Controlling peptide folding with repulsive interactions between phosphorylated amino acids and tryptophan

Article abstract of DOI:10.1021/ja9047575

Phosphorylated amino acids were incorporated into a designed β-hairpin peptide to study the effect on β-hairpin structure when the phosphate group is positioned to interact with a tryptophan residue on the neighboring strand. The three commonly phosphoryl

Full text of DOI:10.1021/ja9047575

Published on Web 09/10/2009
Controlling Peptide Folding with Repulsive Interactions
between Phosphorylated Amino Acids and Tryptophan
Alexander J. Riemen and Marcey L. Waters*
Department of Chemistry, CB 3290, UniVersity of North Carolina,
Chapel Hill, North Carolina 27599
Received June 10, 2009; E-mail: mlwaters@email.unc.edu
Abstract: Phosphorylated amino acids were incorporated into a designed ꢀ-hairpin peptide to study the
effect on ꢀ-hairpin structure when the phosphate group is positioned to interact with a tryptophan residue
on the neighboring strand. The three commonly phosphorylated residues in biological systems, serine,
threonine, and tyrosine, were studied in the same ꢀ-hairpin system. It was found that phosporylation
destabilizes the hairpin structure by approximately 1.0 kcal/mol, regardless of the type of phosphorylated
residue. In contrast, destabilization due to glutamic acid was about 0.3 kcal/mol. Double mutant cycles
and pH studies are consistent with a repulsive interaction as the source of destabilization. These findings
demonstrate a novel mechanism by which phosphorylation may influence protein structure and function.
tyrosine.6 In another native protein model, it has also been
shown that phosphorylation of serine and threonine can have a
Introduction
Phosphorylation of proteins is ubiquitious in cellular processes
as a regulatory control. It is estimated that about one-third of
all human proteins are phosphylated.1 Phosphorylation is a part
of intracellular signal transduction pathways that modulate
cellular proliferation, macromolecule production, and gene
expression.1 Abnormal phosphorylation can be the cause or the
result of many diseases, including cancer and Alzheimer’s
disease.1 Yet, how phosphorylation alters a protein’s function
is still poorly understood and the effect appears to differ from
protein to protein.2 Currently, the most effective method for
studying how phosphorylation effects protein structure and
function is by comparison of crystal structures of phosphorylated
and unphosphorylated proteins.2,3 However, not all proteins
crystallize easily, such as membrane and instrincally disordered
proteins, and a large amount of homogeneous modified protein
is also required for crystallography, thus giving an incomplete
picture of how phosphorylation affects the structure and function
of proteins. The study of phosphorylated residues on structure
in smaller model systems is warranted to obtain a clearer
understanding of how this post-translational modification can
affect structure.
destabilizing affect within an R-helix system, particularly with
phosphothreonine.7,8 The DeGrado laboratory has used phos-
phorylation as a molecular switch to promote the self-assembly
of de noVo designed helical bundles.9 Work from the Zondlo
laboratory has elucidated that the proline-rich regions of the
naturally disordered tau peptide, when phosphorylated, adopts
a polyproline II helix structure.10 Suau and co-workers observed
a structural transition in the intrinsically disordered carboxy-
terminal domain from histone H1 linker protein, where R-helical
character was decreased and ꢀ-sheet character increased de-
pending on the extent of phosphorylation.11 Excised peptides
from phophosphorylated proteins also exhibit conformational
changes upon phosphorylation of various loop structures.12-14
It is becoming apparent that phosphorylation is a versatile post-
translational modification that can induce a wide variety of
structural changes.
Stabilization or destabilizaion via charge-charge interactions
with a phosphorylated residue are to be expected. Phopshoryl-
ated residues can also induce structural changes via metal
binding.15-18 However, to our knowledge, no one has investi-
Recently, a body of information on how phosphorylation
affects local protein structure has emerged from studies in model
peptide systems. Doig and co-workers have shown that phos-
phoserine strongly stabilizes R-helical peptides when positioned
near the N-terminus or when positioned to make a favorable
salt bridge within the helix.4,5 Fujitani and co-worker observed
a stabilization of R-helix structure from a peptide fragment
excised from H+/K+ ATPase through phosphorylation of
(6) Fujitani, N.; Kanagawa, M.; Aizawa, T.; Ohkubo, T.; Kaya, S.;
Demura, M.; Kawano, K.; Nishimura, S.; Taniguchi, K.; Nitta, K.
Biochem. Biophys. Res. Commun. 2003, 300, 223–229.
(7) Szilak, L.; Moitra, J.; Krylov, D.; Vinson, C. Nat. Struct. Biol. 1997,
4, 112–4.
(8) Szilak, L.; Moitra, J.; Vinson, C. Protein Sci. 1997, 6, 1273–83.
