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Structural and Energetic Basis of Carbohydrate−Aromatic Packing
Interactions in Proteins
Wentao Chen,†,‡,§,#Sebastian Enck,‡,§,#,▲Joshua L. Price,†,‡,△David L. Powers,⊥Evan T. Powers,*,‡,§
Chi-Huey Wong,*,‡,§,∥H. Jane Dyson,*,¶and Jeffery W. Kelly*,†,‡,§
†Department of Molecular and Experimental Medicine,‡The Skaggs Institute for Chemical Biology, and§Department of Chemistry,
The Scripps Research Institute, La Jolla, California 92037, United States
⊥Department of Mathematics, Clarkson University, Potsdam, New York 13676, United States
∥Genomics Research Center, Academica Sinica, Taipei 115, Taiwan
¶Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, United States
S Supporting Information
ABSTRACT: Carbohydrate−aromatic interactions mediate
many biological processes. However, the structure−energy
relationships underpinning direct carbohydrate−aromatic
packing interactions in aqueous solution have been difficult
to assess experimentally and remain elusive. Here, we
determine the structures and folding energetics of chemically
synthesized glycoproteins to quantify the contributions of the
hydrophobic effect and CH−π interactions to carbohydrate−
aromatic packing interactions in proteins. We find that the hydrophobic effect contributes significantly to protein−carbohydrate
interactions. Interactions between carbohydrates and aromatic amino acid side chains, however, are supplemented by CH−π
interactions. The strengths of experimentally determined carbohydrate CH−π interactions do not correlate with the electrostatic
properties of the involved aromatic residues, suggesting that the electrostatic component of CH−π interactions in aqueous
solution is small. Thus, tight binding of carbohydrates and aromatic residues is driven by the hydrophobic effect and CH−π
interactions featuring a dominating dispersive component.
Carbohydrates and proteins are the two most abundant
biopolymers in nature, and binding interactions between
them are vital for life.1,2Covalently attached carbohydrates
influence glycoprotein folding, through both intrinsic protein−
carbohydrate interactions3−6and extrinsic chaperone-mediated
mechanisms.7Interactions between carbohydrates and carbo-
hydrate-binding proteins, or lectins, on proximal cells direct
many biological processes,8especially immune system
function.9An understanding of the nature of intra- and
intermolecular protein−carbohydrate interactions is essential
for understanding these processes. Such an understanding
would also improve our ability to, for example, engineer
cellulases to improve their substrate affinity for biofuel
production,10design synthetic lectins for carbohydrate
sensing,11tailor glycomimetic drugs to bind strongly to target
proteins,12or improve the stability and shelf life of protein-
based therapeutics by introducing N-glycosylation sites.4,13
Protein−carbohydrate interactions have been studied ex-
perimentally using peptide-based14and host−guest-based
synthetic model systems.11,15−18The binding of carbohydrates
by lectins and other proteins has also been investigated.19−22
From these studies, it is clear that protein−carbohydrate
interactions are driven by the formation of desolvated hydrogen
bonds, the hydrophobic effect, and CH−π interactions.19,20,23
However, studies aimed at understanding the relative
contributions of each of these forces are rare.14,15Moreover,
such studies are often limited by the difficulty of synthesizing a
series of "hosts" for the carbohydrate "guests", each with
enough diversity to simultaneously quantify the various
contributions to the overall interaction energy. Quantum
mechanical studies of diverse carbohydrate−aromatic inter-
actions have successfully quantified the contributions of
electrostatics, induction, dispersion, etc. to the interaction
energy,24−27but such studies must be performed on molecules
in the gas phase. The balance of forces in aqueous solution may
be different since the net interaction energy for a carbohy-
drate−aromatic binding event in a protein is the difference
between the energies of the protein−carbohydrate interaction
and the sum of the protein−solvent and carbohydrate−solvent
interactions. To experimentally parse the energy of carbohy-
drate−aromatic interactions in aqueous solution, we have
utilized enhanced aromatic sequons, a class of structural
modules that can be placed in proteins to enforce an
intramolecular protein−carbohydrate interaction in the folded
Published: June 7, 2013
April 23, 2013
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The typical sequence, or sequon, that the enzyme
oligosaccharyltransferase recognizes to attach an oligosacchar-
ide onto the side chain amide nitrogen of Asn is Asn-Xxx-Ser/
Thr.28Enhanced aromatic sequons are sequences that include
an aromatic residue that is two residues N-terminal to a sequon
in a type 1 β-turn with a G1 β-bulge (a five-residue enhanced
aromatic sequon) or three residues N-terminal to a sequon in a
type 2 β-turn within a larger six-residue loop (a six-residue
enhanced aromatic sequon).4,29Using the β-sheet-rich 34-
residue WW domain from the human Pin1 protein (Pin WW)
as a scaffold, we previously showed that loop 1 of Pin WW
(residues 16−21) can be converted to enhanced aromatic
sequons with five (sequence: Phe16-Ala18-Asn19-Gly20-Thr21) or
six (sequence: Phe16-Arg17-Ser18-Asn19-Gly20-Thr21) residues,
and that glycosylation of Asn19 with N-acetylglucosamine
(GlcNAc) stabilizes the native state in each case.4,29In both
cases, thermodynamic cycle analysis indicated that most of the
glycosylation-associated stabilization is due to interactions
between the Phe16aromatic ring and the GlcNAc.4,29Thus,
enhanced aromatic sequons within Pin WW could be an ideal
structural setting for probing protein−carbohydrate molecular
recognition. Pin WW can be synthesized chemically, enabling
us to incorporate an array of natural and unnatural amino acids
into the protein−carbohydrate interaction site (position 16 in
the Pin WW sequence) and to use linear free energy
relationships30to quantify the contributions of different forces
to the interaction energy, which can be measured from changes
in the folding free energy of Pin WW.
Peptide Synthesis and Thermal Denaturation. Glycosylated
and nonglycosylated variants of Pin WW were synthesized by solid-
phase peptide synthesis by using a standard Fmoc α-amine protecting
group strategy as described previously.4,29The sequences of the
variants were as follows.
Figure 1. (a) Superposition of the main chains of 20 structures of Pin WW variant 5-1 (PDB code: 2M9E). (b) Superposition of the main chains of
20 structures of Pin WW variant 5-1g, in which Asn19is glycosylated with a single GlcNAc (PDB code: 2M9F). (c) Same as panel a, but for 19
structures of Pin WW variant 6-1 (PDB code: 2M9I). (d) Same as panel b, but for 17 structures of Pin WW variant 6-1g (PDB code: 2M9J). (e)
Enlarged view of loop 1 from Pin WW variant 5-1. (f) Enlarged view of loop 1 from Pin WW variant 5-1g. Chemical shift perturbations of GlcNAc
protons (Δδ) are shown in ppm. (g) Same as panel f, but with GlcNAc, Phe, and Thr rendered in CPK format. (h) Enlarged view of loop 1 from Pin
WW variant 6-1. (i) Enlarged view of loop 1 from Pin WW variant 6-1g. Chemical shift perturbations of GlcNAc protons (Δδ) are shown in ppm.
(j) Same as panel i, but with GlcNAc, Phe, and Thr rendered in CPK format. In all panels, Phe16, Asn19, Thr21, and GlcNAc are shown in gold, cyan,
blue, and magenta, respectively. Images of the structures were made using PyMOL.
