Residual Dipolar Couplings in Short Peptides Reveal
Systematic Conformational Preferences of Individual Amino
Acids
Sonja Alexandra Dames,
†
Regula Aregger,
†
Navratna Vajpai,
†
Pau Bernado,
‡
Martin Blackledge,
‡
and Stephan Grzesiek*
,†
Contribution from the Biozentrum, UniVersity of Basel, Switzerland, and
Institut de Biologie Structurale Jean-Pierre Ebel, Grenoble, France
Received May 23, 2006; E-mail: stephan.grzesiek@unibas.ch
Abstract: Residual dipolar couplings (RDCs) observed by NMR in solution under weak alignment conditions
can monitor average net orientations and order parameters of individual bonds. By their simple geometrical
dependence, RDCs bear particular promise for the quantitative characterization of conformations in partially
folded or unfolded proteins. We have systematically investigated the influence of amino acid substitutions
X on the conformation of unfolded model peptides EGAAXAASS as monitored by their
1
H
Ν
-
15
N and
1
H
R
-
13
C
R
RDCs detected at natural abundance of
15
N and
13
C in strained polyacrylamide gels. In total, 14 single
amino acid substitutions were investigated. The RDCs show a specific dependence on the substitution X
that correlates to steric or hydrophobic interactions with adjacent amino acids. In particular, the RDCs for
the glycine and proline substitutions indicate less or more order, respectively, than the other amino acids.
The RDCs for aromatic substitutions tryptophane and tyrosine give evidence of a kink in the peptide
backbone. This effect is also observable for orientation by Pf1 phages and corroborated by variations in
13
C
R
secondary shifts and
3
J
HNHR
scalar couplings in isotropic samples. RDCs for a substitution with the
â-turn sequence KNGE differ from single amino acid substitutions. Terminal effects and next neighbor
effects could be demonstrated by further specific substitutions. The results were compared to statistical
models of unfolded peptide conformations derived from PDB coil subsets, which reproduce overall trends
for
1
H
Ν
-
15
N RDCs for most substitutions, but deviate more strongly for
1
H
R
-
13
C
R
RDCs. The outlined
approach opens the possibility to obtain a systematic experimental characterization of the influence of
individual amino acid/amino acid interactions on orientational preferences in polypeptides.
Introduction
Weak alignment of molecules dissolved in anisotropic liquid
phases
1
has become a powerful tool to directly monitor average
net orientations and order parameters of individual bonds by
residual dipolar couplings (RDCs). RDCs are proportional to
the ensemble average 〈3 cos
2
(Θ) - 1〉/2, where Θ is the angle
between the internuclear vector and the magnetic field in the
laboratory frame. