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BMC Bioinformatics
Modulation of p53 binding to MDM2: computational studies
reveal important roles of Tyr100
Shubhra Ghosh Dastidar1, David P Lane2and Chandra S Verma*1,3,4
Addresses:1Bioinformatics Institute (A-STAR), 30 Biopolis Street; #07-01 Matrix, Singapore 138671,2Institute of Molecular and Cell Biology
(A-STAR), Proteos, 61 Biopolis Drive, Singapore 138673,3Department of Biological Sciences, National University of Singapore, 21 Lower Kent
Ridge Road, Singapore 119077 and4School of Biological Sciences, Nanyang Technological University, 50 Nanyang Avenue, Singapore 639798
E-mail: Shubhra Ghosh Dastidar - email@example.com; David P Lane - email@example.com;
Chandra S Verma* - firstname.lastname@example.com
*Corresponding author
from Asia Pacific Bioinformatics Network (APBioNet) Eighth International Conference on Bioinformatics (InCoB2009)
Singapore 7-11 September 2009
Published: 3 December 2009
BMC Bioinformatics 2009, 10(Suppl 15):S6doi: 10.1186/1471-2105-10-S15-S6
This article is available from: http://www.biomedcentral.com/1471-2105/10/S15/S6
© 2009 Dastidar et al; licensee BioMed Central Ltd.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Background: The tumor suppressor protein p53 is regulated by the ubiquitin ligase MDM2 which
down-regulates p53. In tumours with overexpressed MDM2, the p53-MDM2 interaction can be
interrupted by a peptide or small molecule to stabilize p53 as a therapeutic strategy. Structural and
biochemical/mutagenesis data show that p53 has 3 hydrophobic residues F19, W23 and L26 that
embed into the ligand binding pocket of MDM2 which is highly plastic in nature and can modulate its
size to accommodate a variety of ligands. This binding pocket is primarily dependent on the
orientation of a particular residue, Y100. We have studied the role of the dynamics of Y100 in p53
Results: Molecular dynamics simulations show that the Y100 side chain can be in "open" or
"closed" states with only the former enabling complex formation. When both p53 and MDM2 are
in near native conformations, complex formation is rapid and is driven by the formation of a
hydrogen bond between W23 of p53 and L54 of MDM2 or by the embedding of F19 of p53 into
MDM2. The transition of Y100 from "closed" to "open" can increase the size of the binding site.
Interconversions between these two states can be induced by the N-terminal region of MDM2 or
by the conformations of the p53 peptides.
Conclusion: Molecular dynamics simulations have revealed how the binding of p53 to MDM2 is
modulated by the conformational mobility of Y100 which is the gatekeeper residue in MDM2. The
mobility of this residue can be modulated by the conformations of p53 and the Nterminal lid region
of MDM2.
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BioMed Central
Open Access
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The tumor suppressing activity of the protein p53 is
down-regulated by the ubiquitin ligase MDM2 which
complexes p53 and targets it for degradation [1]. In
normal cells the p53 protein, which also is a transcrip-
tion factor active at the MDM2 promoter, is maintained
at low levels through this negative feedback loop. In
damaged cells the MDM2-p53 complex is destabilized
through posttranslational modifications, e.g. phosphor-
ylation. This disrupts the complex, frees p53 which then
activates the repair or apoptotic pathways [1]. This has
spawned several studies aimed at to the development of
peptides/small molecules that can displace p53 from its
complex with MDM2 [2-6]. The structural data on the
MDM2-ligand complexes available in Protein Data Bank
(PDB) show a wide structural variation amongst the
several MDM2-inhibitor complexes. These reveal a
highly plastic nature of the binding pocket of MDM2
(Figure 1). The significance of this plasticity in inhibitor
design has been addressed in recent studies [7,8]. One
residue that has been found to play a major role in
modulating this plasticity through the size of the binding
pocket is Y100 [7,9]. The orientation of the Y100 side
chain controls the size of the binding pocket and also
contributes to the stabilization of the ligands in the
complex by enabling stable interactions. Both, the crystal
structure (PDB code 1YCR) [10] and molecular dynamics
(MD) simulations of the complex of MDM2 and a 13
residue fragment of the transactivation domain of p53
show that Y100 points away from the binding pocket
and forms a hydrogen bond (HB) with either the
backbones of E28 or N29 of wild type (WT) p53 peptide.
This stabilizes an unstructured C-terminal region of the
peptide that lies outside the binding pocket [7]. In
addition, there are conformational states with small
molecules/peptides bound where Y100 'points in'
towards the binding pocket and stabilizes the complex
with a different set of interactions [7]. Recently, it was
reported from biochemical measurements that the P27S
mutant of p53 has a higher affinity for MDM2 than wild
type p53. Simulations revealed that in the complex, the
peptide adopts an a-helical conformation at the
C-terminus (which is unstructured in the WT p53) and a
rearrangement of the network of interactions occurs.
There is a dramatic change of the MDM2 surface which is
caused by the reorientations of the L54 and Y100 side
chains. The flip of the latter towards the binding pocket
organizes a cozier fit of the ligand and stabilizes an HB
with the L26 backbone, suggesting an induced fit
mechanism of peptide binding [7].
On the other hand, the apo state of MDM2 as evidenced
by the ensemble of NMR structures (PDB code 1Z1M)
[8] shows that Y100 sits in a deeply buried position (see
Figure 2) which is remarkably different from that in its
complexed state. This suggests a possible coupling of the
dynamics of Y100 with the binding of p53. Interest has
recently been focused on the differences between pre-
organized binding/conformational selection (where for
example the p53 peptide chooses a conformation of
MDM2 from an ensemble, that is optimal to bind that
peptide) or induced binding (for example the p53
peptide binds in some conformation to MDM2, and
the complex then undergoes conformational rearrange-
ments that maximizes the interactions) [11]. To inves-
tigate this we have carried out a series of molecular
dynamics simulations of systems containing MDM2 and
p53 in various states. The main focus is on investigating
the changes that take place in p53 and in MDM2 as they
approach each other. A somewhat similar investigation
but with coarse graining simulations has recently been
reported [12]. The study shows the conformational
changes associated with the transition from uncom-
plexed (apo) state to the complexed states of MDM2
which leads to the diverse conformational states of
We have used molecular dynamics simulations to
examine three different conformations of MDM2: (i)
'Open' state of the conformation taken from the wild
Figure 1
MDM2 in surface representation, taken from various
structures available in PDB, complexed with various
ligands including WT p53 (1YCR), a b-hairpin peptide
(2AXI), small molecule 'nutlin' (1RV1), an 8-mer p53
peptide analogue (2GV2), an optimized peptide
(1T4F) and a 12 residue peptide. PDB codes have been
mentioned at the right bottom of each structure. The
location of Y100 on the surface has been mentioned for the
WT structure. The surface was colored according to the
charges of the surface residues: positive (blue), negative (red)
and neutral (white).
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type (WT) complex (1YCR) with the Y100 side chain
pointing away from the binding pocket; (ii) 'closed' state
- the conformation of MDM2 taken from the complex
with a-helical P27S mutant of p53 with the Y100 side
chain pointing towards the binding pocket; (iii) The
'apo' state - the unliganded state of MDM2, obtained
from the ensemble of NMR structures [8] where Y100 is
in a deeply buried position (see Figure 2).
Other approaches to investigate such phenomena are
Brownian Dynamics simulations [13], Replica Exchange
methods [14-17] and the more recently developed
accelerated molecular dynamics methods [18]. While
the Brownian dynamics methods are well suited for
studying protein-protein associations where proteins are
depicted in a reduced representation [19], the replica
exchange methods can be used to examine pathways of
folding [16]; the accelerated molecular dynamics meth-
ods have been very successful in examining long time
scale processes [18]. In this study, we are attempting to
understand atomic level details of the process prior to
the embedding of p53 into MDM2. Aspects of this
process involve a coupling between folding and binding,
at least of the p53 peptide [20]. We have chosen to run
classical molecular dynamics simulations with several
different starting conditions and as will be seen, these
capture local folding/unfolding events through extensive
surface rearrangements. A major factor that limits the
usage of replica exchange methods here is that the
current system is comprised of around 100 amino acids
and replica exchange methods, particularly in explicit
solvent, can only meaningfully (exhaustively) be applied
to peptides that are up to 40 amino acids long [21-24]
and hence are not suitable for the sort of extensive
surface rearrangements that we sample here. Moreover,
the MDM2 binding site is highly hydrophobic and there
is the possibility that it will rapidly unfold in the REMD
methods. For the same reason, the accelerated molecular
dynamics method was not used.