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Five-residue enhanced aromatic sequon (5-1): Lys6-Leu7-Pro8-Pro9-
Six-residue enhanced aromatic sequon (6-1): Lys6-Leu7-Pro8-Pro9-
The amino acid at position 16 was mutated to other amino acids as
indicated in the text. Asn19(underlined) was modified with an N-
linked N-acetyl glucosamine (GlcNAc) residue in variants in which the
compound number is followed by the letter "g". Thus, 5-1g and 6-1g
have an Asn(GlcNAc) residue at position 19. Mass spectral
characterization of the Pin WW variants is presented in the Supporting
Thermal denaturation profiles were obtained by monitoring the
ellipticity signal at 227 nm from 0.2 to 108.2 °C (for variants of 5-1g)
or from 0.2 to 98.2 °C (for variants of 6-1g) in 2 °C increments on an
Aviv 62A DS circular dichroism spectrometer using quartz cuvettes
with a 1 cm path length. Peptide concentrations were 5−10 μM in 20
mM phosphate buffer (pH 7.0). Thermal denaturation data were fit to
the van't Hoff equation assuming a two-state transition and linear pre-
and post-transition baselines, as described previously.4,29Although the
heat capacities of folding (ΔCp,fold) were used as an adjustable
parameter in these fits, the error in their estimated values is large,
making calculations of folding free energies at temperatures far from
the midpoint temperature (Tm) of the thermal denaturation curve
unreliable. We compensated for this by deriving a consensus value of
the heat capacity of folding for all the glycosylated and non-
glycosylated Pin WW variants from the slope of a plot of their
enthalpies of folding at Tm(ΔHm) versus their Tmvalues (since
dΔHm/dTm= ΔCp,fold; Supporting Information Figure S1).31,32This
approach is justified by the finding that heat capacities of folding are
primarily dependent on protein size, not sequence, and should
therefore change little for variants of a particular protein33,34and by
the observation that the ΔHmvalues for all the Pin WW variants in this
study fall on the same line (Figure S1). The consensus value for
ΔCp,foldwas −0.41 ± 0.02 kcal mol−1K−1, which was used for
calculating temperature-dependent free energies for all Pin WW
Determination of NMR Solution Structures of 5-1, 5-1g, 6-1,
and 6-1g.1H 2D NMR spectra were recorded on Bruker DRX600 or
DMX750 spectrometers at 293 K. Interproton distance restraints were
derived from 2D1H NOESY spectra acquired with a 100 ms mixing
time. To facilitate NMR assignments, we synthesized variants of 5-1,
5-1g, 6-1, and 6-1g, in which13C and15N isotopes were incorporated
into loop 1 by solid-phase peptide synthesis, using the following
isotopically labeled Fmoc-protected amino acids (Sigma-Aldrich):
uniformly13C- and15N-labeled Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH,
and Fmoc-Thr(OtBu)-OH; and uniformly15N-labeled Fmoc-Ala-OH,
Fmoc-Asn(Trt)-OH, and Fmoc-Gly-OH. Structures were calculated in
AMBER35using preliminary coordinates derived from the published
structure of the Pin WW domain.36Variant primary structures of loop
1 were built into the Pin WW structure for 5-1, 5-1g, 6-1, and 6-1g
using NAB.37Details of NMR restraints and structure statistics are
included in Table S1. The lowest energy structures (20 each for 5-1
and 5-1g, 19 for 6-1, and 17 for 6-1g) were analyzed using
PROCHECK-NMR.38The Ramachandran statistics for 5-1 are as
follows: most favored regions, 81.3%; additionally allowed regions,
17.1%; generously allowed regions, 0.6%; disallowed regions, 1.0%.
For 5-1g, the corresponding values are 80.0, 18.8, 0.6, and 0.6%. For 6-
1, the Ramachandran statistics are as follows: most favored regions,
71.5%; additionally allowed regions, 25.6%; generously allowed
regions, 2.6%; disallowed regions, 0.4%; and for 6-1g, the
corresponding values are 72.2, 23.0, 3.5, and 1.3%. All structures
have been deposited in the RCSB Protein Data Bank. Their accession
codes are 5-1, 2M9E; 5-1g, 2M9F; 6-1, 2M9I; 6-1g, 2M9J.
NMR Structures of Pin WW Variants with Enhanced
Aromatic Sequons. To characterize the protein−carbohy-
drate interaction geometry and to ensure that glycosylation
does not change the conformation of Pin WW, we determined
the NMR solution structures of Pin WW variants with five- (5-
1g) and six-residue (6-1g) enhanced aromatic sequons
glycosylated with a single GlcNAc on Asn19and compared
these to the structures of their nonglycosylated counterparts (5-
1 and 6-1, Figure 1 and Table S1). The backbone structures of
nonglycosylated 5-1 and glycosylated 5-1g are very similar
(Figure 1a,b), as are those of 6-1 and 6-1g (Figure 1c,d). The
main differences are in the conformations of the side chains
near the glycosylation site. In nonglycosylated 5-1 and 6-1, the
Phe16side chain prefers a trans conformation (χ1≈ 180°),
while the Asn19side chain is split between gauche−(χ1≈ −60°)
and trans (Figure 1e,h). In glycosylated 5-1g and 6-1g, the
Phe16and Asn19side chains rotate into the gauche−(χ1≈ −60°)
and gauche+(χ1≈ 60°) conformations, respectively, bringing
the α-face of the GlcNAc into contact with the face of the Phe16
aromatic ring (Figure 1f,g and Figure 1i,j).
The planes of the phenyl ring of Phe16and the GlcNAc on
Asn19in 5-1g are almost parallel; the angle between them is
13.9 ± 0.5° (mean ± standard error), and they are separated by
3.97 ± 0.01 Å (Figure 1f,g). The rings are offset by 1.51 ± 0.03
Å, making H5 the closest GlcNAc atom to the Phe16phenyl
ring (2.40 ± 0.01 Å above and 0.78 ± 0.02 Å offset from the
center of the Phe16; Figure 1f). Consistent with the calculated
structure, the H5 resonance is upfield shifted by about 1.2 ppm
(relative to a published reference6) by the Phe16ring current
(Figure 1f). The other protons on the α-face of GlcNAc (H1
and H3) are also shifted but to a lesser degree. The separation
between the GlcNAc and Phe16ring planes is slightly greater in
6-1g than in 5-1g (4.11 ± 0.02 Å), and the angle between them
is greater as well (21.5 ± 0.3°). Thus, despite the slightly
greater separation between the rings, H5 of GlcNAc is as close
to the Phe16ring in 6-1g (2.40 ± 0.02 Å) as it is in 5-1g (Figure
1i,j). Indeed, we find that H5 is also upfield shifted in 6-1g (by
1.7 ppm) as are the other protons on the α-face of the GlcNAc
(H1 and H3) (Figure 1i).
Components of the Intramolecular Protein−Carbohy-
drate Interaction Energy. The interaction energy for
protein−carbohydrate molecular recognition in glycosylated
Pin WW variants is equivalent to the native state stabilization
energy due to glycosylation of enhanced aromatic sequons, or
ΔΔGglyc. ΔΔGglyccan be divided into three components. The
first is the intrinsic effect of glycosylating Asn19(ΔGi), which
probably reflects the conformational preferences of Asn19
versus Asn(GlcNAc)19and the interaction of the GlcNAc
with parts of the protein other than position 16. This is the only
component of ΔΔGglycthat is likely to be different for Pin WW
variants with five- versus six-residue enhanced aromatic sequons
because the φ and ψ angles for Asn19are different in these two
structural contexts (φ = −80.0 ± 2.2°, ψ = 0.9 ± 1.4° in 5-1g vs
φ = −139.3 ± 0.9°, ψ = −19.7 ± 0.5° in 6-1g). The second
component of ΔΔGglycis due to hydrophobic burial of the α-
face of the GlcNAc (which is largely nonpolar20,39) and the side
chain at position 16 (ΔGphob). In proteins, such hydrophobic
burial energies are generally found to correlate with the water−
octanol transfer free energies (ΔGtr, a measure of hydro-
phobicity)40of appropriate analogues of the side chains.41−43
Thus, ΔGphobshould be proportional to ΔGtrfor the position
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16 side chains. The third component of ΔΔGglycderives from
the CH−π interaction between the axial hydrogens projecting
from the α-face of GlcNAc (especially H5) and the aromatic
amino acid side chain at position 16, which is driven by a
combination of dispersion and electrostatic forces44,45
(ΔGCH−π). Dispersion forces refer to interactions between
instantaneous dipoles and multipoles that arise from comple-
mentary polarization of the interacting species' electron charge
distributions46and as such occur between all species. ΔGCH−π
reflects the stronger dispersion interaction of GlcNAc with
aromatic amino acids at position 16 compared to nonaromatic
amino acids. Electrostatic contributions to ΔGCH−πwould be
due to interactions between permanent dipoles or multipoles
on GlcNAc and the aromatic side chain at position 16 and
should therefore be related to the electrostatic properties of the
aromatic ring.
Quantifying the Components of the Protein−Carbo-
hydrate Interaction Energy. To quantify the various
components of ΔΔGglyc, we synthesized 88 glycosylated and
nonglycosylated variants of 5-1g and 6-1g (44 pairs) with an
array of natural and unnatural amino acids at position 16, while
keeping the rest of the glycoprotein structure constant. We
determined the folding free energy for each variant by thermal
denaturation and calculated the change in folding free energy
upon glycosylation (ΔΔGglyc) by subtracting the folding free
energy of each nonglycosylated variant (ΔGfold,nonglyc) from that
of the corresponding glycosylated variant (ΔGfold,glyc; i.e.,
ΔΔGglyc= ΔGfold,glyc− ΔGfold,nonglyc). The ΔΔGglycvalues for
these variants at 60 °C are presented in Table 1 (60 °C was
chosen because it is close to the average thermal transition
midpoints (Tm) of all variants; data at other temperatures, at
which the overall conclusions still hold, are reported in Table
Quantifying Energetic Contributions from the Hydro-
phobic Effect. Linear regression of the ΔΔGglycvalues of the
5-1g variants harboring nonaromatic side chains at position 16
Table 1. Data for Pin WW Variants with Five- or Six-Residue Enhanced Aromatic Sequons in Loop 1 with Sequences Xxx16-
Ala18-Asn19-Gly20-Thr21or Xxx16-Arg17-Ser18-Asn19-Gly20-Thr21, Respectively, in Which Xxx16Is Varieda
aEntries for aromatics and nonaromatics are shaded light orange and light blue, respectively. All energies are reported in kcal mol−1± standard
errors. ΔΔGglycvalues are reported at 60 °C (data at other temperatures are in Table S2). The folding free energies (ΔGfold,glycand ΔGfold,nonglyc) and
other parameters for the individual Pin WW variants on which ΔΔGglycare based are reported in Table S2.bAbu = α-aminobutyric acid; Nva =
norvaline; Cha = cyclohexylalanine; 3,4,5-F3-Phe = 3,4,5-trifluorophenylalanine. Position 16 variants denoted as "pX-Phe" are derivatives of Phe with
the functional group X in the para position of the Phe ring.cΔGtris the water-to-octanol transfer free energy for an appropriate side chain analogue
(Table S2) calculated using Crippen's fragmentation40as implemented in ChemBioDraw Ultra 12.0.dPin WW variants with a "g" following the
number are glycosylated on Asn19with a single GlcNAc.
