91 research outputs found
Binding surfaces positioning in the active and inactive structure, and predicted molecular movement to yield inactive RB1. A., B.
<p>Relative orientation of the functional surfaces in the model of active, nonphosphorylated (A) and inactive, phosphorylated (B) RB1. Cartoon representation of Rb-NP with overlaid transparent surface with RB-N in light blue, RB-P in light-pink. The residues involved in docking LXCXE are shown in yellow, those forming the FXXXV motif are shown in purple and those for EXXXDLFD in cyan. The residues 346â355 which form a helix in unmodified RB-N but are disordered in inactive RB-NP are represented in dark grey <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058463#pone.0058463-Hassler1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058463#pone.0058463-Burke1" target="_blank">[21]</a>, amino acid groups involved in RB-N:P interphase interaction in the inactive conformation in red ([RB-N K136, D139, T140, T142, D145], [RB-P Q736, E737, K740, K729]) and orange [(RB-N L161, K164, L206-E209, L211-I213, F216, E282, E287, N290, N295] [RB-P Q736, E737, K740, K729]). <b>C., D.</b> Cartoon representation of active, nonphosphorylated, (C) and inactive, phosphorylated (D) RB1. RB-N B-fold is coloured in green and RB-P B-fold in purple (this different colour scheme has not been used elsewhere in the paper and is only used here for clarity). The residues 346â355 which are structured in unmodified RB-N and unstructured in inactive RB-NP are represented in dark grey. <b>E.</b> Predicted molecular movement yielding conformational RB1 inactivation. Note surfaces involved in binding LXCXE motif proteins in RB-P (salmon/pink) and the homologous surface involved in FXXXV binding in RB-N (cyan/blue) are collinear in the active (left) but not inactive form (right).</p
RB1 architecture and study design.
<p><b>A.</b> Schematic of RB1 domain structure. RB1 NH2-terminal domain (RB-N, light blue), RB1 pocket-domain (RB-P, raspberry), the position of the twin cyclin folds which form the core of each domain is indicated, RB1 C-terminal region (RB-C, yellow)<b>. B.</b> RB1 constructs used in this study indicating the range of amino-acids covered. In the MBP-RB-NP and MBP-ddRB-NP constructs maltose binding protein (MBP, green) is coupled to the N-terminus of the RB construct, while in ddRB-NP-MBP it is coupled to the C-terminus. In the ddRB-NP, MBP-ddRB-NP and ddRB-NP-MBP constructs two interstitial regions were deleted, corresponding to residues 250â269, the arginine-rich linker (R-linker) of the RB-N domain, and residues 579â643, corresponding to the pocket linker connecting RB-P domain pocket lobes (P-linker). The positions of cyclin-dependent kinase consensus sites in RB-NP are indicated, with sites retained in the ddRB-NP, MBP-ddRB-NP and ddRB-NP-MBP constructs bold and starred. <b>C.</b> Atomic models of the RB-N and RB-P domains, shown in ribbon representations. RB-N left, RB-P right. Cyclin-fold helixes are coloured, RB-N A-fold in cyan, RB-N B-fold in light blue, RB-P A-fold in dark salmon, RB-P B-fold in pink, other helixes and visible loops are shown as grey.</p
Characterisation of RB1 derivatives by small-angle X-ray scattering.
<p><b>A</b>. Experimental and calculated scattering patterns of ddRB<b>-</b>NP (1), MBP-ddRB<b>-</b>NP (2), ddRB<b>-</b>NP-MBP (3). Experimental SAXS data as black dots with black error bars. Lines (red) represent the fits from <i>ab initio</i> models shown in <b>C</b> (ddRB-NP), <b>D</b> (MBP-ddRB-NP) and <b>E</b> (ddRB-NP-MBP). The logarithm of the scattering intensity is plotted as a function of momentum transfer, sâ=â4Ïsin(Ξ/2)/λ where Ξ is the scattering angle and λ is the wavelength of the X-rays (1.5 Ă
). <b>B.</b> Distance distribution functions for ddRB-NP, MBP-ddRB-NP and ddRB-NP-MBP. <b>C.</b> Averaged <i>ab initio</i> models for ddRB-NP obtained using DAMMIN (grey semi-transparent spheres) and MONSA (RB-N blue spheres, RB-P red spheres) superimposed. The models are shown in two different views rotated by 90°. <b>D., E. </b><i>Ab initio</i> models of MBP-ddRB-NP (<b>D</b>) and ddRB-NP-MBP (<b>E</b>) obtained by MONSA. MBP is shown as green, ddRB-NP as grey spheres. The models are viewed as in <b>C</b>. <b>F.</b> Radius of gyration (<i>R<sub>g</sub></i>) distribution obtained by EOM for ddRB-NP. Distributions correspond to a random pool of 10.000 generated structures (blue) and the EOM optimized ensemble (red).</p
KirkwoodâBuff Approach Rescues Overcollapse of a Disordered Protein in Canonical Protein Force Fields
Understanding the function of intrinsically
disordered proteins
is intimately related to our capacity to correctly sample their conformational
dynamics. So far, a gap between experimentally and computationally
derived ensembles exists, as simulations show overcompacted conformers.