We first outline briefly the currently accepted picture of
the mechanism of interaction between p53 and MDM2
in their bound state. Crystallographic, biophysical and
computational studies have traditionally shown that
F19, W23 and L26 are the three critical residues of the
transactivation domain (TA) of p53 which largely
determine the stability of its complex with MDM2
[10,25]. The residues F19 to L25 form an a-helical
segment which has a hydrophobic face that subtends the
side chains of the three hydrophobic residues F19, W23
and L26 which get embedded in the binding pocket of
MDM2. In addition to the hydrophobic interactions
between these 3 residues and MDM2, the W23 side chain
also makes an HB with the backbone of L54 of MDM2,
and this is very critical for the stability of the complex
[7,26]. More recently, it has been demonstrated that
other parts of both p53 and MDM2 are involved in
modulating these interactions [7,12,27]. One residue
whose dynamics appear to potentiate this binding is
Y100 of MDM2 which "gates" the conformation of the
p53 peptide. This residue is conserved across species (see
Supporing Information in [7]). If the p53 peptide is
extended at its C-terminus (as seen in the crystal
structure 1YCR), the side chain hydroxyl of Y100 is
involved in hydrogen bond (HB) with the backbone of
either E28 or N29 of the p53 peptide; however the p53
peptide can also adopt an a-helical conformation at its
C-terminus and this is potentiated by the Y100 hydroxyl
forming an HB with the backbone of L26 which provides
a 'cozier' fit between the p53 peptide and MDM2 [7]. We
set out to investigate the mechanism that governs the
development of these interactions as p53 approaches
MDM2; we are particularly interested in examining the
modulation of the conformational activity of Y100.
Recently, a study has investigated such a process using
targeted simulations of the binding pathway under a
Figure 2
Superimposed structures of 'open' (salmon red),
'closed' (yellow) and 'apo' (purple) states of MDM2.
The residues L54, H96 and Y100 have been shown in sticks
for all conformations and the residue names have been
labeled for the apo-state. The Y100 is deeply buried in the
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coarse grained description [12]. In contrast, here we
examine the process of binding, without directing the
binding, and in an all-atom model with explicit
solvation. The steady behavior of the root mean squared
deviations (RMSD) of MDM2 in the various situations
shows that the simulations are stable (see Figure S1 in
Additional File 1).
p53 and 'open' MDM2
When p53 approaches the 'open' state of MDM2 from a
distance of 3 Å, (trajectory: Mo3A, see Table 1), complex
formation is rapid and the crystallographically observed
reproduced. The W23-L54 HB forms within 300 ps. The
Y100-E28 hbond takes somewhat longer (~2 ns) to form.
Indeed, the complex is stabilized by 10 ns, which suggests
that preorganization of the partners into 'reactive' con-
formations leads to rapid complex formation (Figure 3 and
Additional File 2). This of course was enabled because we
started with the crystal structure conformations (which are
the preorganized conformations). This is in accord with the
models proposed and observed in other simulations for
protein-protein interactions [12,28]. Moreover, the
enthalpy of binding close to what has been reported
elsewhere using the crystal structure of the complex (ΔH~-
56.6 kcal/mol here compared to -54.7 kcal/mol, [7]); the
difference is expected due to the different starting states.
suggestive of a real productive encounter between p53 and
MDM2 starting 3 Å apart. Interestingly Y104, the residue
spatially contiguous to Y100 undergoes side chain mobi-
lities that are larger in magnitude to those in Y100 and yet
are correlated with it (Figure S2 in Additional file 1).
In contrast, when p53 approaches the "open" state of
MDM2 from a separation of ~6 Å (Trajectory Mo6A), it
manages to reach the surface of MDM2 in the vicinity of
the binding pocket (Figure S1 in Additional File 1)
within 0.5 ns. However the interactions are nonspecific
and p53 never manages to embed into MDM2 com-
pletely. The fact that it reaches the surface originates in
the long range electrostatic fields that will no doubt steer
the two molecules [29]. The W23 side chain forms
interactions with the side chain of F55 which appears to
displace p53 to a position slightly away from its native
(or crysatllographically observed) location. At the same
time, Y100 flips in towards the binding pocket; this
presumably happens to minimize the exposure to
Table 1: List of trajectories
Trajectory nameMDM2p53 Length
simulation started from the snapshot taken at 7.5 ns of Mc6A
Apo, model 1
Apo, model 1
Taken from M1n4A at the end of 20 ns
Apo, model 1
Apo, model 2 (Residue 1-119)
Apo, model 4 (Residue 1-119)
10 ns
10 ns
20 ns
20 ns
40 ns
25 ns
20 ns
10 ns
5 ns
20 ns
15 ns
10 ns
The source of the coordinates of the structures has been reported in the text. The RMSD corresponds to the p53 backbone in the starting structures
of the simulations, after superimposing the MDM2 of the on the crystal structure of the MDM2-p53 complex
Figure 3
(a),(b) Snapshots taken at the end of 10 ns simulation
of Mo3A (See Table 1). MDM2 (green) has been shown in
ribbon in 'a' and surface representations in 'b'; and p53 (red)
has been shown in ribbon. The HB between residue pairs
W23-L54, E28-Y100 and the polar interaction between the
side chains of E28 and K51 have been shown with dashed
(black) line in the picture 'a'. (c),(d) Snapshot taken at he end
of Mc3A trajectory (See Table 1)
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solvent of a very hydrophobic binding pocket of MDM2.
Again the mobility of Y104 is correlated with that of
Y100 (Figure S2 in Additional File 1).
p53 and 'closed' MDM2
We next examine the approach of p53 towards MDM2
when the latter is in its 'closed' state. F19 and W23 find
their crystallographically observed positions (trajectory
Mc3AT1, Table 1) within 0.5 ns, but L26 is obstructed by
Y100 which points into the binding pocket (this being
the 'closed' state of MDM2). The HB between the W23
side chain and the L54 backbone is transiently stable
because the W23 is prevented from approaching
sufficiently close to MDM2 (Figure 4 and Additional
File 3). This is because of competition between the W23
side chain and the Y100 side chain for Hbonding with
the backbone of L54 and also because the surface of
MDM2, as defined by an inward-pointing Y100,
occludes p53. Eventually L26 is "kicked out" by Y100
(See Movie in Additional File 3). In the trajectory
Mc3AT1 Y100 is further forced to remain in its closed
conformation by the extended C-terminus of the p53
peptide. To further probe the effect of the restraint
imposed upon Y100, we modified the initial conforma-
tion of the C-terminus of p53. The backbone conforma-
tions of residues 27-28 were changed in a manner that
released the spatial constraint upon Y100. Now we
observe (trajectory Mc3AT2) that Y100 flips out after just
a few nanoseconds and in turn enables p53 to nicely fit
into the binding pocket of MDM2, in a conformation
resembling that of the WT crystal structure (Figure 4 and
Additional File 4); this clearly highlights the effect of the
C-terminal region of p53 on modulating the gating of
Y100 of MDM2. The mobility of Y104 is correlated
strongly with that of Y100; interestingly, when no
binding takes place, the magnitude of fluctuations of
both side chains are similar, while upon binding events,
the mobility of Y104 is correlated with that of Y100 but
the magnitude is larger (Figure S2).
When p53 was placed 6 Å away from the binding pocket
of MDM2 (trajectory Mc6A), again, as seen in the "open"
state described above, p53 reaches the surface of MDM2
in the vicnity of the binding pocket within 0.5 ns. In
contrast to the "open" state, here Y100 flips out to fully
open the binding pocket. Even as F19 attempts to dock,
it provides an anchor that enables W23 and L26 to move
towards their binding pockets. However, local collisions
induce a rotation in the W23 side chain away from the
orientation optimal for its binding. This results in p53
moving away from the surface of MDM2 and the W23
side chain adopts a stacking interaction with F55 of
MDM2. (Figure 5 and Additional Files 5, 6, 7). We
extracted a structure of the p53-MDM2 complex from
Figure 4
(a),(b) Snapshot at the end of 20 ns simulation of
Mc3AT1. Y100 stays in a 'flipped-in' conformation and L26
of p53 (red) is out of the binding pocket, F19 and W23 fits
into their crystallographically observed position and W23
and L54 forms HB; (c),(d) Snapshots at the end of 20 ns
simulation of Mc3AT2. F19, W23 and L26 are fitted into the
binding pocket of MDM2. Critical H-bond between W23 and
L54 is marked with dashed line. Figures in the same row
represent the same structure but differ in ribbon and surface
representations of MDM2 (green).