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versus calculated water−octanol transfer free energies for
appropriate analogues of the position 16 side chains yields a
moderately good fit (R2= 0.69, F = 13.4, p = 0.01) with a slope
of 0.08 ± 0.02 and an intercept of −0.21 ± 0.05 kcal mol−1
(Figure 2a, yellow circles). The fit is not as good for the
corresponding variants of 6-1g with nonaromatic amino acids at
position 16 (Figure 2b, yellow circles, R2= 0.47, F = 7.0, p =
0.03). The slope and intercept of the regression line are 0.10 ±
0.04 and 0.03 ± 0.08 kcal mol−1, respectively. Nonetheless, the
trends for the variants of 5-1g and 6-1g are the same: ΔΔGglyc
increasing as ΔGtrincreases, with similar slopes. The intercepts
indicate the intrinsic effect of glycosylating Asn19in a five- or
six-residue enhanced aromatic sequon (ΔGi), independent of
any interactions with the side chain at position 16.
Glycosylating the Asn is more stabilizing in five- than in six-
residue enhanced aromatic sequons (−0.21 ± 0.05 vs 0.03 ±
0.08 kcal mol−1), consistent with our previous findings.29
ΔΔGglycvalues of the 5-1g variants harboring aromatic side
chains show no correlation with ΔGtr(Figure 2a, blue and red
circles). In contrast, the ΔΔGglycvalues of the 6-1g variants
harboring aromatic side chains show a fair correlation with ΔGtr
(Figure 2b, blue and red circles, R2= 0.53, F = 13.7, p = 0.003)
with a slope of 0.11 ± 0.03 and an intercept of −0.42 ± 0.10
kcal mol−1. Thus, three out of the four cases examined yield
statistically significant correlations between ΔΔGglycand ΔGtr,
indicating that the hydrophobic effect generally makes a
substantial contribution to protein−carbohydrate interactions
in both five- and six-residue enhanced aromatic sequons. In the
remaining case, aromatic variants of 5-1g, the combination of
experimental error, and the innate variability of the protein−
carbohydrate interaction make direct quantification of the
hydrophobic effect difficult. However, it is worth noting that
the contribution of the hydrophobic effect to the protein−
carbohydrate interactions in the nonaromatic variants of 5-1g
and the aromatic and nonaromatic variants of 6-1g are all
similar, as indicated by the similar slopes of their regression
lines between ΔΔGglycversus ΔGtr(0.08 ± 0.02, 0.10 ± 0.04,
and 0.11 ± 0.03, respectively; Figure 2a,b). We therefore expect
the contribution of the hydrophobic effect to the protein−
carbohydrate interactions in aromatic variants of 5-1g to be
similar to that in the other three cases.
The slopes of the correlations between ΔΔGglycand ΔGtr
reflect the strength of the hydrophobic interaction between the
α-face of the GlcNAc and the side chains at position 16 relative
to transfer from water to octanol. It has been shown that the
effects of mutations on protein folding free energies can
correlate with ΔGtrwith slopes close to 1 for fully buried
positions.41−43Our smaller slopes, generally ∼0.1 for the cases
where there is a demonstrable correlation between ΔΔGglycand
ΔGtr, suggest that burying the position 16 side chain under the
α-face of GlcNAc is not as energetically favorable, likely
reflecting the difficulty of burying the nonpolar α-face of
GlcNAc without the destabilizing side effect of burying adjacent
polar hydroxyls.19,47
Quantifying the Energetic Contributions from CH−π
Interactions. The values of ΔΔGglyc predicted by the
regression lines of nonaromatic amino acids in Figure 2a,b
and the ΔGtrof the Phe side chain (−3.4 kcal mol−1) are −0.48
± 0.08 kcal mol−1for 5-1g and −0.31 ± 0.16 kcal mol−1for 6-
1g. However, the experimental values of ΔΔGglycfor the Phe
variants are −0.86 ± 0.04 and −0.80 ± 0.06 kcal mol−1,
respectively (red circles; Figure 2a,b), much larger than
expected from consideration of the hydrophobic effect alone.
The differences between the predicted and measured values
(−0.38 ± 0.09 kcal mol−1for 5-1g and −0.49 ± 0.17 kcal mol−1
for 6-1g) represent the energies resulting from the special
nature of the GlcNAc−aromatic side chain interaction in the
five- and six-residue enhanced aromatic sequons. The proximity
of H5 of GlcNAc and its neighboring axial hydrogens to the
aromatic ring of Phe16in both 5-1g and 6-1g suggests that
those differences are due to CH−π interactions.44To further
probe the electrostatic contribution to the CH−π interaction,
we attempted to fit the ΔΔGglycvalues of Phe, Tyr, and
additional para-substituted position 16 aromatic variants to a
Hammett-type linear free energy relationship:
where B is a constant, x is a variable that measures a
substituent's electronic effect on the aromatic ring, and A is the
sensitivity of ΔΔGglycto x.48Of the various x parameters
available in the literature, we used Hammett's σmand σp.48
The values of ΔΔGglycin Table 1 for variants of 5-1g are fit
poorly by the Hammett relationship with σmand σp(R2= 0.04
and 0.07, respectively; Figure 3a,b). The values of ΔΔGglycfor
variants of 6-1g are also poorly fit by the Hammett relationship
with σmand σp(R2= 0.14 and 0.01, respectively; Figure 3c,d).
Figure 2. (a) Plot of the values of ΔΔGglycvs the calculated water-to-
octanol transfer free energies (ΔGtr) of the appropriate side chain
analogues (Table 1 and Table S2) for Pin WW variants with
nonaromatic or aromatic amino acids at position 16 in a five-residue
enhanced aromatic sequon (variants 5-1g through 5-24g). The values
of ΔΔGglycwhen nonaromatic amino acids, Phe, and other aromatic
amino acids are in position 16 are shown in yellow, red, and blue,
respectively. The line of best fit (straight line) has R2= 0.69, F = 13.4,
p = 0.01, slope = 0.08 ± 0.02, and intercept = −0.21 ± 0.05 kcal mol−1.
(b) Same as panel a, except for Pin WW variants with a six-residue
enhanced aromatic sequon. The line of best fit (straight line) for
nonaromatic residues has R2= 0.47, F = 7.0, p = 0.03, slope = 0.10 ±
0.04, and intercept = 0.03 ± 0.08 kcal mol−1. The line of best fit
(straight line) for aromatic residues has R2= 0.53, F = 13.7, p = 0.003,
slope = 0.11 ± 0.03, and intercept = −0.42 ± 0.10 kcal mol−1. All
reported errors are standard errors of the coefficients. Selected amino
acids are labeled in panels a and b.
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This lack of dependence of ΔΔGglyc on the electronic
properties of the substituents suggests that interactions
involving partial charges or permanent dipoles (i.e., electrostatic
interactions) are not measurable in either the five- or the six-
residue enhanced aromatic sequon.
This result suggests, by a process of elimination, that the
carbohydrate−aromatic interaction in the five- and six-residue
enhanced aromatic sequons must be driven by dispersion
forces.46Several quantum mechanical studies of carbohydrate−
aromatic and other CH−π interactions in the gas phase have
suggested that, while both electrostatics and dispersion forces
contribute to these interactions, dispersion forces contribute
more than electrostatics.24−27,44,45Our results suggest that the
importance of dispersion forces in carbohydrate−aromatic
interactions persists in aqueous solution. However, the
contribution from the electrostatic component to the net
energy of CH−π interactions relative to that from the
dispersion component may be even smaller in aqueous solution
than in the gas phase because any changes in the strength of
CH−π electrostatic interactions due to changes in the electron
density of the aromatic ring are envisioned to be canceled out
by parallel changes in the strength of electrostatic interactions
between water and the aromatic ring in the denatured state.
Three factors contribute to the stabilizing glycan−protein
interactions in five- and six-residue enhanced aromatic sequons,
quantified by ΔΔGglyc: the intrinsic effect of glycosylating Asn19
(ΔGi); the attenuated hydrophobic effect derived from
juxtaposing the side chain at position 16 with the α-face of
the GlcNAc (ΔGphob); and the CH−π interaction when an
aromatic amino acid is at position 16 (ΔGCH−π). The additional
stabilization derived from the aromatic ring−GlcNAc inter-
action is mostly due to dispersion forces in the five- and six-
residue enhanced aromatic sequons. The estimated contribu-
tions of each of these components to ΔΔGglycwhen position 16
is Leu, Phe, or Tyr are illustrated in Figure 4 for the five- and
six-residue enhanced aromatic sequons.