(9) Signarvic, R. S.; DeGrado, W. F. J. Mol. Biol. 2003, 334, 1–12.
(10) Bielska, A. A.; Zondlo, N. J. Biochemistry 2006, 45, 5527–5537.
(11) Roque, A.; Ponte, I.; Arrondo, J. L. R.; Suau, P. Nucleic Acids Res.
2008, 36, 4719–4726.
(12) Megy, S.; Bertho, G.; Gharbi-Benarous, J.; Baleux, F.; Benarous, R.;
Girault, J. P. Peptides 2005, 26, 227–241.
(1) Cohen, P. Eur. J. Biochem. 2001, 268, 5001–5010.
(2) Johnson, L. N.; Lewis, R. J. Chem. ReV. 2001, 101, 2209–2242.
(3) Krupa, A.; Preethl, G.; Srinivasan, N. J. Mol. Biol. 2004, 339, 1025–
1039.
(13) Tokmakov, A. A.; Sato, K. I.; Fukami, Y. Biochem. Biophys. Res.
Commun. 1997, 236, 243–7.
(14) Pons, J.; Evrard-Todeschi, N.; Bertho, G.; Gharbi-BenaroUs, J.;
Benarous, R.; Girault, J. P. Peptides 2007, 28, 2253–2267.
(15) Balakrishnan, S.; Zondlo, N. J. J. Am. Chem. Soc. 2006, 128, 5590–
5591.
(4) Andrew, C. D.; Warwicker, J.; Jones, G. R.; Doig, A. J. Biochemistry
2002, 41, 1897–1905.
(5) Errington, N.; Doig, A. J. Biochemistry 2005, 44, 7553–8.
9
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Riemen and Waters
face of the peptide displaying side chains from the non-hydrogen
bonded residues in a two-stranded ꢀ-sheet.24 On the NHB face,
the side chains of residues are oriented closer to cross-strand
residue side chains, thus giving a larger contribution to hairpin
stability through side chain-side chain interactions than those
residues on the hydrogen bonded (HB) face.24 Serine was
replaced with threonine and tyrosine at position 2 and their
phosphorylated analogs were used to determine the significance
of the phosphorylated residue (Figure 1b). Unfolded control
peptides 1-9 were synthesized in which each peptide consisted
of either residues 1-7, composing of the N-terminal arm and
turn of the ꢀ-hairpin, or residues 6-12, consisting of the
C-terminal arm and turn (Figure 1c). Cyclic peptides 10-14
were synthesized as fully folded controls for each of the
ꢀ-hairpins. Cyclization was achieved by a disulfide bond
between cysteine residues at the N- and C-termini of the peptides
(Figure 1d).
SW-1 Peptide Structural Studies. NMR spectroscopy was
used to determine to what extent the SW-1 peptide and the
phosphoserine-containing peptide pSW-1 fold into ꢀ-hairpin
structures. Downfield shifting of g0.1 ppm of the R-protons
(HR) along the peptide backbone relative to unfolded values
indicates ꢀ-hairpin structure.25 SW-1 was found to have a
majority of HR shifts above 0.1 ppm compared to the unstruc-
tured controls, except for the two terminal residues, which are
typically frayed in ꢀ-hairpins, and Asn6, which is located within
the turn of the hairpin (Figure 2a). This Asn is typically upfield
shifted from the unstructured control value due to its conforma-
tion in the turn. Downfield shifting of backbone amide
hydrogens in HB positions relative to random coil values also
indicates ꢀ-sheet structure, which is seen for Val3, Val5, and
Ile10 (Figure 2b). NHB residues Thr4, Thr9, and Trp11 are also
significantly downfield shifted. NOESY data further confirmed
that this peptide forms the predicted ꢀ-hairpin structure (see
Supporting Information).
Figure 1. (a) Schematic diagram of the designed ꢀ-hairpin peptides.
Interstrand hydrogen bonding and relative orientation of the side chains
are indicated. (b) Structure of the phosphorylated amino acids. (c) Sequences
of unstructured control peptides. (d) Sequences of the cyclic control peptides
for the fully folded state. The underline residues form a disulfide bond.
gated whether phosphorylation can result in a repulsive interac-
tion with a hydrophobic or aromatic group that could affect
peptide structure. Herein we present the destabilizing effect of
phosphorylated amino acids due to repulsive interactions with
tryptophan (Trp) within a designed ꢀ-hairpin peptide. These
studies help to further delineate the range of structural changes
that may occur due to phosphorylation.
The pSW-1 peptide was synthesized with a phosphoserine
replacing the serine in SW-1 and characterized by NMR
spectroscopy to study the effect of phosphoserine on the
ꢀ-hairpin structure. The HR shifts of pSW-1 are not as
downfield shifted as SW-1 peptide, many of which are <0.1
ppm, indicating that incorporation of a phosphoserine causes a
destabilization of the ꢀ-hairpin structure in this sequence (Figure
2a). A decrease in amide backbone shifts is also seen at the
hydrogen-bonded positions when compared to SW-1 (Figure
2b). NOESY data of pSW-1 show no long distance NOEs,
indicating little to no ꢀ-hairpin structure. The lack of long
distance NOE’s is indicative of a highly dynamic peptide, which
may still sample a ꢀ-hairpin conformation but spends most of
its time in an unstructured state.
Results and Discussion
System Design. A set of 12-residue peptides designed to
autonomously fold into ꢀ-hairpins in aqueous solution was used
to investigate the effect of phosphorylation on a side chain-side
chain interaction with a cross-strand Trp residue on structure.
Features that influence folding include the turn sequence, the
ꢀ-sheet propensity of the strand residues, and the side chain-side
chain interactions.19 All of the ꢀ-hairpins peptides contain the
sequence VNGK to promote a favorable type I′ turn, which has
been shown to promote ꢀ-hairpin structure.20-23 The SW-1
peptide system was designed to study the effect of phosphoryl-
ation on a modestly folded ꢀ-hairpin sequence containing a
serine residue in position 2 that is directly cross-strand from a
tryptophan in position 11 on the non-hydrogen-bonded (NHB)
face of the hairpin (Figure 1a). The NHB face is defined as the
Circular dichroism (CD) experiments were also performed
to confirm that there is a loss of structure with incorporation of
phosphoserine (Figure 2c). Since the SW-1 peptide is only
modestly folded, there is a large negative signal at 197 nm
typical of random coil, but there is also a shoulder at 215 nm
that indicates ꢀ-sheet structure. For pSW-1, this shoulder at
215 nm is not observed and a larger random coil signal at 197
nm is present, which is consistent with the NMR data,
reinforcing that pSW-1 has little defined structure.
(16) Settimo, L.; Donnini, S.; Juffer, A. H.; Woody, R. W.; Marin, O.
Biopolymers 2007, 88, 373–385.
(17) Liu, L. L.; Franz, K. J. J. Biol. Inorg. Chem. 2007, 12, 234–47.
(18) Huq, N. L.; Cross, K. J.; Reynolds, E. C. J. Pept. Sci. 2003, 9, 386–
392.
(19) Searle, M. S. J. Chem. Soc.-Perkin Trans 2 2001, 1011–1020.
(20) Hughes, R. M.; Waters, M. L. J. Am. Chem. Soc. 2005, 127, 6518–9.
(21) Blanco, F. J.; Jimenez, M. A.; Herranz, J.; Rico, M.; Santoro, J.; Nieto,
J. L. J. Am. Chem. Soc. 1993, 115, 5887–5888.
(24) Syud, F. A.; Stanger, H. E.; Gellman, S. H. J. Am. Chem. Soc. 2001,
(22) RamirezAlvarado, M.; Blanco, F. J.; Serrano, L. Nat. Struct. Biol. 1996,
3, 604–612.
123, 8667–8677.
(25) Espinosa, J. F.; Munoz, V.; Gellman, S. H. J. Mol. Biol. 2001, 306,
(23) Sharman, G. J.; Searle, M. S. Chem. Commun. 1997, 1955–1956.
397–402.
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A R T I C L E S
Figure 2. (a) HR chemical shift differences: SW-1 (blue bars, X ) serine) and pSW-1 (red bars, X ) phosphoserine) from random coil peptides in pD 7.0
buffer. The Gly bars reflect the HR separation in the hairpin. (b) Backbone amide chemical shifts of SW-1 and pSW-1. (c) Circular dichroism spectra
comparison of SW-1 (blue) and pSW-1 (red) at 25 °C in 10 mM sodium phosphate pH 7.0 buffer.
Table 1. Fraction Folded and ∆G of Folding for ꢀ-Hairpin
Peptidesa
fraction folded
(Gly splitting)b
fraction folded
(HR)c
∆G folding
(kcal/mol)
∆∆G
(pXW-1 - XW-1)
peptide
SW-1
0.41((0.01) 0.40((0.04)
0.23((0.05)
1.53((0.05)
-0.03((0.09)
0.28((0.09)
0.2((0.1)
pSW-1 0.10((0.01) 0.07((0.02)
1.3
0.31
0.9
1.0
QW-1
EW-1
TW-1
0.49((0.01) 0.50((0.1)
0.39((0.01) 0.3((0.2)
0.43((0.01) 0.40((0.06)
pTW-1 0.21((0.01) 0.12((0.06)
YW-1
pYW-1 0.62((0.02) 0.5((0.3)
1.1((0.2)
0.88((0.02) 0.82((0.07) -0.90((0.04)
0.1((0.2)
a Values calculated from data obtained at 25 °C, 50 mM potassium
phosphate-d2, pD 7.0 (uncorrected), referenced to DSS. b Error
determined by chemical shift accuracy on NMR spectrometer. c Average
of the HR values from Val3, Val5, Orn8, and Ile10. The standard
deviation is in parentheses.
Figure 3. pH study of fraction folded of SW-1 (blue) and pSW-1 (red).
Fraction folded was determined by NMR glycine splitting in buffers with
the pD indicated at 20 °C.
The extent of folding to a ꢀ-hairpin by SW-1 and pSW-1
peptides was quantified using two methods. The first method
utilizes the extent of HR downfield shifting relative to random
coil controls and fully folded control as previously described
(see Experimental Procedures). The second method utilizes the
extent of the diastereotopic glycine HR splitting located in the
turn of the hairpin relative to glycine HR splitting observed in
the fully folded control (see Experimental Procedures). SW-1
was found to be 40% folded and pSW-1 only 10% folded using
both methods (Table 1). The extent of destabilization due to
phosphoserine was calculated to be approximately 1.3 kcal/mol.
The destabilization that occurs when the phosphoserine is
incorporated in this hairpin is believed to result from an
unfavorable interaction between the phosphate group and the
tryptophan indole ring directly across from it. This destabiliza-
tion may be caused by repulsion of the negatively charged
phosphate and the electron rich indole ring of cross-strand
tryptophan, or through steric clash between the large phosphate
group and the tryptophan, or a combination of both.
pH Studies. To determine whether the destabilization was
caused by electrostatic repulsion of the phosphate group with
the electron-rich indole ring of tryptophan or a steric clash of
the phosphate group with the tryptophan, a pH study was
performed on SW-1 and pSW-1. By varying the pH and thus
the charge on the phosphate group, a change in fraction folded
will indicate whether there is an electronic component to the
destabilization. A comparison of the fraction folded verses pH
for both SW-1 and pSW-1 is given in Figure 3. It was observed
that, as the pH increases, the SW-1 fraction folded is constant,
while the pSW-1 fraction folded decreases. Interestingly, the
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A R T I C L E S
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destabilization of ꢀ-hairpin. Indeed, it is interesting to note that
phosphorylation has no impact whatsoever on folding of the
SV-1/pSV-1 peptides, which provides further support that the
nature of the destabilization is primarily electronic rather than
steric.
EW-1 and QW-1 Peptide Studies. To further investigate the
electrostatic destabilization of hairpin, the peptide EW-1 was
studied with a glutamic acid at position 2 replacing the
phosphoserine. The peptide QW-1 was used as a neutral analog
for direct comparison to EW-1. NMR characterization showed
that QW-1 is in fact more stable than EW-1 at pH 7: EW-1 is
24% folded while QW-1 is 50% folded (Figure 5a, Table 1).
Comparison of the CD spectra of EW-1 and QW-1 in pH 7
phosphate buffer also indicates that QW-1 is more folded than
EW-1 (Figure 5b). This again shows that placing a negatively
charged species cross-strand from tryptophan in this hairpin
system is destabilizing. A pH study demonstrates that EW-1
exhibits pH dependence similar to pSW-1 (Figure 6). At lower
pH, EW-1 becomes more folded, just as with pSW-1. In
contrast, QW-1 is independent of pH. Interestingly, when
protonated, glutamic acid is more stabilizing than glutamine.
The same was not true for pSW-1 and SW-1, which may
suggest a greater role for steric repulsion in the case of pSW-1
than EW-1.
Figure 4. Double mutant cycle diagram for interaction between cross-
strand phosphoserine and tryptophan. The cross-strand interaction between
phosphoserine and tryptophan is determined by subtracting the stability of
B and C from that of A and D.
Table 2. Double Mutant Cycle Data for pSW-1 and Mutants at pH 4a
peptide
fraction foldedb
∆Gf (kcal/mol)
∆∆G(pXW) (kcal/mol)
A
B
C
D
pSW-1
SW-1
pSV-1
SV-1
0.23((0.01)
0.41((0.01)
0.22((0.02)
0.22((0.02)
0.74((0.05)
0.21((0.05)
0.75((0.05)
0.75((0.05)
0.53
TW-1 Peptide Studies. To determine if phosphothreonine,
another commonly phosphorylated amino acid, has the same
effect as phophoserine, the TW-1 peptide system was designed
with the same ꢀ-hairpin scaffold as SW-1, but with threonine
positioned directly cross-strand from tryptophan, as in the SW-1
peptide (Figure 1). NMR analysis of the HR chemical shifts of
TW-1 and pTW-1 indicate that destabilization occurs when Thr
is phosphorylated (Figure 7a) to a similar extent as was observed
for SW-1 and pSW-1. TW-1 is about 40% folded, while pTW-1
is only 12% folded, giving a destabilization of approximately
0.9 kcal/mol (Table 1). The CD spectra of TW-1 and pTW-1
corroborate the NMR data, indicating that pTW-1 is less stable
than TW-1, with TW-1 having a more pronounced shoulder at
215 nm than pTW-1, and pTW-1 having a larger minima at
197 nm (Figure 7b).
a Conditions: 20 °C, 50 mM sodium acetate-d4, pD 4.0. b ∆G of
folding was calculated from the fraction folded using eq 3. Error
determined by chemical shift accuracy on the NMR spectrometer.
fraction folded at pH 2.5 and 4.7 is unchanged for pSW-1, which
correlates to a -1 charge on phosphate, while at pH 7.4 the
fraction folded is lower and the phosphate group now has a -2
charge, and a higher fraction folded is observed at pH 1.2, where
the phosphate group is uncharged. As the charge state of the
phosphoserine increases, the degree of folding decreases,
indicating that the charge of the phosphate is a predominant
contributor to the destabilization of the ꢀ-hairpin. However,
sterics appear to play some role in the destabilization, as pSW-1
is less folded than SW-1 even in a neutral charge state of the
phosphoserine.
Double Mutant Studies. To determine whether the phospho-
serine-tryptophan interaction is the major destabilizing interac-
tion, a double mutant cycle was performed (Figure 4).25 In the
double mutant cycle, both of the interacting residues are mutated
individually and together. The single mutants, B and C, disrupt
the side chain-side chain interaction in A but may result in
other changes that affect the stability of the ꢀ-hairpin, such as
the ꢀ-sheet propensity. The double mutant, D, corrects for all
unintended changes that affect the ꢀ-hairpin stability. Thus, the
sum of the stabilities of peptides A and D minus those of the
single mutants, B and C, provides the side chain-side chain
interaction of phosphoserine and tryptophan. The peptides SW-1
and pSV-1 were used as individual mutants and SV-1 was
synthesized as the double mutant. pSV-1 contains a phospho-
serine at position 2 and a valine at position 11 to replace the
tryptophan, and SV-1 contains a serine at position 2 and a valine
at position 11. The fraction folded and ∆G of folding for all
the peptides in the double mutant cycle are given in Table 2.
These studies were preformed in a pD 4 buffer solution due to
low solubility of SV-1 and pSV-1 at pD 7. The interaction of
the phosphoserine-tryptophan was calculated to be a destabi-
lization of 0.5 kcal/mol, which is the same as the overall
destabilization between SW-1 and pSW-1 at pH 4, therefore
indicating that this interaction is the direct cause of the
YW-1 Peptide Studies. The effect of tyrosine phosphorylation
within the ꢀ-hairpin model system was also explored. NMR
analysis of YW-1 indicates that incorporation of the tyrosine
cross-strand from the tryptophan stabilizes the hairpin (Figure
8a), which is about 82% folded. The incorporation of phospho-
tyrosine results in a destabilization of 1.0 kcal/mol of the hairpin,
which is about 60% folded (Table 1). CD spectra confirmed
the change in stability between YW-1 and pYW-1, showing a
strong ꢀ-sheet minimum at 215 nm and no random coil minima
at 197 nm for YW-1, while pYW-1 has a smaller minimum at
215 nm than YW-1 and a minimum at 197 nm (Figure 8b).
The larger degree of folding of YW-1 and pYW-1 relative
to their SW-1 and TW-1 analogs is due to a favorable interaction
between tyrosine and the tryptophan. It has been previously
shown that aromatic groups cross-stand in NHB positions of
ꢀ-hairpins have a stabilizing effect via edge-face aromatic
interactions.26,27 NMR characterization of the aromatic region
of YW-1 suggests that Tyr2 interacts with Trp11 in an edge-
-face interaction as well, with the ortho-proton of Tyr2 directed
toward the face of Trp11. The ortho and meta hydrogens on
(26) Tatko, C. D.; Waters, M. L. Protein Sci. 2003, 12, 2443–2452.
(27) Cochran, A. G.; Skelton, N. J.; Starovasnik, M. A. Proc. Natl. Acad.
Sci. U.S.A. 2001, 98, 5578–5583.
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Figure 5. (a) HR chemical shift differences: QW-1 (blue bars) and EW-1 (red bars) form random coil peptides. Values calculated from data obtained at
25 °C, 50 mM potassium phosphate-d2, pD 7.0. The Gly bars reflect the HR separation in the hairpin. (b) Circular dichroism spectra comparison of SW-1
(blue) and pSW-1 (red) at 25 °C in 10 mM sodium phosphate pH 7.0 buffer.
Tyr) when it is cross-strand from Trp. Experimental measure-
ment of an unfavorable interaction can be more difficult than
measuring a favorable interaction. The ꢀ-hairpin system,
however, allows for an intramolecular method of measuring an
unfavorable interaction between naturally occurring residues.
A pH dependence was demonstrated, in which protonation of
the acidic residue increases the stability of the hairpin, consistent
with a repulsive electrostatic interaction. A similar pH-dependent
destabilization of the folded state was found for Glu across from
Trp, although the magnitude of destabilization was less.
Substituting different phosphorylated residues (serine, threonine,
tyrosine) in the ꢀ-hairpin peptide sequence has a similar
destabilizing effect of 0.9-1.3 kcal/mol on the ꢀ-hairpin
structure at pH 7, in which the phosphate is a dianion. This is
interesting because tyrosine analog YW-1 is considerably more
stable than TW-1 and SW-1, yet the amount of destabilization
caused by incorporation of phosphorylated residue is roughly
the same. In contrast, the magnitude of the repulsive Glu-Trp
interaction is 0.3 kcal/mol, which is similar to the magnitude
of the phosphoSer-Trp interaction at pH 4, in which the
phosphate is a monoanion. It seems that in this hairpin system
the phosphate-aromatic interaction is not significantly depend-
ent on the residue that the phosphate group is attached; the
charge state has a larger influence on the magnitude of
destabilization.
Figure 6. pH study of fraction folded of QW-1 (blue) and EW-1 (red).
Fraction folded was determined by NMR glycine splitting in buffers with
the pH indicated at 20 °C.
the tyrosine are not equivalent, with one ortho proton signifi-
cantly upfield shifted (Figure 9). The observation of two sets
of ortho and meta protons indicates restricted rotation of the
aromatic ring of Tyr. In the less folded pYW-1, the ortho-proton
on Tyr is shifted about half as much as in YW-1. The lesser
extent of aromatic proton shifting is consistent with these
residues interacting less in the destabilized hairpin.
These results are all consistent with a repulsive electrostatic
interaction as the driving force for destabilization of the folded
state that may be described as an unfavorable anion-π
interaction,28 although a repulsive charge-hydrophobic interac-
tion cannot be ruled out at this time. Such unfavorable
interactions in a naturally occurring system have not previously
been investigated. This type of interaction represents a novel
mechanism by which phosphorylation may destabilize a par-
ticular protein structure, change its structure, or prevent a protein
binding event. This effect on structure is significant given the
ubiquitous nature of protein phosphorylation in signal trans-
duction and aberrant phosphorylation in many disease states.
Although Trp is not an extremely common amino acid, the same
type of repulsive interaction can be expected with Phe, albeit
likely weaker, as has been observed in other comparisons of
Since the YW-1 hairpin has a very high stability due to the
favorable contacts of the tyrosine, the overall hairpin stability
may not be directly destabilized by the interaction of the
phosphotyrosine with tryptophan. To address this issue, a double
mutant cycle was performed as described above, where valine
replaces tryptophan in the appropriate mutant peptides. As
mentioned above, the double mutant cycle was performed at
pH 4 due to solubility limitations of the valine-containing
peptides at pH 7. The double mutant study found that the
destabilization due to the interaction between phosphotyrosine
and tryptophan is approximately 0.4 kcal/mol at pH 4 (Table
3). This destabilization is close to the total energy lost upon
phosphorylation of Tyr at pH 4 (i.e., pYW-1 relative to YW-
1), which is 0.6 kcal/mol.
Discussion
The studies described above demonstrate destabilization of
ꢀ-hairpin structure upon phosphorylation of a residue (Ser, Thr,
(28) Schottel, B. L.; Chifotides, H. T.; Dunbar, K. R. Chem. Soc. ReV.
2008, 37, 68–83.
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Riemen and Waters
Figure 7. (a) HR chemical shift differences: TW-1 (blue bars) and pTW-1 (red bars) form random coil peptides. Residue X is either threonine (TW-1) or
phosphothreonine (pTW-1). Values calculated from data obtained at 25 °C, 50 mM potassium phosphate-d2, pD 7.0. The Gly bars reflect the HR separation
in the hairpin. (b) Circular dichroism spectra comparison of TW-1 (blue) and pTW-1 (red) at 25 °C in 10 mM sodium phosphate pH 7.0 buffer.