Figure 5
(a) Snapshots taken at the end of 20 ns simulation of
Mc6A. W23 of p53 (red) is out of the binding pocket. (b)
Snapshot taken at the end of 22 ns of the simulation of the
trajectory Mc6A7ns, showing the crystallographically
observed binding mode of the side chains F19, W23 and L26
and W23-L54 HB is formed.
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this trajectory which whose conformation had the lowest
deviation from the crystallographic complex (RMSD ~4
Å), and that had Y100 pointing "out"; this was subject to
an MD simulation (Trajectory Mc6A7ns). This structure
forms the crystallographically observed interactions in
less than a nanosecond, including the W23-L54 HB
(Figure 5b and movie Additional File 7). Again Y104
mobility is correlated with that of Y100 (Figure S2 in
Additional File 1). Here we see some evidence of the
presence of an intrinsic harmonicity with open-closed
states of the Y100 appearing at a timescale of ~3-4 ns.
p53 and 'apo' MDM2
Thus far we have used the complexed state of p53-
MDM2 determined crystallographically to seed the
starting conformations of our various investigations.
Given that the structure of MDM2 is available in its
uncomplexed or "apo" state from NMR [8], we decided
to use this as our starting state for MDM2. In all the 24
conformations of MDM2 available, the Tyr100 points
inwards; this is expected as this minimizes the exposure
of the very hydrophobic binding pocket of MDM2 and
also enables the HB between the backbone of Leu54 and
the side chain hydroxyl of Tyr100. When p53 was placed
near the binding pocket of MDM2 (trajectory M1n4A),
surprisingly p53 loses its helicity. This appears to be due
to the fact that the side chains of MDM2 in this NMR-
derived state are not optimally located to interact with
p53 (which the crystallographic state does). However, as
the system explores the conformational states, we soon
observe that MDM2 interacts with W23 and F19 of p53
and its binding pocket is induced to open (Figure 6).
Although this does not lead to the crystallographically
observed state of the complex, W23 and F19 do embed
in the binding pocket which leads to stabilization of the
hydrophobic pocket of MDM2. F19 gets packed by
several hydrophobic residues including I61, V75, F91,
V93 and F86 (Figure 6c). The interesting observation is
that although the orientation of the side chains of F19
and W23 change to a non native conformation, we do
not see dissociation of the complex. It appears that at the
initial stage of complex formation, helical conforma-
tions of p53 can initiate complex formation that can be
seeded by an initial insertion of F19 into the hydro-
phobic pocket. This in turn is complemented by some
expansion of the overall pocket of MDM2 that enables
W23 to get embedded (See Movies in Additional Files 8).
To examine these later stages of binding we take the last
(at a time of 20 ns) snapshot of the above trajectory and
replace p53 (which has been somewhat distorted
conformationally from its native bound conformation)
by p53 taken from the crystal structure. We find that
despite p53 being in its optimal conformation, in these
simulations Y100 (which points in toward the binding
pocket) does not enable p53 to embed and indeed
pushes it away. In addition, the side chains of H96 and
R97 obstruct the C-terminus of p53. Once again, Y100 is
constrained by the presence of the C-terminal end of the
TA of p53 (as we saw earlier in the case of p53 at 3 Å
from the closed state) and cannot flip out to create space
for W23 to embed. This indicates that prior to the
binding of p53, the opening of the hydrophobic pocket
of MDM2 by transition of Y100 to its 'open' conforma-
tion appears to be a key step to facilitate this binding.
The interesting feature is that the mobility of Y104 is
now dependant upon the position of the lid. The movies
in the additional files 9, 10, 11 show that that once the
lid opens, Y100 goes into an open state but Y104 is still
constrained by the lid and does not open.
The TA of p53 is known to be largely intrinsically
disordered with some parts of it adopting a helical
conformation upon binding to MDM2. This brings three
side chains (F19, W23 and L26) of p53 to be displayed
on the same face of the helix thus enabling them to
embed into a hydrophobic binding pocket of MDM2.
Experimental data show that the unliganded state or the
apo-state of MDM2 (derived using NMR, [8]) is quite
different from its complexed state (derived using crystal-
lography, [10]). What is not understood well is the
process of binding of p53 and MDM2 to each other,
mediated by these conformational states: is it pre-
organization prior to binding or is it reorganization
after binding (or induced fit). To test these, we have
carried out a series of MD simulations that mimic the
approach of p53 to MDM2. We avoided the simulation
of folding/unfolding transitions of p53 during the
simulations [20] and assumed that when p53 is very
close to MDM2, stereo-chemical constraints would
demand that the helical conformation of p53 be
dominant in its interactions. We have mostly focused
on the plasticity of MDM2 and the dynamics of Y100.
The structural data shows that the plasticity of the
binding pocket of MDM2 is mainly determined by the
orientation of Y100 (Figures 1, 2), with spatially
contiguous Y104 correlated with Y100 in mobility.
When p53 is in its native (or crystallographically
observed) state and MDM2 is "open", binding occurs
with only small local reorganizations as side chains
reorient minimally to maximize interactions. The size of
the binding site, as determined from the solvent
accessible surface area (data not shown) varies from
~2250 Å2to ~2450 Å2as the MDM2 transits from a
relatively closed apo state to the p53 bound state. The
general fluctuations of MDM2 (Figure S3 in Additional
File 1) are conserved in pattern across the various
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simulations but the magnitudes vary. This is under-
standable because the peptide and MDM2 modulate
each other and this will certainly change depending on
their relative orientations, with the largest mobility
witnessed in the presence of the lid (but absence of
peptide). The one outlying feature is the high mobility of
the 30-45 region when p53 is actually binding to MDM2
in approximately the crystallographically observed
mode. While this region is distal to the p53 binding
side, nevertheless the fact that it is high compared to the
equilibrium dynamics [7] suggests that real equilibrium
has not been achieved (this is ok for the purposes of our
current study which only aims to examine the processes
that occur as p53 approaches MDM2). When p53 is
distant from MDM2, Y100 can assume both "in" and
"out" states, with only the "out" state enabling binding.
The binding process requires an initial encounter
complex that is driven by nonspecific forces where
electrostatic steering plays a major role. This is then
accompanied by either the embedding of Phe19 or W23,
that act as anchors across which the other two residues
can be embedded. Both these residues, W23 and L26
require that Y100 is in an "out" conformation. This
would enable the sidechain of W23 to make an HB with
the backbone of L54 (which otherwise makes an HB with
the hydroxyl of Y100). When Y100 is "in", it also
p53 binding
(f) (g)
Figure 6
(a) Surface representation of the Model 1 of the NMR ensemble (1Z1M), showing the absence of a well defined
pocket. This conformation has been used to run M1n4A after placing the p53. (b) MDM2 surface is showing an well defined
pocket after simulation in presence of p53 (M1n4A), p53 has not been shown for clarity. (c) Hydrophobic Packing of F19 of
p53 (red) in the binding pocket of MDM2 (green) at the end of 20 ns simulation (M1n4A) and (d) the complex same
conformation with MDM2 in surface representation. Y100 is deeply buried, pointing towards the core of MDM2. (e) shows the
snapshot from M1n4WT at the end of 5 ns simulation, which does not form the HB between W23 and L54; (f),(g) are the
snapshot taken at the end of 10 ns simulation of the trajectory of M1n6A, shown in two different representations.
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occludes L26 from embedding. At the same time, when
Y100 is "in", the C-terminal region of the TA of p53 also
plays a critical role in modulating the dynamics of Y100
by preventing its transition to "open" states (at least in
the timescales of these simulations).
The NMR ensemble shows that the "apo" states of
MDM2 have Y100 "in" [8]. This is to minimize the
exposed hydrophobic surface of the binding pocket.