The quantification of the structure−energy relationships
presented herein for protein−carbohydrate molecular recog-
nition was made possible by applying a powerful tool from
classical physical organic chemistry (linear free energy relation-
ships) to an extensive set of protein−carbohydrate interaction
energies measured within the structurally well-defined context
of enhanced aromatic sequons. There are, however, two caveats
to the quantification. First, introducing a mutation into a
protein that perturbs an interaction of interest may result in a
structural reorganization of the native state. The overall effect
of the mutation on the folding free energy is then the sum of
the change in the interaction energy due to the mutation and
the reorganization energy if there are any structural changes.
The reorganization energy is always negative (i.e., thermody-
namically favorable), so the effect of the mutation on the
folding free energy must be considered a lower bound on the
interaction energy. In our case, however, we expect the
reorganization energies to be minimal because the loop 1
structures of a given class are quite robust, as illustrated by
Figure 5. Alignment of the main chain in loop 1 of the lowest
energy calculated structure of 5-1g (sequence: Met15-Phe16-
Ala18-Asn19-Gly20-Thr21-Val22) with that of a Pin WW variant
(PDB code: 2F21)49with the same loop type (a type 1 β-turn
with a G1 β-bulge), but a different sequence (Met15-Ser16-Ala18-
Asp19-Gly20-Arg21-Val22) shows that the three amino acid
differences have little effect on the loop structure (Figure 5a).
Analogously, alignment of the main chain in loop 1 of the
lowest energy calculated structure of 6-1g (sequence: Met15-
Phe16-Arg17-Ser18-Asn19-Gly20-Thr21-Val22) with that of wild-
type Pin WW (PDB code: 1PIN),50which has the same loop
type (a type 2 β-turn within a six-residue loop) but a different
sequence (Met15-Ser16-Arg17-Ser18-Ser19-Gly20-Arg21-Val22),
demonstrates only slight perturbations of the structure (Figure
Figure 3. (a,b) Plots of ΔΔGglycfor variants of 5-1g with para-substituted aromatic amino acids at position 16 vs σmand σp, respectively. The lines of
best fit (straight lines) have R2= 0.04 and 0.07, respectively. (c,d) Plots of ΔΔGglycfor variants of 6-1g with para-substituted aromatic amino acids at
position 16 vs σmand σp, respectively. The lines of best fit (straight lines) have R2= 0.14 and 0.01, respectively. Values of ΔΔGglycfor 5-1g and 6-1g
are shown in red.
Journal of the American Chemical Society
dx.doi.org/10.1021/ja4040472 | J. Am. Chem. Soc. 2013, 135, 9877−9884
Page 7
Second, we are employing the intramolecular protein−
carbohydrate interactions in our Pin WW variants to ascertain
features of protein−carbohydrate interactions in general, both
intra- and intermolecular, which may have different geometries.
The characteristics of protein−carbohydrate interactions are
likely to depend on geometry. However, the facial interaction
between a carbohydrate and the position 16 amino acid side
chain in enhanced aromatic sequons is a common type of
protein−carbohydrate interaction, especially for aromatic
amino acid side chains.20,21,51Thus, the behavior of the
protein−carbohydrate interaction in the enhanced aromatic
sequon should be representative of a broad class of protein−
carbohydrate interactions. Furthermore, while the intramolec-
ular protein−carbohydrate interaction in enhanced aromatic
sequons cannot replicate certain aspects of intermolecular
protein−carbohydrate interactions, in particular, the effect of
the loss of translational entropy, the attractive forces that drive
protein−carbohydrate interactions should be the same whether
the interaction is intra- or intermolecular.
Figure 4 also shows that, although contributing significantly
to protein−carbohydrate packing, the hydrophobic effect
cannot drive strong protein−carbohydrate interactions the
way it drives protein folding or the binding of other types of
ligands; the contributions from the hydrophobic effect are
never more than 44% of ΔΔGglycfor 5-1g or 6-1g. Thus, tight
binding of carbohydrates requires either multivalency52or a
protein−carbohydrate binding site rich in desolvated hydrogen
bonds (the energetics of which were not addressed in this
study) and aromatic amino acids.20,23The arabinose binding
protein of Escherichia coli is a naturally occurring example of
these strategies.53
We have utilized a robust protein scaffold to study the
molecular origin of energetically favorable carbohydrate−
aromatic packing interactions in aqueous solution. Both the
hydrophobic effect and CH−π interactions are found to
contribute significantly to the overall interaction energy. The
observation that the electronic nature of aromatic groups has
little effect in dictating the strength of carbohydrate−aromatic
interactions in aqueous solution suggests that the electrostatic
component of CH−π interaction energies is likely small relative
to the dispersion component, possibly for reasons outlined at
the end of the Results section.
The structure−energy relationships presented herein provide
a framework for understanding the energetic basis of protein−
carbohydrate molecular recognition in aqueous solution (as
opposed to the computationally more accessible gas phase) and
will serve as benchmarks for the molecular mechanics force
fields used in simulations of systems with intra- and
intermolecular protein−carbohydrate interactions.54Improving
these force fields will in turn improve our ability to design and
optimize protein−carbohydrate interactions, facilitating the
development of industrial and therapeutic applications that
rely on these interactions.
Plot of ΔHmvs Tmfor the determination of ΔCp,foldfor the Pin
WW variants, NMR, and refinement statistics and thermody-
S Supporting Information
Figure 4. Estimated composition of components of ΔΔGglycfor 5-1g
and 6-1g variants. Values of ΔΔGglycfor five- and six-residue enhanced
aromatic sequons with Leu, Phe, or Tyr at position 16 were broken
down into three components: (1) the inherent effect of glycosylating
Asn19(ΔGi); (2) the energy of hydrophobic burial of the Asn19
GlcNAc and the position 16 side chain (ΔGphob); and (3) the energy
of the CH−π interaction between the GlcNAc and aromatic amino
acid side chains at position 16 (ΔGCH−π). The first component is the
intercept of the best-fit lines for nonaromatic amino acids in Figure
2a,b for five- or six-residue enhanced aromatic sequons, respectively.
For Leu, the second component is calculated by subtracting the first
component from ΔΔGglyc. For Phe and Tyr, it is calculated by
multiplying their ΔGtr(Table 1) by the slope of the best-fit lines for
nonaromatic amino acids in Figure 2a,b for five- or six-residue
enhanced aromatic sequons, respectively. The third component,
ΔGCH−π, is calculated by subtracting the first and second components
from ΔΔGglyc. Although, as discussed in the main text, experimental
error and the innate variability of the protein−carbohydrate interaction
energetics prevent precise determination of the contribution of the
hydrophobic effect to ΔΔGglycfor aromatic variants of 5-1g, we expect
the hydrophobic effect to be roughly the same as for nonaromatic
variants of 5-1g. This uncertainty is indicated by the dashed line
separating the hydrophobic and CH−π contributions in the bars for
the cases where Phe or Tyr occupy position 16 in the five-residue
enhanced aromatic sequon.
Figure 5. (a) Alignment of the main chain in the loop 1 region from
the lowest energy calculated structure of 5-1g (magenta; sequence:
Met15-Phe16-Ala18-Asn(GlcNAc)19-Gly20-Thr21-Val22; the GlcNAc is
not shown for clarity; PDB code: 2M9F) with that of a Pin WW
variant, the structure of which was determined previously by X-ray
crystallography (cyan; PDB code: 2F21)49that has the same loop type
(a type 1 β-turn with a G1 β-bulge) but three different amino acids in
its sequence (Met15-Ser16-Ala18-Asp19-Gly20-Arg21-Val22). Main chain
RMSD = 0.6 Å for loop 1. (b) Alignment of the main chain in the loop
1 region from the lowest energy calculated structure of 6-1g (magenta;
sequence: Met15-Phe16-Arg17-Ser18-Asn(GlcNAc)19-Gly20-Thr21-Val22;
the GlcNAc is not shown for clarity; PDB code: 2M9J) with that of
wild-type Pin WW (cyan; PDB code: 1PIN),50which has the same
loop type (a type 2 β-turn within a six-residue loop) but three different
amino acids in its sequence (Met15-Ser16-Arg17-Ser18-Ser19-Gly20-Arg21-
Val22). Main chain RMSD = 0.9 Å for loop 1. Images of the structures
were made, and RMSD values were calculated, using PyMOL.
Journal of the American Chemical Society
dx.doi.org/10.1021/ja4040472 | J. Am. Chem. Soc. 2013, 135, 9877−9884
Page 8
namics data from fitting CD-monitored thermal denaturation
data of Pin WW variants. This material is available free of
charge via the Internet at http://pubs.acs.org.
Corresponding Author
email@example.com; email@example.com; firstname.lastname@example.com.
edu.tw; email@example.com
Present Addresses
▲S.E.: BASF SE, Ludwigshafen D-67056, Germany.
△J.L.P.: Department of Chemistry and Biochemistry, Brigham
Young University, Provo, UT 84602.
Author Contributions
#W.C. and S.E. contributed equally to this work.
The authors declare no competing financial interest.
We thank Dr. Gira Bhabha (Scripps) and Dr. Gerard Kroon
(Scripps) for help with the NMR experiments, Dr. Pritilekha
Deka and Dr. Sangho Park (Scripps), Professor Robert J.
Woods, and Dr. B. Lachele Foley (University of Georgia,
Complex Carbohydrate Research Center) for help with the
structure calculations, and Dr. Sarah R. Hanson, Dr. Elizabeth
K. Culyba, and Dr. Amber N. Murray for helpful discussions.
This work was supported by the Skaggs Institute for Chemical
Biology, the Lita Annenberg Hazen Foundation, and by
National Institutes of Health Grants GM051105 (J.W.K.),
GM071862 (H.J.D.), and AI072155 (C.-H.W.). J.L.P. was
supported in part by NIH postdoctoral fellowship F32
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text:
Article 113 After the office of Leadership, the President is the highest official in the country. His is the responsibility for implementing the Constitution and acting as the head of the executive, except in matters directly concerned with (the office of) the Leadership. Article 114 The President is elected for a four-year term by the direct vote of the people. His re-election for a successive term is permissible only once. Article 115 The President must be elected from among religious and political personalities possessing the following qualifications: Iranian origin; Iranian nationality; administrative capacity and resourcefulness; a good past-record; trustworthiness and piety; convinced belief in the fundamental principles of the Islamic Republic of Iran and the official religion of the country. Article 116 Candidates nominated for the post of President must declare their candidature officially. Law lays down the manner in which the President is to be elected. Article 117 The President is elected by an absolute majority of votes polled by the voters. But if none of the candidates is able to win such a majority In the first round, voting will take place a second time on Friday of the following week. In the second round only the two candidates who received greatest number of votes in the first round will participate. If, however, some of the candidates securing greatest votes in the first round withdraw from the elections, the final choice will be between the two candidates who won greater number of votes than all the remaining candidates. Article 118 Responsibility for the supervision of the election, of the President lies with the Guardian Council, as stipulated in Article 99. But before the establishment of the first Guardian Council, however, it lies with a supervisory body to be constituted by law. Article 119 The election of a new President must take place no later than one month before the end of the term of the outgoing President. In the interim period before the election of the new President and the end of the term of the outgoing President, the outgoing President will perform the duties of the, President. Article 120 In case any of the candidates whose suitability is established in terms of the qualifications listed above should die within ten days before polling day, the elections will be postponed for two weeks. If one of the candidates securing greatest number of votes dies in the intervening period between the first and second rounds of voting, the period for holding (the second round of) the election will be extended for two weeks. Article 121 The President must take the following oath and affix his signature to it at a session of the Islamic Consultative Assembly in the presence of the head of the judicial power and the members of the Guardian Council:
"In, the Name of God, the Compassionate, the Merciful, I, as President, swear, in the presence of the Noble Qur'an and the people of Iran, by God, the Exalted and Almighty, that I will guard the official religion of the country, the order of the Islamic Republic and the Constitution of the country; that I will devote all my capacities and abilities to the fulfillment of the responsibilities that I have assumed; that I will dedicate myself to the service of the people, the honor of the country, the propagation of religion and morality, and the support of truth and justice, refraining from every kind of arbitrary behavior; that I will protect the freedom and dignity of all citizens and the rights that the Constitution has accorded the people; that in guarding the frontiers and the political, economic, and cultural independence of the country I will not shirk any necessary measure; that, seeking help from God and following the Prophet of Islam and the infallible Imams (peace be upon them), I will guard, as a pious and selfless trustee, the authority vested in me by the people as a sacred trust, and transfer it to whomever the people may elect after me."
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text:
Some rooms in your house may get too much of a sun exposure and they might be too hot for people to live in, these rooms usually are rooms with larger windows.
In the summer the sun, mainly coming from the south side of your house, penetrates your house window's and rises the internal house temperature – this will make your electric bill go up as you will need to use much more energy to cool the space.
There's am easy and inexpensive solution to this :
Solar film when installed correctly can reduce the heat in the rooms in the summer and help warm the room in the winter.
Kevin Koval has been installing these film for more than 10 years, he says that the most important thing in installing solar films on windows is to clean the window properly before attempting to apply the film on the window.
Adding solar film has many benefits
The main purpose when installing the film is to block UV, heat, and glare.
the added value of the film on the glass is that if somebody is trying to break the glass in order to get into your house and steal, the solar film will act as a protection layer and will make it exteremly impossible to break it.
The quality films are completely clear and can last over 30 years – so you won't be left in the dark once the are coating your glass.
How Much a Solar Film Installation Cost?
- The cost is dependent on the type of solar film you will choose to use
- The Price usually varies from 6 to 14 dollars per square foot.
- The total cost is then determined by the number of windows and the size of the windows.
- New technology also includes film the helps retain heat during the winter.
- Solar Film will reject heat during the summer.
- Federal tax credits is available for up to 10% rebate of the cost or $500.


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Hi. I am new. I did martial arts when I was 18-19. I am now a 26 year old man. Recently I like MMA. I added it to my list fighting events I enjoy watching. MMA has made me want to start martial arts again and train MMA.
P.S I was unhappy Sonnen lost. |
Good to have you on board.
thanks. Hey where abouts in Norway do you train ? Is TMA and MMA very popular over there ?
Welcome to the forum :)
Welcome! I'm new here as well. Enjoy!
Welcome :D
Hello everyone,
I am newbie in this forum. I am pleased to be part of this community. Hope everyone is okay and having fun while learning here. |
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This iReport is part of an assignment:
Summer of the supermoons
About this iReport
- Posted August 11, 2014 by
- MaryMcAvoy Follow
North Andover, Massachusetts
North Andover, Massachusetts
More iReports you should see
Fly Me To The Moon
Mary McAvoy has been photographing for nearly ten years, primarily nature shots. She posts her photos at http://www.TheRipestPics.com and at http://www.sublimedays.com. "I took this photo of the supermoon on July 12, 2014. I had no idea there was a jet crossing between the moon and me. It was a stunning surprise to see the jet in the photo when I downloaded the images I took that night (this one at 10:35 p.m.) just north of Boston, Massachusetts (and Logan Airport). I had seen something cross the lens during one shot but I thought it was a mosquito caught in the lens hood. I took this photo with my Nikon D3200, handheld, 300mm, f/5.6, ISO 800, 1/1000."
#supermoon #July12supermoon #marymcavoy #NikonD3200 #handheldphotography #moon #flymetothemoon
#supermoon #July12supermoon #marymcavoy #NikonD3200 #handheldphotography #moon #flymetothemoon
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The Sims 3 Plus Showtime includes both The Sims 3 and The Sims 3 Showtime expansion pack in one! Create your great sim and roam the streets of downtown Sunset Valley, or venture into a new world of...More
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54 year old man | Garden City Park, NY, USA | Seeking women 40-54 within 20 miles of New Hyde Park, NY
You can just be yourself, everyone else is taken.
Sharing the adventures of life is so much better than experiencing them alone. What else can I say? Would rather not write my life's story here. Lot's to tell. The perfect match? We'll know it when we feel it. Let's talk.
I have Dogs
Some other viewpoint
Caribbean, Las Vegas, Europe. Will try new places.
Like films of all kinds. Travel. Horse back riding. Flying airplanes. Love to go to the gym and stay in shape. Weights and trend mill my fav.
Movies, reading, travel. Gym. Horses. Dinner. Anything outdoors. Nature!!
Read a wide variety of books from novels to science. Last one = The Scientific American Book of Love, Sex, & The Brain! Wow!
Him | What he is looking for | |
Appearance | ||
Height: | 3'0" (91cms) | 5'0" (152cms) to 6'0" (182cms) |
Body type: | Athletic and toned | Slender, Athletic and toned |
Eyes: | Brown | No preference |
Hair: | Dark brown | No preference |
Lifestyle | ||
Smoke: | No Way | No Way |
Drink: | Moderately | No preference |
Occupation: | Education / Teacher / Professor | No preference |
Will tell you all about it. |
Income: | I'll tell you later | No preference |
Relationship: | Never Married | Never Married, Widow / Widower, Currently Separated, Divorced |
Have kids: | No | Yes, and they sometimes live at home, No, Yes, and they live away from home, Yes, and they live at home |
Wants kids: | No, but it's OK if my partner has kids | No, but it's OK if my partner has kids |
Background | ||
Ethnicity: | White / Caucasian | Latino / Hispanic, Native American, Pacific Islander, White / Caucasian |
Italian |
Faith: | Spiritual but not religious | No preference |
Who knows what's up there! |
Languages: | English | English |
Education: | Graduate degree | No preference |
MS in Earth Science. |


text: Italian Police Investigate Burglary of Virtual Home Thief steals $140 in furnishings from house in Facebook game By Nick McMaster, Newser Staff Posted Oct 27, 2010 4:00 PM CDT STORY COMMENTS (20) (Newser) – Italian police are investigating a bizarre theft: the complete ransacking of a virtual home. A Palermo woman says a digital thief emptied her seven-room home in the Facebook game Pet Society, leaving only her virtual cat behind. The damage? $140 in payments for furniture, paintings, mirrors, and pet outfits, the AFP reports. "I don't think it matters that the flat only exists in Facebook," she says. "It is real to me and I have suffered a real loss." The Italian police have responded with a probe into the "illegal and aggravated entry" into her email, a charge that carries between 1 and 5 years in prison, MSNBC reports. Read more on the virtual theft here. A user's virtual home in Pet Society, a Facebook game. (Flickr) A user's virtual home in Pet Society, a Facebook game. (Flickr) A user's virtual home in Pet Society, a Facebook game. (Flickr) ADVERTISEMENT(Click Prev or Next to continue viewing images.) « Prev« Prev | Next »Next » Slideshow AFP (Source Grid ») MSNBC (Source Grid ») Crime & Courts • Technology • World Facebook • virtual economy • virtual currency • Pet Society SHARE YOUR TAKE: Check out another Hilarious story: Obama: I Can't Wait 'to See Who Koch Bros.... 16% 68% 3% 7% 3% 3% To report an error on this story, notify our editors.


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Franklin — Wheaton Franciscan Hospital-Franklin has been named the 2013 Business of the Year by the South Suburban Chamber of Commerce.
The chamber bestowed its awards to businesses in Oak Creek, Franklin and the surrounding south suburban communities during its Annual Awards Dinner on Jan. 30.
More than 170 South Suburban Chamber members, business leaders and elected officials attended the dinner, held at the Crowne Plaza Hotel.
Wheaton Franciscan received the Business of the Year Award for its success, its contributions to the local community and to the community at large.
"This is a huge honor that reflects on our teamwork and dedication to the communities we serve," said Sheila Gansemer, vice president of patient care services for Wheaton Franciscan-Franklin, who accepted the award.
In July of 2013, the hospital opened a new 60,000-square-foot medical office building to house a range of primary care physicians and specialists; the expansion helped create space for the next phase of the hospital's construction, which will include more inpatient beds, operating rooms, gastrointestinal laboratories and a cardiac catheterization laboratory.
The hospital also offers multiple programs for community involvement, including its Mental Health Community Education Series and the STRIDES (Student Transitions Resulting in the Development of Essential Skills) program. The company also promotes health events, such as the Franklin Bike Rodeo and the Franklin Family Health Night.
"I am so proud to have a team that has this high level of commitment," said Wheaton-Franciscan Central Market President Coreen Dicus-Johnson. "Our goal is to continue to build on this excellent level of care."
The other finalists for Business of the Year were FastSigns from Greenfield and Toppers Pizza in Franklin.
Pride in Premises
The 2013 Pride in Premises Award was given to the Staybridge Suites Milwaukee Airport South, located in Franklin.
The Pride in Premises Award is given to a company or organization that has demonstrated care for their buildings, physical environment and surroundings.
"Staybridge Suites Milwaukee Airport South has taken its position on South 27th Street and Ryan Road and used the natural plantings, cattails and shrubs to create a welcoming entrance to the hotel," said Barbara Wesener, executive director of the chamber. "Areas that previously were overgrown are now mulched, a fire pit serves as a gathering spot and the environmentally friendly parking lot all contribute to the well-designed premises of the Staybridge Suites."
"We thought the outdoor fire pit would be a great feature for our guests who wanted to enjoy the outdoors," Staybridge General Manager Mark Hansen. "And our parking lot now has brick pavers, rather than asphalt, that allow water to filter through. We're always looking for ways to make our surroundings more beautiful."
Dental Associates in Franklin and the Hilton Garden Inn Milwaukee Airport were runners-up for the Pride in Premises Award.
Member of the Year
The chamber also named Mary Starr as its 2013 Member of the Year.
Starr, a Franklin resident, is the co-owner and executive vice president of the Greenfield insurance risk-management agency The Starr Group and has served two terms on the chamber's board of directors.
"I was very surprised and honored," Starr said. "It was something I wasn't expecting at all."
Starr has served as the chairwoman of the Events Committee, which is responsible for the chamber's two largest fundraisers: the golf outing and the annual awards dinner. She also helped establish the chamber's Health and Wellness Council.
"I'm very passionate about wellness and wanted to bring it to the other business members in the chamber," Starr said. "I wanted to give back to a community that has already given me so much."
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text: YAMAHA V-MAX (1991-2004) Review Published: 24 November 2006 Sections: MCN Review Owners' Reviews Photo Gallery Facts & Figures History & Versions Bikes For Sale "An ill-handling, raw powered Rottweiller of a motorcycle" "An ill-handling, raw powered Rottweiller of a motorcycle" At a glance 1198cc - 140 bhp 35 mpg - 100 miles range Insurance group: 15 of 17 Compare insurance quotes now Medium seat height (765mm) Overall Rating 4 out of 5 Mad Max. There, that tells you everything you need to know about the Yamaha VMX1200 V-Max, an ill-handling, raw powered Rottweiller of a motorcycle. The full power 140bhp model is the only one worth having, so skip the early 90s UK spec models with a feeble 95bhp. Ride Quality & Brakes 2 out of 5 The Yamaha VMX1200 V-Max handles like a drunk office girl on a photocopier...and is just as dangerous. The frame seems to flex, the back end squirms under the engine's power and the front end loads up with the Yamaha VMX1200 V-Max's substantial 280kgs wet weight under braking. In a word, scary. Engine 5 out of 5 The Yamaha VMX1200 V-Max has a beautiful, almost liquid power delivery from just above tickover, with a crazed step up in poke from about 4000rpm as the V-Boost fuel charge system kicks in. The Yamaha VMX1200 V-Max motor puts a grin on the face of the most jaded speedfreak. It punches you in the guts with its sheer torque. Build Quality & Reliability 4 out of 5 You can turbocharge the motor, add nitrous, almost anything really and the V-4 lump will take it. The Yamaha VMX1200 V-Max's chassis is the weaker half of the motorcycle by far and as it was designed to race the strips of California, the V-Max can suffer if ridden too often in British rain. Insurance, running costs & value 3 out of 5 The Yamaha VMX1200 V-Max is a cult machine and good used examples go for very decent money. Many are customised and feature mad performance tweaks. The V-Max was never a big selling motorcycle when brand new, so supply of original examples will never meet demand, prices will keep rising. Don't bother buying a restricted early 90s UK model. Find a Yamaha V-Max for saleInsurance group: 15 of 17 – compare motorcycle insurance quotes now. Equipment 2 out of 5 The Yamaha VMX1200 V-Max's 15 litre fuel tank can run dry in 90 miles, the pillion seat is really just a bum stop for the rider and the brakes and suspension are indifferent, even by 90s standards. Apart from a few cosmetic tweaks Yamaha did almost nothing to develop the V-Max throughout a near 20 year lifespan. Bit poor frankly. Owners' Reviews 1 owner has reviewed their YAMAHA V-MAX (1991-2004) and rated it in a number of areas. Read what they have to say and what they like and dislike about the bike below. We're currently improving the way this section works, which means we've had to suspend the submission of new owners' reviews for a short period. Please check back soon. Summary of Owners' Reviews Overall Rating 5 out of 5 Ride Quality & Brakes 5 out of 5 Engine 5 out of 5 Build Quality & Reliability 4 out of 5 Value & Running Costs 5 out of 5 Equipment 5 out of 5 5 out of 5 OLD RELIABLE 18 November 2007 by murphit Having owned and ridden my Max for the last ten years in all conditions, including snow and ice, I find that comments of bad handling to be misplaced. Understand and respect what is under you and the machine csn return fast safe rides. No I'm not a... Read more slow rider, I use the Max to the Max. Reliable, fun, respected by others, reputation justified, only down side is the quality of finish, but with some effort this is easy to overcome. I still look for a replacement for the Max but fail to find a machine that has the ability to come close to it's stance, torque, power, and