Figure 8. (a) HR chemical shift differences: YW-1 (blue bars) and pYW-1-1 (red bars) form random coil peptides. Residue X is either tyrosine (YW-1)
or phosphotyrosine (pYW-1). Values calculated from data obtained at 25 °C, 50 mM potassium phosphate-d2, pD 7.0. The Gly bars reflect the HR separation
in the hairpin. (b) Circular dichroism spectra of YW-1 (blue) and pYW-1 (red) at 25 °C in 10 mM sodium phosphate pH 7.0 buffer.
Table 3. Double Mutant Cycle Data for pYW-1 and Mutants at pH 4a
peptide
fraction foldedb
∆Gf (kcal mol-1
)
∆∆G(pXW) (kcal mol-1
)
A
B
C
D
pYW-1
YW-1
PTyrV-1
TyrV-1
0.72((0.01)
0.88((0.01)
0.33((0.01)
0.40((0.01)
-0.56((0.05)
-1.16((0.05)
0.42((0.05)
0.24((0.05)
0.42
a Conditions: 20 °C, 50 mM sodium acetate-d4, pD 4.0 (uncorrected).
b ∆G of folding was calculated from the fraction folded using eq 3.
Error determined by chemical shift accuracy on the NMR spectrometer.
unfolding of ankyrin repeats 1 and 2 of the tumor suppressor
p19INK4d upon phosphorylation31 and induction of folding upon
hyperphosphorylation of tau protein in Alzheimer’s disease, for
example.10
Lawrence and co-workers have used a similar repulsive
interaction to design reporter peptides for protein tyrosine
kinases that produce a fluorescent signal upon phosphorylation
of tyrosine.32 These tyrosine kinase substrates contain a tyrosine
that quenches the fluorophore pyrene in close proximity through
an aromatic interaction. Upon phosphorylation of the tyrosine,
a fluorescent signal is generated due to the disruption of the
Figure 9. Upfield shifting of Tyr protons in YW-1 and pYW-1 relative to
random coil values determined from control peptides 5 and 6.
Trp and Phe interactions in peptide model systems.29,30 Since
polyphosphorylation is common in proteins, one can envision
an additive destabilizing effect of multiple phosphorylations.
This sort of additive effect has been proposed in the partial
(29) Tatko, C. D.; Waters, M. L. J. Am. Chem. Soc. 2004, 126, 2028–
2034.
(31) Low, C.; Homeyer, N.; Weininger, U.; Sticht, H.; Balbach, J. ACS
Chem. Biol. 2009, 4, 53–63.
(30) Laughrey, Z. R.; Kiehna, S. E.; Riemen, A. J.; Waters, M. L. J. Am.
Chem. Soc. 2008, 130, 14625–14633.
(32) Lawrence, D. S.; Wang, Q. ChemBioChem 2007, 8, 373–378.
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14086 J. AM. CHEM. SOC. VOL. 131, NO. 39, 2009

Controlling Peptide Folding
A R T I C L E S
mM NaOAc-d3, 24 mM AcOH-d4, 0.5 mM DSS, or pD 7.0
(uncorrected) with 50 mM KPO4D2, 0.5 mM DSS. Samples were
analyzed on a Varian Inova 600-MHz instrument. One dimensional
spectra were collected by using 32K data points and between 8
and 128 scans using 1.5 s presaturation. Two dimensional total
correlation spectroscopy (TOCSY) and nuclear Overhauser spec-
troscopy (NOESY) experiments were carried out using the pulse
sequences from the Chempack software. Scans in the TOCSY
experiments were taken from 16 to 32 in the first dimension and
from 64 to 128 in the second dimension. Scans in the NOESY
experiments were taken from 32 to 64 in the first dimension and
from 128 to 512 in the second dimension with mixing times of
200-500 ms. All spectra were analyzed using standard window
functions (sinbell and Gaussian with shifting). Presaturation was
used to suppress the water resonance. Assignments were made by
using standard methods as described by Wu¨thrich.33 All experiments
were run at 298.15 K (HR shift) or 293.15K (double mutant and
pH studies).
aromatic interaction, allowing these peptides to act as a
fluorescence reporter of tyrosine kinases. It was hypothesized
that the charged and bulky phosphate in combination with a
less electron rich π system of phosphotyrosine was responsible
for the disruption of the tyrosine-pyrene interaction. These
fluorescence reporter peptides appear to undergo a similar
repulsive electrostatic interaction as is observed in YW-1 and
pYW-1, in which the π-π interaction is disrupted by
phosphorylation.