What is interesting is that these "apo" states, derived
from NMR, had their N-terminal 24 residues (the lid)
removed prior to simulations (in presence of p53 in the
system). If the lid is included in the simulations (in
absence of p53), there are two scenarios (Figure 7 and
Additional Files 9, 10, 11). In some of the structures, the
lid is localized over the binding site and pushes against
Y100 and even during simulations, Y100 remains "in".
However, there are some structures where the lid is
localized away from the binding pocket. Simulations of
these structures show that the restraint of the lid on Y100
is rapidly released and Y100 assumes an "out" con-
formation (Figure 7 and Additional Files 9, 10, 11). The
p53 binding
p53 binding
p53 binding
Figure 7
(a),(b) Snapshot taken at the end of 20 ns simulation of 'apo' state only (Trajectory M1n). Y100 has flipped out. (c),
(d) Snapshot taken at the end of 15 ns simulation of the 'apo' state of MDM2 containing the lid covering the MDM2 surface
(residue 1-119, Trajectory M1n119). Y100 remains in the deeply buried situation and no well defined binding pocket has been
observed. (e), (f) Snapshot taken at the end of 15 ns simulation of the 'apo' state of MDM2 containing the lid but not in contact
with the MDM2 surface. Y100 flips out and well defined binding pocket has been observed. Figures in each row correspond to
same structure but in two different representations: ribbon and surface. Surfaces have been shown for residues 25-109 only.
The lid residues (1-24) have been shown in purple color.
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same happens of the lid is deleted and then simulated in
the absence of p53. This once again highlights the
importance of the restraints that the local environment
places upon the gating of Y100. Y100 in turn modulates
the size of the binding pocket and also controls the local
polarity by releasing the L54 backbone as a potential
hydrogen bonding partner (for W23). Thus this suggests
that a re-organization or induction to "open" and
"closed" from "apo" states may be induced by the lid
(or other factors). These in turn can be modulated by the
presence of p53 and can either directly bind p53 (open
Y100) or when p53 can induce an open state from a
closed state by the conformational dynamics of the
C-terminal end of the TA domain. This starts assuming
significance biologically for several reasons: (a) the
control of the size/polarity of the binding pocket by
Y100 can affect the binding of p53 (and related proteins
such as p73) and small molecules. We have shown how
the orientation of Y100 modulates the thermodynamics
of the peptidic inhibitors [7], a feature also demon-
strated in other computational studies [30,31]; (b) the
increasing importance of the MDM2 lid region, which is
strictly conserved in mammals, in modulating the
binding of p53 [32,33]; it is possible that by gating
the dynamics of Y100, the lid region may influence the
kinetics of binding of p53. The lid is known to compete
only very weakly with peptidic inhibitors of MDM2 [34].
However to our knowledge, there is no published data
available on the kinetics of peptide binding as modu-
lated by the lid region in MDM2. The work of Worall
et al. [33] has shown that the lid dynamics mostly
modulate the ligase activity of the MDM2 which involves
interactions of the lid with a region away from the p53
TA binding N-terminal region. It is clear that there must
be a correlation between lid dynamics and substrate
access as has been also found in several other systems as
lactate dehydrogenase, lipases, adenylate kinases [35] (c)
these questions assume greater significance with recent
observations pointing to the increasingly complex
interactions between MDM2 and p53 [30,31,36,37].
While our findings of the role of Y100 in modulating the
equilibrium dynamics of the complex are in agreement
about by ourstudies, i.e.,the modulation of the dynamics
of the peptide and protein even before the binding event
has taken place, is particularly relevant to understanding
insights into the development of peptidic therapeutics
whose rates of binding may be tunable.
Molecular dynamics simulations have revealed how the
binding of p53 to MDM2 is modulated by the
conformational mobility of Y100 which is the gatekeeper
residue in MDM2. They also reveal how the mobility
of this residue itself can be modulated by the conforma-
tions of p53 and by the conformations of the Nterminal
lid region of MDM2.
The 'open' state of MDM2 (residues 25-109) was
obtained from the crystal structure of the MDM2-p53
complex (RCSB entry 1YCR, resolved at 2.6 Å) [10]. The
'closed' state of MDM2 (residues 25-109) was obtained
from our previous work [7]. The structure of the 'apo'
state was chosen as Model 1 of the NMR ensemble [8]
and although this structure consists of residues 1-119, we
chose only residues 25-109 to be consistent with
the other structures of MDM2 that have been used in
the current and previous work. In order to examine the
dynamics of the approach of p53 and MDM2 towards
each other, we carry out all-atom simulations with
MDM2 and p53 separated by varying distances (Table 1).
We have also carried out simulations of MDM2 alone
with its N-terminal 1-24 residues included as well. The
CHARMM22 force field [38] was used to represent the
systems. Each system was solvated using TIP3P water
After brief energy minimizations, each system was heated
to 300 K followed by equilibration under constant
pressure and temperature. Then molecular dynamics
(MD) simulations at constant temperature and volume
were carried out on each system for periods varying
between 5-40 ns, yielding a total sampling time of ~200
ns. SHAKE [39] was applied to freeze the vibration of
bonds involving hydrogen atoms, thus enabling a 2fs
integration time step to be used. The Berendsen thermo-
stat [40] was applied to keep the temperature constant.
Simulations were carried out using the CHARMM
package. Enthalpy was calculated using the MMGBSA
approximations, with the GBSW [41,42] implicit solvent
model. Figures were prepared using PYMOL [43] and
movies were generated using VMD [44].
Competing interests
Theauthorsdeclarethat theyhavenocompetinginterests.
Authors' contributions
SGD and CSV conceived the idea, SGD carried out
the calculations and analysis. SGD and CSV wrote the
manuscript. DPL discussed, advised and edited the
Other papers from the meeting have been published as
part of BMC Genomics Volume 10 Supplement 3, 2009:
Eighth International Conference on Bioinformatics
(InCoB2009): Computational Biology, available online
BMC Bioinformatics 2009, 10(Suppl 15):S6http://www.biomedcentral.com/1471-2105/10/S15/S6
Page 9 of 11
(page number not for citation purposes)
Page 10
at http://www.biomedcentral.com/1471-2164/10?
Additional material
Additional file 1
PDF format contains RMSD plots, average fluctuations, information of
sampling of key side chain dihedrals etc.
Click here for file
Additional file 2
Movie file of Trajectory Mo3A, red ribbon represents p53 and green
ribbon represents MDM2. Few important side chains have been shown
in 'sticks'. H-bonds appear as dashed lines.
Click here for file
Additional file 3
Movie file of Trajectory Mc3AT1, coloring scheme is same as M1, The
surface representation of this movie is available at http://web.bii.a-star.
Click here for file
Additional file 4
Movie file, Trajectory Mc3AT2, shows the conformational transition of
Y100 and p53 binding. The same movie but having MDM2 in 'surface'
representation is available at http://web.bii.a-star.edu.sg/~shubhragd/
Click here for file
Additional file 5
Movie file, Trajectory Mc6A.
Click here for file
Additional file 6
Movie file, Trajectory Mc6A, side view
Click here for file
Additional file 7
Movie file, Trajectory M1n4A7ns
Click here for file
Additional file 8
Movie file, Trajectory M1n4A
Click here for file
Additional file 9
Movie file, Trajectory M1n, showing dynamics of Model 1 of NMR
ensemble after deletion of residues outside the range of 25-109. The
conformational change of Y100 side chains is observed.
Click here for file
Additional file 10
Movie file, Trajectory M2n119. Shows the dynamics of MDM2 when
the lid is close to the binding pocket. The Y100 has been shown in purple
color for clarity. The Movie shows that Y100 stays in a deeply buried
state when the lid is present.
Click here for file
Additional file 11
Movie file, Trajectory M4n119. Shows the dynamics of MDM2 when
the lid is away from the binding pocket. Y100 flips and points away from
the binding pocket.
Click here for file
This work was supported by the Biomedical Research Council (Agency for
Science, Technology and Research), Singapore. We thank Ted Hupp of
Edinburgh University for discussions.
This article has been published as part of BMC Bioinformatics Volume 10
Supplement 15, 2009: Eighth International Conference on Bioinformatics
(InCoB2009): Bioinformatics. The full contents of the supplement are
available online at http://www.biomedcentral.com/1471-2105/10?