all round suitability. I have ridden 350+ miles in a day and enjoyed every mile, and still walked normally when I got off. My missus when a pillion passenger does not have to perch on a pad high above me. Basically it's as mean as a junk yard dog, and it's rear end dangely bits. Overall Rating 5 out of 5 Ride Quality & Brakes 5 out of 5 Engine 5 out of 5 Build Quality & Reliability 4 out of 5 Value & Running Costs 5 out of 5 Equipment 5 out of 5 Read all 1 owners' reviews in full Facts & Figures Model info Year introduced 1991 Year discontinued 2004 Original price - Warranty term (when new) Two year unlimited mileage Running costs Insurance group 15 of 17 Annual road tax £80 Annual service cost - Performance Max power 140 bhp Max torque 117.7 ft-lb Top speed 160 mph 1/4-mile acceleration 10.9 secs Average fuel consumption 35 mpg Tank range 100 miles Specification Engine size 1198cc Engine type 4 stroke, V4, 5 gears Frame type Steel cradle type Fuel capacity 15 litres Seat height 765mm Bike weight 262kg Front suspension Preload Rear suspension Preload Front brake Twin 282mm disc Rear brake 290mm disc Front tyre size 110/90 x 18 Rear tyre size 150/90 x 15 History & Versions Model history 1986: Yamaha VMX1200 V-Max launched in USA.1991: Restricted version arrives in UK.1996: Full 140bhp version of the Yamaha VMX1200 V-Max appears in UK.1999: Cosmetic upgrade with `carbon look' bodywork.2004: Yamaha VMX1200 V-Max discontinued in UK. Other versions None. Photo Gallery Yamaha VMX1200 V-Max motorcycle review - Riding Yamaha VMX1200 V-Max motorcycle review - Side view Yamaha VMX1200 V-Max motorcycle review - Top view Yamaha VMX1200 V-Max motorcycle review - Side view Yamaha VMX1200 V-Max motorcycle review - Engine Yamaha VMX1200 V-Max motorcycle review Yamaha VMX1200 V-Max motorcycle review - Brakes Yamaha VMX1200 V-Max motorcycle review - Top view Yamaha VMX1200 V-Max motorcycle review - Brakes Yamaha VMX1200 V-Max motorcycle review - Riding Yamaha VMX1200 V-Max motorcycle review - Riding Yamaha VMX1200 V-Max motorcycle review - Riding Other pages you might like ... NewsArticles VideoVideos ReviewsBike reviews For saleBikes for sale Yamaha celebrates 30 years of VMAX with carbon version A new VMAX Carbon model has been added to the range to celebrate 30 years since the famous bike... Celebrate the VMAX's 26th birthday by buying a watch! What better way to celebrate the 26th anniversary of the Yamaha VMAX than buying a VMAX Chronograph... Exclusive photo: finished MotoCzysz E1pc revealed, claiming V-MAX-beating acceleration He's teased with dyno videos, but this is the first time American superbike auteur Michael Czysz has... First Yamaha V-maxs hit dealer floors Yamaha dealer Flitwick Motorcycles have taken delivery of the first three V-Maxs in the UK, with... What should we test on Yamaha's new V-Max? You could help MCN with its test of Yamaha's all-new V-Max by telling us what you want to know about... 2009 Yamaha V-Max official details and pictures Yamaha has officially unvelied the 2009 V-Max and MCN can now reveal the official specifications and... New 2009 Yamaha V-Max: Official pictures This is the first official picture ever seen of Yamaha's new 2009 V-Max - and you can see many more... Tokyo Show: Yamaha V-Max The Tokyo Motorcycle Show has opened to the public today, and one of the new models is the Yamaha... Leno's V-Max sells for £75,000! American TV star, Jay Leno, sold his customised V-Max in a Las Vegas charity auction for £75,079... Video: Yamaha V-Max 2-minute test The V-Max is what happens when you mix 1679cc and nearly 200 horsepower in a motorcycle that's one... YAMAHA V-MAX (2008-on) "A thrill ride of epic proportions - the V-Max redefines the word 'torque'" MCN Rating: 4 out of 5 4 owners' ratings:4.3 out of 5 All related reviews YAMAHA V-MAX (2004/54) £7,495 Clwyd YAMAHA V-MAX (1997/R) £4,999 Kent YAMAHA V-MAX (2008/58) £7,999 Surrey YAMAHA V-MAX (1993) £3,000 Lancashire YAMAHA V-MAX £19,995 Merseyside YAMAHA V-MAX £18,745 Lancashire YAMAHA V-MAX (2015) £18,884 Lincolnshire YAMAHA V-MAX (2015) £18,745 Derbyshire YAMAHA V-MAX (1999/T) £4,995 Oxfordshire YAMAHA V-MAX £15,749 Fife All related bikes for sale


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Product Description
Bilstein B6 HD Rear Shock for 1999, 2000, 2001, 2002, 2003 and 2004 Cobra Mustangs.
Upgrade your Cobra with Bilstein 46 B6 HD Series rear shocks for increased performance. This shock features a firm, yet comfortable ride that is expected from a performance car. The heavy duty shock is great for the street and occasional track event. After installing Bilstein struts, your Mustang will gain additional traction and enhanced stability. The new found performance will be accompanied by an exceptionally comfortable street ride. Bilstein tuned the B6 HD shock specifically for the Cobra's independent rear suspension.Bilstein 46mm B6 HD Series shocks improve handling and stability without sacrificing ride comfort. Super damping abilities make Bilsteins ideal for the driver who demands superior performance while maintaining an exceptional street ride. Mono-tube construction ensures greater damping force and better handling with its large effective area of the working piston. Bilstein B6 shocks are for those that expect more damping power from their shock absorbers but don't want to opt for an all-out sports model. Bilstein road and track tests their shocks for fine tuning.
Bilstein entered the motorsports industry over 50 years ago and is still a leader in suspension components. Not only does Bilstein make aftermarket performance shocks, but they also produce OEM shock absorbers. Many makes and models utilize factory Bilstein shocks including certain Ford Mustangs. Bilstein provides performance parts for many types of racing including Fomrula One, rally racing, 24 hours at LeMans, and NASCAR.
Sold individually.
Installation Instructions
Product Questions
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Long sleeve tunic
[No refunds] of the item is shown will be long-term stock and.
Pays close attention to regarding the storage of inventory, while during product registration no dirt or product description different notation rank or if there is no damage. And it agreed upon purchase and sale and consider them, so the return is prohibited. Please note that.
We ask product sent to you, by your risk always payout will be.
Carrier storage time (approximately 7 days from shipping center arrival) will be returned if you do not receive even beyond the product. The damage suffered by the company this will be invoiced.
For more information-Click hereFrom
Long sleeve tunic
Material |
100% cotton |
Color |
White |
Notation size |
38 |
For more sizes |
Length: 81 cm Shoulder width: 56 cm Sleeve length: approx. 50 cm Width: 49 cm Hem width: 50 cm |
For more product information |
It is a bit gray, or white. Seen color lossthough in the back will feel also, so please check for more size, images etc., consider please. |
Shop contact information |
This product is the EC inventory. By ordering only the EC inventory method of payment credit card, cod, shortest next day shipment is possible. EC inventory list here. |
JP Post EMS | Your order will be shipped to you via Japan Post EMS. Japan Post is a speedy delivery service that delivers to more than 120 countries. With an effective tracking system and insurance program, EMS is able to offer a secure delivery worldwide. You can track your package with the tracking code provided and it will be delivered within a week. See estimated shipping fee |
Credit Card | We accept credit cards. The following credit can be used: •Visa •MasterCard •American Express •JCB •others |
PayPal | We accept PayPal. After receiving the order confirmation e-mail, please proceed with the PayPal payment. Note *Please note that if payment through PayPal is not confirmed by the date indicated in the e-mail, your order will be automatically cancelled. |
This product cannot be shipped to United States from the store.
Please contact the store for further information.


text:
Reckless Getaway for Android is very popular and thousands of gamers around the world would be glad to get it without any payments. And we can help you! To download the game for free, we recommend you to select your phone model, and then our system will choose the most suitable apk files. Downloading is very simple: select the desired file and click "download free Reckless Getaway apk", then select one of the ways you want to get the file. Just a few easy steps and you are enjoying full version of the game for tablet or phone!
Game description: Reckless Getaway:
Each level begins with a robbery. Driving a car at a huge speed you should leave from a pursuit with a lot of the stolen money. On your way there will be not only civil cars, but also police cars and the whole protecting cordons. Smash everything on the way. It is possible to receive bonuses for the crippled police cars, for pushed off in an abyss civil — too. You have not only to reach to the finish, but also gain as many points as possible. In the game you are expected by 16 levels, the game is filled with infinite drive, physics is executed perfectly in spite of the fact that it differs from cars behavior in reality. Picture is bright and juicy. Music is vigorous and cheerful.
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Keyword Tag Cloud
Alternative Data Analytic Models Automation Big Data Branch Banking Cost Reduction Credit Card Credit Card Regulations Credit Risk Policy Cross-sell Customer Friction Customer Intimacy Customer Service debit card Decisioning Demand Deposit Account Financial Crisis Fraud Generation Y Innovation Instant Prescreen IT Investment Lifestyle Banking Mobile Banking Online Banking Risk Management Security Transparency Trust UnderbankedBlogs We Love
Topic Archives: Operational Efficiency
Is Your Collections Process Insane?
Topics: Credit Risk Management, Operational Efficiency
Tagged: Collections, Risk Management
Insanity, it's often said, is trying the same thing over and over and expecting a different result. Nowhere is this axiom truer in financial services than in the collections process.