Increasing evidence suggests that the solvent plays a crucial role
in shaping the ensembles of intrinsically disordered proteins and
has led to several attempts to modify water parameters and thereby
favor proteinâwater over proteinâprotein interactions.
This study tackles the problem from a different perspective, which
is the use of the KirkwoodâBuff theory of solutions to reproduce
the correct conformational ensemble of intrinsically disordered proteins
(IDPs). A protein force field recently developed on such a basis was
found to be highly effective in reproducing ensembles for a fragment
from the FG-rich nucleoporin 153, with dimensions matching experimental
values obtained from small-angle X-ray scattering and single molecule
FRET experiments. KirkwoodâBuff theory presents a complementary
and fundamentally different approach to the recently developed four-site
TIP4P-D water model, both of which can rescue the overcollapse observed
in IDPs with canonical protein force fields. As such, our study provides
a new route for tackling the deficiencies of current protein force
fields in describing protein solvation
Single particle analysis of electron microscope images of MBP-ddRB-NP. A.-F.
<p>3D reconstruction of unmodified MBP-ddRB-NP. <b>A.</b>, <b>B</b>. Single particle reconstruction for unmodified MBP-ddRB-NP. Calculated density map of MBP-ddRB-NP, shown as surface representations in grey related by a 90<sup>o</sup> rotation. <b>C.</b> 3D reconstruction in mesh representation oriented as in <b>B</b> with the docked structures of the RB-N and RB-P domains (PDB codes 2QDJ and 3POM) shown as cartoons colour-coded as follows: RB-N domain lobe A -cyan, lobe B -light blue; RB-P domain lobe A -dark salmon and lobe B â pink. <b>D., E.</b> Segmented densities shown as solid surface representation with overlaid surface representation of the unmodified RB-NP 3D reconstruction in mesh. The density attributed to the MBP tag is shown in light green, that attributed to RB-N in light blue and to RB-P in light pink. <b>F.</b> Docked structures of the RB-N and RB-P domains (PDB codes 2QDJ and 3POM) without density mesh, shown as cartoons and colour-coded as in C. <b>G.âL.</b> 3D reconstruction of phosphorylated MBP-ddRB-NP. <b>G., H</b>. 3D reconstruction shown as a grey surface in two orthogonal views. <b>I.</b> 3D reconstruction in mesh representation oriented as in <b>H</b> with the docked structures of inactive RB-NP (PDB code 4ELJ) shown as cartoons colour-coded as follows:-. RB-N domain lobe A -cyan, lobe B -light blue; RB-P domain lobe A -dark salmon and lobe B â pink. <b>J., K.</b> Segmented densities shown as solid surface representation with overlaid surface representation of the 3D reconstruction in mesh<b>.</b> Same colour coding as in <b>D</b> and <b>E. L.</b> Docked structures of inactive RB-NP (PDB code 4ELJ) without density mesh, shown as cartoons colour-coded as in I.</p
Unusual Structural Morphology of Dendrimer/CdS Nanocomposites Revealed by Synchrotron X-ray Scattering
Low-resolution structure of CdS nanoparticles (NPs) grown in the
presence of the third-generation rigid polyphenylenepyridyl dendrimers
(PPPDs) is analyzed by synchrotron small-angle X-ray scattering (SAXS)
and transmission electron microscopy (TEM). The combination of rigidity
of the PPPDs and a high local concentration of chelating nitrogens
strongly interacting with growing CdS NPs yields anisometric particles
instead of conventional spherical ones. The scattering data from the
free PPPDs and from the composite CdS/PPPD NPs are interpreted in
terms of 3-D models revealing a peculiar morphology of the nanocomposite
whereby the PPPDs enclose the CdS NPs like a âflattened ball-in-handsâ.