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text:
What's Happening, Manhattan?
Summer may not officially be here, but the city of Manhattan and its surrounding areas are ready to go, with numerous events planned for this weekend.
- Wine in the Wild on June 7th at 7pm
Hosted by the Friends of the Sunset Zoo, Wine in the Wild is an annual fundraiser for the Sunset Zoo. Visitors will have the chance to explore different exhibits at the zoo, all while enjoying live music and a variety of wines. General admission tickets can be purchased in advance for $45, or for $55 the day of. Saturday also marks the grand opening of the outdoor gibbons exhibit, which is home to two white-handed gibbons and has been a work in progress since April 2012.
Paws on Poyntz on June 7th from 8am to 1pm
The 13th annual Weenie Dog Races will be taking place downtown this weekend, starting with registration on the patio of AJ's Pizza. Events will include the Drag Dogs and Divas Fashion Show, followed by the Mutt Strutt and the actual races to end the morning-long celebration. Proceeds from the event will go to benefit the Purple Power Animal Welfare Society.
- Saturday Storytelling at The Wonder Workshop
While The Wonder Workshop Children's Museum has great programs and activities going on year-round, this Saturday marks the beginning of the Storytelling in the Garden series. Join storytellers from 10:30 to 11:15 AM every Saturday from June 7th to June 28th to hear stories read in both English and Spanish.
- Family Storytime at Manhattan Public Library
Join us Saturday morning from 11 to 11:30 for a storytime about the sense of sight. Miss Jessica will read a silly book about blind mice and the children will get to pretend to be bears going over a mountain. There is no pre-registration required for this storytime and it will be held in the lower atrium.
- Science Saturday at People's Grocery
A collaboration between our young adult librarian and adult services librarians, the Science Saturday series involves exploring the science of everyday life. This Saturday's session will be held at People's Grocery, with their staff teaching how to make Mexican food. Cooking will start at 10am and pre-registration is recommended.
Latest posts by Amber (see all)
- Nonfiction for Young Readers – March 22, 2015
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text:
Reviewed By Jamie B (Monroe, OH) Reviewed for a 2004 Acura TL —3/18/2015 5:19 AM
Installed these 2 days ago and i couldn't be happier. Great peddle feel and they look fantastic!!
Reviewed By dwight H (WESTMINSTER, CO) Reviewed for a 2012 Acura TL —10/16/2014 6:54 AM
The Stop Tech rotors are a perfect replacement for the OEM Acura rotors. The ceramic pads in the kit fit the calipers correctly. After the recommended break in the brakes stop much better than the original factory brakes. This kit was a nice upgrade to the factory brakes.


text:
Reduce stress to increase productivity
Here are some tips for improving your time management skills from the October issue of Mayo Clinic Women's HealthSource:
Plan each day. Abiding by a schedule minimizes conflicts and last-minute tasks. Create a to-do list each day with the most important tasks at the top. Even if you don't complete the list, you'll know your time was spent constructively.
Delegate. Your team members are qualified to help you with certain tasks. Don't be afraid to ask for help.
Take time to do a quality job. Doing a task the right way might take longer, but you'll save time in the long run by avoiding mistakes.
Practice the 10-minute rule. Take 10 minutes each day to work on those tasks you just dread. Once you start a task, you might find yourself pushing through until you're finished.
Evaluate how you're spending your time. Keep a diary for three days to track the things you're accomplishing. Look for areas where time could be spent more wisely.
Get plenty of exercise and sleep. You'll feel and look better, and improved focus and concentration will lead to increased efficiency.
Take a time management course. Employers, community colleges, and community education programs often offer these classes.
Take a break when needed. Too much stress can spoil any attempts at getting organized. So take a walk, do some stretches, or read a magazine.
If stress becomes too overwhelming, don't hesitate to ask for help. Consider discussing your situation with a doctor or mental health professional.
Hot topics on dvm360
Pol on defense as Michigan veterinary board discusses negligence charges
Controversial reality TV veterinarian calls his approach 'common sense.'
Photo gallery: The top 10 veterinary schools in America, according to U.S. News
U.S. News & World Report ranks programs for the first time since 2011.
Front Desk Disasters, Episode 3: Dude looks like a lady
Everyone's favorite receptionist is at it again. Would you handle this situation differently?
Video: Flea hideouts in the house
Parasitology expert Michael Dryden, DVM, MS, PhD, reveals prime hideouts for fleas—and gives tips to clear them out of clients' homes for good.
Veterinarians: Your clients are going to Google with these cat questions
You might be surprised by what your clients are researching. Plus, get an educational client handout.


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Reverend Dr. Hyung Jin Moon explains the fundamental beliefs of the Unification Church (Tongligyo) and introduces Reverend Dr. Sun Myung Moon, known as True Father.
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And Now Marcus Wilson Has Quit!
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I'm 310,748 - you've got to go !
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text:
Imran Khan's daughter Imara is his stress buster after a hectic shoot schedule.
Imran Khan is having a "positive experience" filming "Katti Batti", thanks to his "wonderful co-star" Kangana Ranaut.
Reportedly, Ekta Kapoor and Imran Khan will be coming together for a film tentatively titled 'Bhoole Se Naam Na Lo Pyaar Ka'.
The parents of deceased students who lost their children in the school tragedy chanted 'Go Imran Go'.
Imran Khan was previously married to British socialite Jemima Khan and has two sons.
Imran was previously married to Jemima 'Goldsmith' Khan and has two sons with her, Sulaiman Isa and Kasim.
'Amplifier' singer Imran Khan says the new-age rappers might have become a rage with the listeners but they have not given quality music to Bollywood.
It is a distant dream and, more than that, they know in their hearts they will never have it.
The two sides have agreed to resume formal talks from Sunday after they were suspended in August end.
The PTI call to shutdown Faisalabad earlier this week ended in violence.
Violent clashes in different areas of Faisalabad on Monday had left an activist dead.
Major trade organizations refused to become a part of the 'shut down' plan.
Imran demanded Nawaz Sharif government to initiate an impartial probe into the alleged rigging in 2013 general elections.
Can the Pakistan Supreme Court accept that those elected by the people cannot be dismissed from parliament for 'impiety'?
An anti-terrorism court issued warrants against Imran Khan and fiery cleric Tahirul Qadri.


text:
Olympia (15-0) managed only four hits but pulled out its second consecutive 3-2 baseball victory in the Florida League High School Invitational Showcase on Wednesday.
Brandon Hadley scored two runs and added an RBI double for the Titans against Miami Florida Christian at Sanford Memorial Stadium. Zack Mishriki added two hits.
Olympia will conclude pool play with an 11:30 a.m. game against Deltona Trinity Christian today.
The tournament final is set for 7 Friday night.Read more
Bishop Moore defeated host Lyman 3-1 (25-22, 19-25, 25-23, 28-26) in boys volleyball behind 42 assists from Nicholas O'Grady.
Domanik Stratford added 24 kills and 11 digs for the Hornets (6-0).
Zach Rynning had 23 kills for the Greyhounds (8-2).
The Hornets used aggressive serving which disrupted Lyman's passing game.
In the final set, most points were scored by side-outs. Server sophomore Garth Verdaflor went down with cramps midway through the set.
When both teams were tied at 20 apiece, the Hornets went on a game-winning run.
Read more
Apopka's boys volleyball team won the Jungle Queen Invitational in Fort Lauderdale over the weekend.
The Blue Darters (9-1) went undefeated over five tournament matches and beat host Cardinal Gibbons 3-1 (25-22, 23-25, 25-20, 25-23) in the Gold Division championship. They also took down Miami Sunset, West Boca Raton, Pine Crest and Wellington along the way.
Schylar Lillethorup, son of Apopka coach Jamie Lillethorup, led the team with 66 kills and 80 assists during the tournament. Michael Jamieson added 58 kills, nine aces and libero Brian Kennedy had 62
Apopka will play on the road against Dr. Phillips (10-0) at 7 p.m. Tuesday for first place in the West Metro Conference.Read more
Olympia continues its undefeated stretch as the Titans defeated host Winter Park 4-1 on Friday.
Michael Byrne pitched six innings and tallied 12 strikeouts for the Titans. J.P. Colton was 2-for-3 for Winter Park (7-7). Olympia is now 13-0.
