15 research outputs found
Stability of Nucleosomes Containing Homogenously Ubiquitylated H2A and H2B Prepared Using Semisynthesis
Post-translational modifications (PTMs) of histones are
an essential
feature in the dynamic regulation of chromatin. One of these modifications,
ubiquitylation, has been speculated to directly influence the stability
of the nucleosome, which represents the basic building block of chromatin.
Here we report a strategy for the semisynthesis of site-specifically
ubiquitylated histone H2A (uH2A). This branched protein was generated
through a three-piece expressed protein ligation approach including
a traceless ligation at valine. uH2A could be efficiently incorporated
into nucleosomes, thereby opening the way to detailed biochemical
and biophysical studies on the function of this PTM. Accordingly,
we used uH2A, as well as a previously generated ubiquitylated H2B,
in chaperone-coupled nucleosome stability assays to demonstrate that
the direct effect of ubiquitylated histones on nucleosomal stability
is in fact modest
Investigating the Dynamics of Destabilized Nucleosomes Using Methyl-TROSY NMR
The nucleosome core
particle (NCP), comprised of histone proteins
wrapped with âŒ146 base pairs of DNA, provides both protection
and controlled access to DNA so as to regulate vital cellular processes.
High-resolution structures of nucleosomes and nucleosome complexes
have afforded a clear understanding of the structural role of NCPs,
but a detailed description of the dynamical properties that facilitate
DNA-templated processes is only beginning to emerge. Using methyl-TROSY
NMR approaches we evaluate the effect of point mutations designed
to perturb key histone interfaces that become destabilized during
nucleosome remodeling in an effort to probe NCP plasticity. Notably
the NCP retains its overall structural integrity, yet relaxation experiments
of mutant nucleosomes reveal significant dynamics within a central
histone interface associated with alternative NCP conformations populated
to as much as 15% under low salt conditions. This work highlights
the inherent plasticity of NCPs and establishes methyl-TROSY NMR as
a valuable compliment to current single molecule methods in quantifying
NCP dynamic properties
Examples for the determination of radial magnification errors.
<p>(A) Radial intensity profile measured in scans of the precision mask. Blue lines are experimental scans, and shaded areas indicate the regions expected to be illuminated on the basis of the known mask geometry. In this example, the increasing difference between the edges corresponds to a calculated radial magnification error of -3.1%. (BâD) Examples for differences between the experimentally measured positions of the light/dark transitions (blue circles, arbitrarily aligned for absolute mask position) and the known edge distances of the mask. The solid lines indicate the linear or polynomial fit. (B) Approximately linear magnification error with a slope corresponding to an error of -0.04%. Also indicated as thin lines are the confidence intervals of the linear regression. (C) A bimodal shift pattern of left and right edges, likely resulting from out-of-focus location of the mask, with radial magnification error of -1.7%. (D) A non-linear distortion leading to a radial magnification error of -0.53% in the <i>s</i>-values from the analysis of back-transformed data. The thin grey lines in C and D indicate the best linear fit through all data points.</p
Analysis of the rotor temperature.
<p>(A) Temperature values obtained in different instruments of the spinning rotor, as measured in the iButton at 1,000 rpm after temperature equilibration, while the set point for the console temperature is 20°C (indicated as dotted vertical line). The box-and-whisker plot indicates the central 50% of the data as solid line, with the median displayed as vertical line, and individual circles for data in the upper and lower 25% percentiles. The mean and standard deviation is 19.62°C ± 0.41°C. (B) Correlation between iButton temperature and measured BSA monomer <i>s</i>-values corrected for radial magnification, scan time, scan velocity, but not viscosity (symbols). In addition to the data from the present study as shown in (A) (circles), also shown are measurements from the pilot study [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref027" target="_blank">27</a>] where the same experiments were carried out on instruments not included in the present study (stars). The dotted line describes the theoretically expected temperature-dependence considering solvent viscosity.</p
Absence of a long-term trend in <i>s</i><sub><i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i></sub>-values of the BSA monomer with time of experiment for the three kits (blue, green, and magenta).
<p>Highlighted as bold solid line is the overall average, and the grey area indicates one standard deviation.</p
Corrected best-fit apparent monomer molecular mass from integration of the <i>c</i>(<i>s</i>) peak when scanned with the absorbance system (green) and the interference system (magenta).
<p>Only data with rmsd less than 0.01 OD or 0.01 fringes were included. The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median is displayed as a vertical line.</p
Observed fraction of dimer (as a ratio of dimer peak area to the sum of monomer plus dimer peak areas).
<p>The box-and-whisker plot indicates the central 50% of the data as solid line and draws the smaller and larger 25% percentiles as individual circles. The median displayed as vertical line. The mean and standard deviations are 18.5% ± 1.1% for the absorbance system, and 19.0% ± 2.1% for the interference system.</p
Examples of transient changes in the console temperature reading during the SV experiment, as saved in the scan file data.
<p>For comparison, the maximum adiabatic cooling of -0.3°C would be expected after approximately 300 sec, recovering to the equilibrium temperature after approximately 1,200 s (see Fig 3 in [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126420#pone.0126420.ref033" target="_blank">33</a>]).</p
Histogram and box-and-whisker plot of <i>s</i>-values of the BSA monomer after different corrections: Raw experimental <i>s</i>-values (black, with grey histogram), scan time corrected <i>s</i><sub><i>t</i></sub>-values (blue), rotor temperature corrected <i>s</i><sub><i>20T</i></sub>-values (green), or radial magnification corrected <i>s</i><sub><i>r</i></sub>-values (cyan), and fully corrected <i>s</i><sub><i>20T</i>,<i>t</i>,<i>r</i>,<i>v</i></sub>-values (red with red histogram).
<p>The box-and-whisker plots indicate the central 50% of the data as solid line and draw the smaller and larger 25% percentiles as individual circles. The median for each group is displayed as a vertical line.</p
Example for the analysis of absorbance data from the sedimentation velocity experiment of BSA.
<p>(A) Absorbance scans (symbols) and best-fit <i>c</i>(<i>s</i>) model at different points in time indicated by color temperature. (B and C) Bitmap and overlay of the residuals of the fit. (D) <i>c</i>(<i>s</i>) sedimentation coefficient distribution showing peaks for monomer, dimer, trimer, and traces of higher oligomers.</p