The sizes of the CdS NPs found by SAXS are in good agreement with
the TEM data. The presented approach to elucidate particle morphology
should open the ways of detailed characterization of the modern composite
materials from the SAXS data
Reconstruction of Quaternary Structure from Xâray Scattering by Equilibrium Mixtures of Biological Macromolecules
A recent
renaissance in small-angle X-ray scattering (SAXS) made
this technique a major tool for the low-resolution structural characterization
of biological macromolecules in solution. The major limitation of
existing methods for reconstructing 3D models from SAXS is imposed
by the requirement of solute monodispersity. We present a novel approach
that couples low-resolution 3D SAXS reconstruction with composition
analysis of mixtures. The approach is applicable to polydisperse and
difficult to purify systems, including weakly associated oligomers
and transient complexes. Ab initio shape analysis is possible for
symmetric homo-oligomers, whereas rigid body modeling is applied also
to dissociating complexes when atomic structures of the individual
subunits are available. In both approaches, the sample is considered
as an equilibrium mixture of intact complexes/oligomers with their
dissociation products or free subunits. The algorithms provide the
3D low-resolution model (for ab initio modeling, also the shape of
the monomer) and the volume fractions of the bound and free state(s).
The simultaneous fitting of multiple scattering data sets collected
under different conditions allows one to restrain the modeling further.
The possibilities of the approach are illustrated in simulated and
experimental SAXS data from protein oligomers and multisubunit complexes
including nucleoproteins. Using this approach, new structural insights
are provided in the association behavior and conformations of estrogen-related
receptors ERRα and ERRγ. The possibility of 3D modeling
from the scattering by mixtures significantly widens the range of
applicability of SAXS and opens novel avenues in the analysis of oligomeric
mixtures and assembly/dissociation processes
Solution Behavior of the Intrinsically Disordered NâTerminal Domain of Retinoid X Receptor α in the Context of the Full-Length Protein
Retinoid X receptors (RXRs) are transcription
factors with important
functions in embryonic development, metabolic processes, differentiation,
and apoptosis. A particular feature of RXRs is their ability to act
as obligatory heterodimerization partners of class II nuclear receptors.
At the same time, these receptors are also able to form homodimers
that bind to direct repeat separated by one nucleotide hormone response
elements. Since the discovery of RXRs, most of the studies focused
on its ligand binding and DNA binding domains, while its N-terminal
domain (NTD) harboring a ligand-independent activation function remained
poorly characterized. Here, we investigated the solution properties
of the NTD of RXRα alone and in the context of the full-length
receptor using small-angle X-ray scattering and nuclear magnetic resonance
spectroscopy. We report the solution structure of the full-length
homodimeric RXRα on DNA and show that the NTD remains highly
flexible within this complex
Description of the proteins and complexes compared in the text and figures.
<p>Description of the proteins and complexes compared in the text and figures.</p
Scattering curves of Ca<sup>2+</sup>-free, Ca<sup>2+</sup>-bound and MPT-bound S100A4.
<p>Differences in the WT (<i>cyan</i>) and the Ca<sup>2+</sup>-bound WT (<i>red</i>) are mainly observed at 0.15 Ă
<sup>â1</sup>â0.25 Ă
<sup>â1</sup> (A). The scattering profile of the Î13 mutant (<i>purple</i>) changes less upon Ca<sup>2+</sup>-binding (<i>orange</i>) (B). Typical one-dimensional <sup>1</sup>H NMR spectra for the studied systems acquired at 700.17 MHz, zoomed to the aliphatic proton region. From bottom to top: buffer; WT; WT-Ca<sup>2+</sup>; Î13; Î13-Ca<sup>2+</sup> (C). Typical examples of EOM <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097654#pone.0097654-Bernado1" target="_blank">[20]</a> extended (D) and compact S100A4 models (E). Distribution of the radii of gyration of the generated model ensemble (<i>black</i>) for ensemble optimization method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0097654#pone.0097654-Bernado1" target="_blank">[20]</a>, the best ensemble fitting the Ca<sup>2+</sup>-free (<i>cyan</i>) and Ca<sup>2+</sup>-bound (<i>magenta</i>) WT S100A4 SAXS data (F). Scattering curve differences between the MPT-bound WT (<i>red</i>) and the MPT-bound Î13 mutant (<i>orange</i>) are more diffuse (G).</p
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