"It's just been solid defense and pitching," Olympia coach Chuck Schall said. "They [Winter Park] actually hit the ball pretty good. We just spread their hits out."
Olympia hitters had three of their runs scored with two outs. Cameron Montgomery had a base hit that brought in two runners, and Zach Mishriki had a base hit of his own that brought in another runner.
"Moving forward, we need to continue to be a stronger defense," Schall said. "The key to everything is defense."
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Kyla Allison of Wekiva and Astaja Tyghter of Colonial have been selected as the girls basketball players of the year in their team's respective divisions of the Metro Conference.
Allison, a 5-foot-10 senior, was chosen as the top player in the West Division after helping the Mustangs reach the Class 7A state championship game. She averaged 18.6 points, 6.2 assists and 5.4 rebounds per game.
Tyghter, a 5-9 senior and the East Division's top player, averaged a double-double (22.3 ppg, 11.2 rpg) for Colonial, which lost in the 8A state final.
Wekiva (Allison, Ciara Cumbie) and Jones (Angela Jernigan, Korneila Wright) were the only schools with two first-team players in the West Division. Besides Tyghter, Chelsea Johnson made Colonial the only East school with two first-teamers.Read more
After a long search for a new head football coach, Ocoee has its man.
Ben Bullock, who led Mount Dora to an 8-3 district runner-up season in 2014, is coming to Ocoee and will work closer to his Orange County home.
Bullock, who was a Division III college assistant for Brockport and then Hartwick in his native New York for a total of nine years prior to joining Mount Dora's staff as defensive coordinator in 2012, said he will be on campus at Ocoee on March 30 following spring break.
"It's exciting," he said. "There's a great community that we're going to get more involved with and there's a lot of potential. The Ocoee Bulldogs Pop Warner system is very good."
Bullock replaces Dale Salapa, who stepped down several days after the Knights closed out a 2-8 season last fall.
Ocoee opened in 2005 and has had two winning seasons – 2006 (8-3) and 2009 (7-4).
The Knights appeared at points to be close to naming veteran head coaches Chip Gierke and Mike Cullison to fill the vacancy....
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Lutron MS-OP600M-WH Motion Sensor, Maestro Dimmer with Occupancy Sensor, 600W, Multi-Location - White
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Do Cabinets need to 'Line Up'?
Hi everyone! I have been lurking and reading for quite a while, but am new to posting on here.
My husband and I are building our first home, which also will be our forever home. So knowing that we have one chance to get it right has resulted in us over-thinking almost everything! :-)
I am currently working on the kitchen cabinet layout and would love your input. My primary concern is functionality. I have been working on the cabinet layout. What I am coming up with as being the most functional for where I want to put stuff doesn't result in the wall cabinets and base cabinets (mostly drawers) lining up very well. For example, in a 48" space I would have 2 24" wall cabinets above an 18" 3 drawer base and a 30" 3 drawer base. There would be examples like this in several areas of the kitchen. Does that make any sense? Do you think it would look funny? or Am I overthinking the whole issue?
I wouldn't even attempt to make them line up, and didn't. Honestly, I didn't even think about that when planning my layout.
I just went and checked my 'inspiration' pics, and I'd say 9 times out of 10, they don't line up, so yup, I think you're over thinking :) It's okay, we all do it with some aspect during this crazy reno business!
I may try to create a focal point in one area by balancing the bases to the wall cabinets. ItÂs more important for the kitchen to be functional. Make sure the reveals around windows are equidistant. Cabinets above the range & sink must be centered. A good designer should always be able to create a custom looking kitchen with stock cabinets.
Don't try to match the lower widths with the upper widths.
It is best to create a nice balanced arrangement on the upper, independent of the lower. Think about a typical lower run, which might include a 33" sink base, an 18" trash pullout and a 24" DW. The upper would look a mess if you matched these varying widths.
I would say it's considered standard practice to try to have the vertical lines of the cabinets line up. So it's not so much that if you have a 24 and then a 12 below that you have to mirror that up top, but that it would be considered ideal to have a 36" on the wall. That helps to provide a cleaner line. I have very few upper cabinets but they do line up and I feel that ads to the flow. With my cabinet doors especially because they are 42" tall and have a strong vertical frame.
That said, I want to say that I consider that a TEENY part of the planning process. Part of where you weigh all the choices. There are numerous places I've been/worked on that don't have the line up that I feel totally great about. I also feel like with drawers rather than doors below it's much less of an issue.
I strongly disagree with morgne. I think it's a very bad idea to try to get your upper cabinets to line up with your lower cabinets -- makes no sense functionally -- and it's definitely not standard practice with any of the kitchen designers that I know.
The upper cabinets and lowers are not on the same plane, since the upper cabinets are 12-13 and the lower are 24 deep. So, the run of upper cabinets, if the kitchen has any corners, is 12" longer than the lower run.
Obviously, related areas such as range to hood or the area around the sink will align, but for the rest of it, I think a nice rhythm of upper doors makes the most sense visually.
I have done two (contemporary) kitchens where the range hood is completely hidden, and the upper bank of cabinets is just four equally sized doors, which do not align perfectly with the range below, and no one has ever really noticed that, they just comment on the clean design.
None of my cabinets line up, and I wouldn't have it any other way. Indeed, I would be wary of any kitchen "designer" that pushed for the lining up of upper and lower cabinets because it so greatly limits the utility of a kitchen. To me, that's a rookie error: something that looks good on your *paper* floorplan but limits functionality. In your finished kitchen, no one will notice whether they line up or not, so why give up so much to do it?
(BTW, I feel the same way about goofy shaped islands.)
Thanks everyone for the input! Most of the kitchens I see around here do line up, so I think there is at least some following for Morgne's logic. However, nobody I know has ever used a KD. It is good to know that I don't have to limit myself by those restrictions because I really want a truly functional kitchen.


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Discussion in 'Fly Swaps' started by Thomas Mitchell, May 7, 2009.
Thanks guys, they already went out but that's cool of you anyway.
I should have thought of it before but next time I host a swap, if there are any extras, I think I'll distribute them so that everyone gets an extra that someone else tied. I should have thought of it this time but my brain was too fried from a tough week at work.
If anyone got the wrong fly box or I managed some other CF, just let me know.
The gallery is now complete and everyone should have gotten a PM from me with a USPS delivery confirm number in case the flies don't show.
Also - I'm interested in your feedback about this sort of 'on time' swap format.
I liked it a lot, especially the suggested mail-in date. Made sure everyone was on their toes.
i got my flies and they look great! great on time delivery and great flies!!!
ill be sure to join more of your swaps
Thomas, I got my flies today and they look better than they do in the pictures...which is saying a lot. Super tasty flies guys!! Great job!
I also really like the 'on time' swap format, like it really should be. I hosted a swap this winter and had some really late stragglers, and it ended up being much later than I would have liked. So next time for me is going to be the "OCD Thomas" style :thumb:
I can't wait to get out and fish these.
Got my package today. Everything looked great.
D3's purple muddler that I got had perfect proportions, in my humble opinion. (I already took the tags off, but I think it was yours)
You guys just about doubled the amount of spey flies in my inventory.
Thomas, thanks again! Really liked the straight-forwardness right out of the gate! Great flies everyone, can't wait to get out and fish them. :thumb:
Got my set today. Great job by all and great job by the swapmaster.
This was my first swap and I had a lot of fun. Lots of great flies, can't wait to get back to the nortwest and try them out.
Awesome ties and swap. Thanks gents!! I agree they are definitely even better in person!
Also got mine today, very nice looking flies. I really like David's purple skater, simply an awsome tie.
Got mine and they all look great! thanx everyone! can't wait to try um all out!
Thanks all. It was a good swap.
I've got an idea for another one that'll come in late summer as we get ready to fill our boxes for Fall/Winter.
Received mine yesterday.
Beautiful flies guys! All of them are very impressive and I myself, can't wait to try them out!:thumb:
Thank you Tom for running the show...
very nice ties folks. You have inspired me to start tying for my trip to the Klamath this fall. I might have to join the next swap.
Great flies guys.