Conclusion
We have shown that incorporation of phosphorylated residues
cross-strand from tryptophan in a ꢀ-hairpin results in the
destabilization of the hairpin structure. The nature of this
interaction is primarily due to an unfavorable interaction between
of the negatively charged phosphate group and the electron rich
indole ring of tryptophan. The magnitude of the destabilization
is dependent on pH and ranges from 0.4 to ca. 1 kcal/mol. These
findings demonstrate a novel mechanism by which phosphoryl-
ation may influence protein structure and function and have
implications for phosphorylation-dependent signaling. Further
studies are currently being conducted investigating the position-
dependence of the destabilizing phosphate-tryptophan as well
as the effect of multiple phosphorylations in alternative ꢀ-hairpin
systems.
Determination of Fraction Folded. To determine the unfolded
chemical shifts, 7-mers were synthesized as unstructured controls
and cyclic peptides were synthesized for the fully folded state. The
chemical shifts for residues in the strand and one-turn residue were
obtained from each 7-mer peptide. The chemical shifts of the fully
folded state were taken from the cyclic peptides. The fraction folded
on a per residue bases was determined from eq 1.
fraction folded ) [δobs-δ0]/[δ100-δ0]
(1)
Experimental Procedures
where δobs is the observed HR chemical shift, δ100 is the HR
chemical shift of the cyclic peptides, and δ0 is the HR chemical
shift of the unfolded 7-mers. The overall fraction folded for the
entire peptide was obtained by averaging the fraction folded of
resides Val3, Lys8, and Ile10. These residues are in hydrogen-
bonded positions have been shown to be the most reliable in
determining fraction folded.34 The overall fraction fold was also
determined using the extent of HR glycine splitting observed in
the turn residue Gly10 given in eq 2.
Synthesis and Purification of Peptides. Peptides were synthe-
sized by automated solid-phase peptide synthesis on an Applied
Biosystems Pioneer peptide synthesizer using Fmoc-protected amino
acids on a PEG-PAL-PS resin. Fmoc-[N]-protected and Benzyl-
[O]-protected phosphoserine, phosphothreoine, and phosphotyrosine
were purchased from AnaSpec. Activation of amino acids was
performed with HBTU and HOBT in the presence of DIPEA in
DMF. Peptide deprotection was carried out in 2% DBU (1,8
diazabicyclo[5.4.0]undec-7-ene) and 2% piperidine in DMF for
approximately 10 min. Extended cycles (75 min) were used for
each amino acid coupling step. All control peptides where acetylated
at the N-terminus with 5% acetic anhydride and 6% lutidine in
DMF for 30 min. Cleavage of the peptide from the resin was
performed in 95:2.5:2.5 trifluoroacetic acid (TFA):ethanedithiol [or
triisopropylsilane (TIPS)]water for 3 h. Ethanedithiol was used as
a scavenger in for sulfur-containing peptides. TFA was evaporated
and cleavage products were precipitated with cold ether. The peptide
was extracted into water and lyophilized. It was then purified by
reverse-phase HPLC, using a Vydac C-18 semipreparative column
and a gradient of 0 to 100% B over 40 min, where solvent A was
95:5 water:acetonitrile, 0.1% TFA and solvent B was 95:5 aceto-
nitrile:water, 0.1% TFA. After purification, the peptide was
lyophilizedtopowderandidentifiedwithESI-TOFmassspectroscopy.
Cyclization of Peptides. Cyclic control peptides were cyclized
by oxidizing the cysteine residues at the ends of the peptide via
stirring in a 10 mM phosphate buffer (pH 7.5) in 1% DMSO
solution for 9-12 h. The solution was lyophilized to a powder and
purified with HPLC using the method described above.
fraction folded ) [∆δGlyObs]/[∆δGly100
]
(2)
where ∆δGly Obs is the difference in the glycine HR chemical shifts
of the observed, and ∆δGly 100 is the difference in the glycine HR
chemical shifts of the cyclic peptides.
The ∆G of folding at 298 K for the peptides was calculated using
eq 3, where f is the fraction folded.
∆G ) -RT ln(f/(1 - f))
(3)
Acknowledgment. We gratefully acknowledge support from the
National Institutes of Health (GM 071589) and the National Science
Foundation (CHE-0716126).
Supporting Information Available: NMR assignments and
NOEs. This material is available free of charge via the Internet
at http://pubs.acs.org.
JA9047575
CD Spectroscopy. CD spectroscopy was performed on an Aviv
62DS circular dichroism spectrophotometer. Spectra were collected
from 260 to 185 nm at 25 °C, with 1 s scanning.
NMR Spectroscopy. NMR samples were made to a concentra-
tion of 1 mM in D2O buffered to pD 4.0 (uncorrected) with 50
(33) Wu¨thrich, K. NMR of Proteins and Nucleic Acids; Wiley: New York,
1986.
(34) Syud, F. A.; Espinosa, J. F.; Gellman, S. H. J. Am. Chem. Soc. 1999,
121, 11577–11578.
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