The Right Treatment at the Right Time
The goal in collections is to efficiently deal with delinquent accounts in a way that will minimize your … Continue reading →
Triage: From Battlefields to Banks
Topics: Operational Efficiency, Technology
Tagged: Automation, Loan Origination, Triage
At Zoot, we frequently have discussions with financial institutions (FIs) on the virtues of automation. All of Zoot's solutions are focused on making business processes as automated and data-driven as possible. However, the decision to automate a business process—like the process of originating a loan—is never an easy one. There are a huge number of … Continue reading →
Finding the Right Balance: The Pursuit of a Better Loan Origination System
Topics: Customer Acquisition and Retention, Customer Service, Operational Efficiency
Tagged: Customer Service, Loan Origination System
Bankers are trying to find the right balance for their business, but it can be difficult, especially when working with a legacy loan origination system (LOS). Many think they need to sacrifice efficiency to improve customer service. With a modern LOS they can have both—no sacrifices need to be made in the pursuit of a … Continue reading →
Attributes: The Cornerstone of Sound Credit Policy
Topics: Credit Risk Management, Operational Efficiency, Technology
Tagged: Attributes, Automation, Cost Reduction, Credit Risk Policy
You've heard the term cornerstone before, right? The word comes from the concept of setting the first stone in the construction of a masonry foundation. It is the most important "brick" since it is the reference by which all other bricks will be set for the entire building. Many precautions are taken to ensure that … Continue reading →
From Prussia with Love: Agility as a Competitive Advantage
Topics: Operational Efficiency, Technology, Trends
Tagged: Cost Reduction, Credit Risk Policy, Financial Crisis, Innovation
In 1806, at the Battle of Jena-Auerstedt, the Prussian Army got crushed by Napoleon. Using carefully planned and flexible troop dispositions, Napoleon was able to run circles around the slower Prussians. As a result, the French were able to deal a critical blow to the Kingdom of Prussia leading to its subjugation for the next … Continue reading →
What Products Could My Bank Offer to the Underbanked?
Topics: Account Opening, Credit Risk Management, Customer Acquisition and Retention, Customer Experience, Fraud, Operational Efficiency, Regulations, Technology, Trends
Tagged: Alternative Data, Credit Card, Customer Friction, Generation Y, Underbanked
Perhaps the most perplexing question facing bankers who desire to serve the underbanked consumers is what products would actually meet their needs. Fortunately, research on this topic is already being done and it has been proved that mainstream banks can successfully and profitably offer appealing products to this valuable market. The only significant barrier is … Continue reading →
What are underbanked consumers looking for?
Topics: Account Opening, Credit Risk Management, Customer Acquisition and Retention, Customer Experience, Fraud, Operational Efficiency, Regulations, Technology, Trends
Tagged: Alternative Data, Credit Card, Customer Friction, Generation Y, Underbanked
The following is a synopsis of a presentation from the CFSI Underbanked Financial Services Forum. I wanted to share these insights with you because I believe many financial institutions could benefit from expanding their offering in a similar way.
George Hodges of New Market Partners reported on a study conducted for Regions Bank. A key … Continue reading →
What are Alternative Financial Services?
Topics: Account Opening, Credit Risk Management, Customer Acquisition and Retention, Customer Experience, Fraud, Operational Efficiency, Regulations, Technology, Trends
Tagged: Alternative Data, Credit Card, Customer Friction, Generation Y, Underbanked
Two common themes I've heard from bankers are that there is a lack of innovation in financial services and that banks already provide all of the products consumers need. In this column, I will argue that neither of these claims is true. There are clear needs in the marketplace for products not being delivered by … Continue reading →
Want Loyalty? Start with the Customer
Topics: Customer Experience, Operational Efficiency
Tagged: Credit Card, Customer Friction, Customer Service
My last blog post focused on creating a retail experience that drives customer loyalty beyond rewards. The bottom line is that knowing what is important to your customers and responding to their needs is better than surpasses simply offering a rewards card when it comes to building loyalty. That sounds logical, but there are many … Continue reading →
A Healthier View of Bank Channels
Topics: Customer Experience, Operational Efficiency, Trends
Tagged: Branch Banking, Customer Service, Mobile Banking, Online Banking
The debate over bank channels has reached an interesting place. Depending on whom you ask, branch banking is either a relic of a time gone by or an invaluable component of a successful retail banking strategy. The truth, as usual, is somewhere in between these extremes.
I recently heard a very healthy perspective on the … Continue reading →


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Results: 1Comments by: Nightstrikergp |
File: MikScrollingBattleText04-15-08 |
I've been using Mik's for awhile no...
Posted By: Nightstrikergp
I've been using Mik's for awhile now and have nothing but priase for it. However after re-installing wow and updating my addons again I installed the latest version and unfortunately it seems to be causing a problem.
It works great but the problem is, it's also telling me not only my combat, it's also scrolling all the players an...


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Originally written on
Redleg Nation
Last updated 6/25/14
No sailing, no guitar, no golf? Say it ain't so, Bronson!
Arroyo has probably been my favorite player the last few years, but I don't even like to write about him anymore. That contract extension Walt Jocketty handed him last off-season still makes me sick to my stomach. I'll just keep my fingers crossed that Bronson can rebound somewhat next year, even if I'm not very confident about it.
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{{else}}Success! Thanks for repurchasing.
{{/if}} {{else}} {{#if Expired}}Increase my visibility!
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{{else}} {{/if}} {{/if}}You can now invite anyone to meet at your favorite Starbucks.
Limited-time feature
41 year old woman | Severn, MD, USA | Seeking men 35-48 within 50 miles
Active within 24 hours
I love to explore, try new restaurants, new interesting places. Would be even more fun with someone that enjoys some of the same things
I am looking to meet new people and find someone that I can get to know in my free time. Preferably someone who would enjoy doing things outdoors - hiking or biking and laughing. Ultimately, I would like to meet someone that I have enough in common with that we have things to talk about, but plenty of differences to make the conversation interesting.
Cycling, Golf, Skiing, Walking / Hiking
I exercise 3-4 times per week.
I like Cats, Dogs, Fish
One profile said "I'll tell you later" under pets. I'm thinking boa constrictor AND tarantula and he's possibly a person of interest in at least one missing person case.
Biking, hiking, movies. skydiving. laughing.
The Girl with the Dragon Tattoo, Consumer Reports and National Geographic magazines, just started Black Holes and Baby Universes by Stephan Hawking.
Her | What she is looking for | |
Appearance | ||
Height: | 5'3" (160cms) | 5'6" (167cms) to 6'6" (198cms) |
Body type: | Athletic and toned | Slender, About average, Athletic and toned |
Eyes: | Blue | No preference |
Hair: | Dark blonde | No preference |
Lifestyle | ||
Smoke: | No Way | No Way, Occasionally, Cigar aficionado, Yes, but trying to quit |
Drink: | Social Drinker | Social Drinker, Regularly, Moderately |
Occupation: | Technical / Science / Computers / Engineering | No preference |
Civil Engineer... Construction Management |
Income: | $100,001 to $150,000 | $100,001 to $150,000, $150,001+ |
Relationship: | Never Married | Never Married, Widow / Widower, Divorced |
Have kids: | No | No |
Wants kids: | Not sure | Definitely, Someday, Not sure, Probably not, No |
Background | ||
Ethnicity: | White / Caucasian | White / Caucasian |
Faith: | Christian / Catholic | Agnostic, Atheist, Christian / Catholic, Christian / LDS, Christian / Protestant, Spiritual but not religious, Christian / Other |
Languages: | English | English |
Education: | Bachelors degree | Bachelors degree, Graduate degree, PhD / Post Doctoral |


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Nigel Hawthorne | ... | Himself - Narrator (unknown episodes, 1997) |
Series Writing credits | ||
Richard Overy | (unknown episodes, 1997) |
Series Original Music by | |||
Stanislas Syrewicz | (4 episodes) | ||
Amazing film! Although I have to disagree about the comment appeared here, mistakenly stated the wrong fact that the movie shows that Russia has been obsessed with war, as the great patriotic war was about people defending their home and families, unwillingly drawn into the battle of their lives by the fascists. the movie shows,above everything I think, the borders of human behavior under tremendous pressure, and the brilliancy of of some against the banality of others, often in higher positions, which caused many logically impossible situations, and made the turn of events amazingly chaotic. I enjoyed it very much, along with great editing and script, writing rare videos and excellent soundtrack are a beautiful bonus.
"Gore Vidal's American Presidency" | "Russia" | "The Nazis: A Warning from History" | "Battle of the Atlantic" | "Cold War" |
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Episode guide | Full cast and crew | Company credits |
External reviews | IMDb TV section | IMDb UK section |
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The following cases were adjudicated over two days of District Criminal Court on Dec. 28 and 31 in Bolivia.
Codes: PG, pleaded guilty; PNG/NG, pleaded not guilty, found not guilty; PNG/G, pleaded not guilty, found guilty; BCDC, Brunswick County Detention Center; NCDOC, North Carolina Department of Corrections.
Friday, Dec. 28
Judge Jerry A. Jolly presided over the following cases with prosecutor Quintin McGee and courtroom clerk Jennifer Hearn:
Allen Jackson Goodman, PG misdemeanor larceny.
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