text: Well like I said, its a just war on our part because we didn't want this to happen, but it did, we were attacked first, and the terrorist were holding up in Afghanistan, Iraq, Iran, Egypt, and other areas of the middle east, we really had no choice in the matter. Yes, the majority of the people in the area wanted nothing to do with this, but the problem was is that's where the bad guys were. I feel bad for the innocent people's situation, I really do, but the terrorist were and still are using the general population as a shield to hide in. Sadam also needed to be taken down, the over all population are doing so much better now that he is finely gone, slowly but surly they will be able to live their lives with better opportunities, as well as women in the area are gaining more and more freedoms. Yes there are areas were women are having it tougher then others but that's because these areas are still stuck and reluctant to change their old man and women role way of thinking. Their economy is doing better, their infestructur is doing better, they have way more freedoms then before. There are still problems there with their mundane bickering over the two mager Muslim religious practices and thinking on who is better, but many there are looking past that and wanting what's best for their country and how to better every one. The war on terror is also a peace keeping war as well as a reconstruction of Iraq. Its true that this will take a very long time and be costly, but we can't leave until the job is done because then we wouldn't be taking responsibility that we took upon our selves when we got involved in this. Also the end result will better every one when we accomplish this. But if we do pull out, that means all that we have done, all the lives lost all the money the hardship and everything else would have been for nothing. I don't know about you guys but the very thought of all the American soldiers and our allies, and of cores the innocent casualties who risked it all to help better their country by helping us or who were just trying to live their lives, all these people having their deaths be for nothing makes me sick. If we pull out, all of these people then gave their lives for nothing.


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Masterpiece in the Making
Today's Truth
And I am convinced and sure of this very thing, that He Who began a good work in you will continue until the day of Jesus Christ [right up to the time of His return], developing [that good work] and perfecting and bringing it to full completion in you (Philippians 1:6, AMP).
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It took four years of fresco painting for the Italian Renaissance sculptor, painter, architect, and poet Michelangelo di Lodovico Buonarroti Simoni, to finish the ceiling of the Sistine Chapel. Commonly known as Michelangelo, his time painting was mostly spent alone, on his back, …


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Ripon officers hit the playground for recess
Police in Ripon are going to recess.
According to Sgt. Steve Merchant, the department started a new school visitation program in January that has been a success.
Rather than waiting for a call of something happening, officers daily visit schools in an effort to promote positive interactions with school staff, students and parents.
When they can, officers visit during recess, so they can interact with children on the playground. On Wednesday, officer Ken Husman visited Weston Elementary School, giving out badge stickers to children.
He said the school check program is making officers more approachable.
"Meeting the kids on their playground and making them feel safe is great," Husman said in a news release.
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This weekend the international program took the study abroad students to Cambridge. It was only about 2 hours away from London and was well worth the trip. We toured the campus and town all day and had a great time. It was amazing to see one of the oldest universities in Europe and one of the most prestigious. We shopped, ate, and admired all day long. There was a river surrounding the whole town where the college image students rowed tourists around in gandola type boats that looked really cool but we all thought it was a bit too cold for water activities. The chapels and libraries were truly breathtaking and it was such an amazing experience.
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Ninth week in London
Yonsei Sarangeo!
I think I am still in the honeymoon phase of my study abroad trip. I LOVE IT!!! Everything is great, my roommate is awesome, my friends are totally my style of friends, the food is great and cheap, the people are nice and understanding of my lack of communication skills, and classes start tomorrow. I am a little nervous, but also excited, because I have been out of school for almost two months or more, so I miss it. Orientation was amazing and mentors club was fun. Mentors club is a club I got invited to before I arrived in Korea. It consisted of having a current student from Yonsei be a mentor for a whole group of international students. I think there were about 25 students in my cell and my mentor. Let me just say, I am so excited to go to a sport event here at Yonsei. The school has a cheer team, that is amazing! The whole student body is involved with the cheering. There were about maybe ten or more dances we did that involved cheering. It made me think that we weren't as cheery as they are over here. The cheering is called AKARAKA, it is huge and amazing and I will post a link on the bottom so you can see what I am talking about. I also want to point out that although I don't know enough Korean maintain a conversation with someone, yesterday I was able to get medicine, get myself from one place to another, and also buy a computer. It felt like a great accomplishment and I wouldn't have been able to do it without the help of my friend. We both made a great accomplishment yesterday with our minor knowledge of the Korean language. Please feel free to ask questions if you guys have any and talk to you guys next week!!!
Eighth week in London
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On my eighth week here, the international program took us on a trip to York and the North of England for a weekend trip. The bus ride was very very long and it seemed like it would never end but … Continue reading
Seventh week in London
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In my seventh week here, I booked a ticket with a tour company to go to Stonehenge and the Roman baths! It was amazing! It was only about a two hour drive from London to Stonehenge which was really nice. … Continue reading
My sixth week in London
This was reading week for me so all I did was catch up on reading and essay writing. The classes here are so different but I really like the way they do it here. You only go to each class once a week but for two or three hours, sometimes four. This is so convenient because if I took a week off to travel, I'd only miss each class once. Also, I don't feel like I am spending all of my time in class. This is nice because I have more time to do the things I like to do here in London. This was especially nice during reading week because I was able to just travel around London and check some sights off my list. image
Late Pre-departure
Hello my name is Daisy Romero I am in Korea right now and the time is 2:39 on a Sunday. I was so caught up on getting all stuff together as well as hanging out with friends in family before I left, that I completely spaced about the blog. However, I do have a journal that I write in so I can tell you exactly what I was feeling before I left to Korea. Here it goes.
It is Wednesday, a day before I leave. I am still in like lala land, I feel like nothing is really going on. I just feel like I am on a mini vacation from work and tomorrow I have to go back. Yesenia (my friend since middle school) keeps telling me, "It's like we are in High school again. Hanging out with no worries about work and just doing what we want." I was like this is going to end soon and I am going to be on my way to Korea, but for some reason I don't feel any different. There is no anxiety attacks, or worries right now. Maybe once I get there I will feel the culture shock and all that, but right now I am just like "meh". Last week I was worrying about my departure, what am I going to do with no work?, what about the language barrier? will my roommate like me? what if I don't like her? this is going to be hard. However, as the days went by my worries went away. Daisy you are going to be fine, you will make friends to help you with the language barrier, you can turn on that customer service (i work retail so I can be really customer friendly when it is needed) when you meet your roommate, and you will go into clubs for that extra time you have on your hands or exercise. Let's do this Daisy! you have been waiting three months for this you are ready.
Yes, even in writing I talk myself, just for motivational purposes. So that was what I was feeling before I left I will post something new on Thursday my time to let you guys know about my experiences this week. Until then Annyong!
Fifth week in London
My fifth week here was spent starting my essays. Here, instead of having assignments throughout the term, you just go to lectures and take notes for ten weeks and then the last week you write an essay that makes up 100% of your grade. So there's a lot of pressure toward the end of the term. This is why I've decided to start now! I also visited the science museum and Hyde park. It was actually sunny this weekend which made London so beautiful! It finally felt like home! image
Fourth week in London
image This week I went with a group of international students on a tour of the Hampton Court Palace. We had a great time touring the gardens and apartments of the Tudors. It was a lot of fun but I hope to go back in the spring when the gardens are in bloom. Also it would be much more enjoyable when it's not freezing outside. It was great to go outside of London and see so much history. Next will be a tour of the North of England and York, which should be really cool.
I'm pretty much completely adjusted to the culture and differences here and just settling in.
Second week in London
image I'm now in my second week at Roehampton and other than scheduling issues and equivalency questions, I am doing much better. I have settled in quite a bit and am learning to enjoy life here on campus. I was able to visit central London and experience being a tourist in this beautiful city. I have to walk 25 minutes to class which is hugely different from at western Oregon where I'd walk 5 minutes to class. The classes here are much bigger and much more intimidating but I thinknill be alright. Now that I've settled, staying here seems much more doable. I'm looking forward to all of the experiences I will have here in the next few months.
First week in London
I've been in London for two days now but it feels like I've been here for weeks already. The first day was great! I toured around campus and the roehampton area. I was very excited and happy to be here but I didn't get much sleep. Then today, I had a really hard time dealing with what I'm assuming was culture shock. It's probably also because I'm very tired, but I was so overwhelmed when trying to pick out groceries it was unreal. Everything is just so dif
ferent and I don't recognize brands or what is a good deal or not. So I gave up and went to a cafe to grab a bite to eat. I looked on the menu for anything that sounded familiar. I realized that this was me shutting out the other culture. I used to think that I wouldn't have culture shock in London because it's not that different from America, but I was so wrong. It's completely different in so many ways. And when I realized that, I freaked out a bit. So I called my mom, took a nap, and feel much better. I also feel that I'm paranoid about American stereotypes and being treated poorly but so far it hasn't been a problem. I think by next week I'll be settled in much better and be coping with the culture shock well.


