>th</sup> of the total cell length) were classified as stage 3 (sporulating, right panel). (b) SpoIIIE clusters were automatically detected and classified depending on their size and composition. FWHM, full width at half maximum. (c) Analysis of the cluster size distribution versus the number of single-molecule events shows two distinct cluster types: PALM-limited clusters (red dots) have a size equal or smaller (∌45 nm FWHM) than the resolution of PALM in our conditions and contain a large number of events (>1,000), whilst dynamic clusters (orange dots) are large (>100 nm FWHM) and contain fewer events (<1,000). (d) The size of PALM-limited clusters is independent of cell cycle stage and the most typical size is ∌45 nm FWHM. (e) PALM-limited cluster sizes as a function of imaging time (total time used to image each single cluster) show that these clusters are extremely stable.</p

    SpoIIIE clusters assemble asymmetrically in sporulation septa.

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    <p>(a) PALM imaging of SpoIIIE (green dots) in sporulating cells overlaid with an epi-fluorescence image of the membrane (white, top panel). Intensity profiles across the direction perpendicular to the septum were used to determine the precise localization of the septal plane, which was used to calculate the distance of each single-molecule detection to the center of the septum and to automatically partition the cell into forespore (yellow) and mother cell (red). Using this partition, individual PALM-limited clusters and single-molecule events were classified as belonging to the mother cell or the forespore compartments. (b) Histogram of PALM-limited cluster localizations with respect to the center of the septum (red columns) in sporulating cells with flat septa and undergoing DNA translocation (<i>N</i> = 43). Black dotted line indicates the position of the septum. A Gaussian distribution was fitted to the data (blue solid line). SpoIIIE PALM-limited clusters preferentially localize on the mother cell side of the sporulation septum. (c) Histogram of single-molecule localizations in PALM-limited clusters (<i>N</i> = 43) in sporulating cells undergoing DNA translocation, and Gaussian fit (blue dotted line). In sporulating cells, the distribution of SpoIIIE with respect to the septum is asymmetric and biased towards the direction of the mother cell. (d) Histogram of localizations of single molecules in PALM-limited clusters of dividing cells (<i>N</i> = 71) and Gaussian fit (blue dashed line). During division, the distribution of SpoIIIE with respect to the septum is symmetric and unbiased. (e) Distance of SpoIIIE PALM-limited clusters to the center of asymmetric septa versus the amount of translocated DNA. Open blue circles represent individual distance values for individual clusters, and black squares the average distance for all clusters detected at each particular percentage of DNA translocated. The relative distance increases linearly with the amount of DNA in the forespore until ∌45% of DNA translocated, and thereon remains constant at ∌50 nm. Solid black line is a guide to the eye. (f) The size (FWHM) of individual PALM-limited clusters in the directions parallel or perpendicular to the asymmetric septa were calculated and plotted against each other to evaluate cluster symmetry (squares). The overwhelming majority of clusters are symmetric (green squares) with only a small minority being longer in the direction parallel to the septum (black squares). The dotted line is a guide to the eye.</p

    (A) Immunoprecipitation experiments in WT PC3 cells or in cells overexpressing CD9 (PC3/CD9) or a nonpalmitoylated form of CD9 (PC3/CD9)

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    Biotin-labeled cells were lysed in Brij97 and incubated with anti-CD9, anti-CD81, or anti-α5 antibodies (the latter is used as a negative control). Immunoprecipitated proteins were detected using peroxidase-coupled streptavidin. (B) Immunofluorescence images of PC3/CD9 living cell basal membrane by TIRF microscopy at 37°C. Cells were incubated with the anti-CD9 Cy3B-conjugated antibody SYB-1 (middle; green in the merge image) and with various antibodies labeled with Atto647N (left; red in the merge images) and raised against (top to bottom) CD81, CD9P-1, the α5 chain of integrin, CD55, or CD46. Bars, 10 Όm.<p><b>Copyright information:</b></p><p>Taken from "Single-molecule analysis of CD9 dynamics and partitioning reveals multiple modes of interaction in the tetraspanin web"</p><p></p><p>The Journal of Cell Biology 2008;182(4):765-776.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518714.</p><p></p
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