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Climb aboard the next-generation of personal transport: we ride Toyota's I-REAL
October 26, 2007
October 26, 2007 Gizmag leapt at the opportunity to take Toyota's I-REAL personal mobility concept for a spin at the Tokyo Motor Show yesterday. The I-REAL uses three wheels (two at the front and one at the back), in low-speed mode, shortening its wheelbase to allow it to manoeuvre naturally among pedestrians at similar eyesight height without taking up a large amount of space; in high-speed mode the wheelbase lengthens to provide a lower center of gravity and better driving performance.
"The seating position was remarkably comfortable and the joystick controls fell to hand immediately. Handling was surprisingly intuitive – when compared to other personal mobility devices such as the Segway, which is itself by no means difficult to master, the I-REAL was a breeze to pilot" , said Gizmag's Mike Hanlon of his first impressions of the I-REAL. "Simply, it's quiet, it's very easy to use - it can be used at strolling pace and you can travel alongside somebody who's walking very slowly and hold a conversation with them very easily, but when given full throttle it can sprint away much faster than a human being can hope to hit full pace running".
Still no definite details on just when this machine will go into production, but judging by the incredible reaction the vehicle attracts and hints dropped in the company's PR – "I-REAL, I will be real" - we expect to see Toyota moving swiftly towards the commercialization phase.Share
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Last month, a bipartisan group of legislators introduced the so-called "Innovation Act of 2015" (H.R. 9) in the U.S. House of Representatives. That bill proposes numerous reforms to the U.S. patent system, including...more
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Been doing alot or reading and research on lights as I want to do alot of night riding this fall. I am leaning towards the Serfas lights , anyone use them and have any input good or bad?
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New to Typophile? Accounts are free, and easy to set up.
Chennai Slab is an extension of the original Chennai. Chennai Slab is a simplified slab serif with over sixty OpenType alternates. These alternates include alternates for the ball terminals, unique simplified alternates and more traditional capital forms. Use Chennai Slab when you need a fun and versatile slab serif. The sans-serif Chennai makes a great complement. Six weights are available.
Awesome typeface, great work! Looks like a mix between Archer and Grover Slab...
Good looking work :)


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NYC Parks Events Calendar
Get up, get out, and do something! Just about any day of the year, you can find something fun, informative, and healthy to enjoy at an area park. Feel free to browse the listings below, or use the search tools to find an event that matches your interests and location.
Viewing 11,801–11,810 out of 15,796 events
from October 14, 2012 (x) to April 25, 2026
Saturday, February 7, 2015
Citizenship Classes for Adults
at Poe Park Visitor Center (in Poe Park), Bronx
12:30 p.m.–1:30 p.m.
In this class, students will review sample citizenship questions as well as practice English comprehension, pronunciation, and listening skills.
Category: Education
Shelter Building Workshop
at Blue Heron Nature Center (in Blue Heron Park), Staten Island
1:00 p.m.
Develop skills that will help you create shelter from natural and man-made objects.
Category: Education, Nature, Kids
Victorian Valentine Workshop
at Bartow-Pell Mansion Museum (in Pelham Bay Park), Bronx
1:00 p.m.–3:00 p.m.
Show that you care—not with a boring, store-bought card, but with a handmade, full-of-charm, sure-to-woo original creation.
Category: Arts & Crafts, History, Kids, Valentine's Day
Written in the Rocks Lecture
at Wave Hill, Bronx
1:00 p.m.–2:00 p.m.
The bedrock of Wave Hill is composed of the "roots" of once majestic mountains that rivaled the Rockies in scale and grandeur.
Category: Education, Nature, Talks
Le Petit Zen Art - Adults
at Poe Park Visitor Center (in Poe Park), Bronx
1:30 p.m.–3:00 p.m.
Participants will create small works of art utilizing multiple mediums.
Category: Arts & Crafts, Art
Central Park Tour: Northern Welcome Tour
at East 106th Street Entrance (in Central Park), Manhattan
2:00 p.m.–2:45 p.m.
See the park's northernmost highlights on this walk from Conservatory Garden, along the Harlem Meer and past the North Woods.
Category: Education, History, Tours
Paper Arts & Crafts
at Poe Park Visitor Center (in Poe Park), Bronx
2:00 p.m.–3:30 p.m.
Participants of all ages have fun creating paper arts and crafts. Projects may include origami, kirigami, decoupage, and collage.
Category: Arts & Crafts
Remembering Yesterday: Queens and Its World's Fairs
at Kingsland Homestead (in Margaret I. Carman Green - Weeping Beech), Queens
2:30 p.m.–4:30 p.m.
Remembering is both a collective and a personal activity. In this exhibition, both are on display in relation to two World's Fairs that took place in Flushing, Queens in…
Category: Art, History
Winter Walk in Pelham Bay Park
at Bartow-Pell Mansion Museum (in Pelham Bay Park), Bronx
3:00 p.m.–5:00 p.m.
Our event is currently full and the registration is closed.
Category: Nature, Tours, Talks
Sunday, February 8, 2015
2014 Emerging Artist Fellowship Exhibition at Socrates Sculpture Park
at Socrates Sculpture Park, Queens
8:00 a.m.–6:00 p.m.
The annual Emerging Artist Fellowship Exhibition is a cornerstone of the park's visual arts program and is widely acclaimed for the ambition, breadth & innovation of the works on view.
Category: Art, Waterfront
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Related Information
- Get information on upcoming events directly in your inbox by signing up for our Email Newsletters.
- Know about an event that's not on the calendar? Send us a tip!
- Planning an event and considering hosting it in a park? Make sure to read our Planning an Event page.


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El ángel y el loco. Acrílico (80x80). Colección particular
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Conchita Mencia's curator insight, March 23, 2014 3:29 PM
Preciosa profecía ¡ volvamos a nuestros orígenes !
Ricard Lloria's curator insight, February 19, 12:57 PM
Realmente lleno de sentido, emociones y sobre todo , vida.
corneja's curator insight, September 24, 2014 6:32 AM
Interesting this link between fire and storytelling. The post say that the power of the flame is reproduced in our homes through fireplaces and candles.


text: 23.03. Match schedule The match schedule will be publicized on our webpage on Monday March 30th. Please notice, that we can not quarantee it, it can change, so the right one you will get by your arrival. 12.03. Trimona – another partner of the tournament Trimona, the company which products are handball glue and cleaning compound, is again the partner of the tournament this year. In these indoors, where using of handball glue is allowed, is possible to use only Trimona sort. The organizer doesn't provide the glue, but limited amount of it will be available by the Hall Supervisors. 10.03. HUMMEL is again partner of the tournament As in previous years, HUMMEL is official partner of PHC. Danish producer of sporting goods especially for indoor sports is a leading supplier to many handball clubs. During the Prague Handball Cup you can buy goods for special tournament prizes in "Hummel" stands. Stand No.1 - Tournament Center in Galerie Harfa (sportswear, balls, bags, accessories, fashion clothes) - Friday, Saturday, Sunday 10 am -10 pm. Stand No.2 - Sports Complex HAMR (wide range of products aimed at children) Friday, Saturday 10 am -6 pm. Stand No.3 - Sports Arena Sparta - Podvinný mlýn (megastand with an attractive offer of the goods) final day, Monday, 9 am -4 pm. You can find complete offer on www.hummel-kluby.cz 02.03. Tournament's T-Shirts Like every year the participants will have the possibility to purchase unique tournament T-Shirt with this year 's logo in the front and the listing of all participating clubs on the back. They will be for sale in the tournament centre and limited number of them (and only the blue) also in the playing halls. To ensure that you will receive your T-Shirts directly on arrival and to secure your size, you can pre-order them by contacting the organizer by e-mail. This year´s edition of T-shirts is in blue (size XS - XXL), very limited edition is also in pink colour (only 200 pieces, size XS - XL). The price for T-shirt is 300 CZK or 12 EUR.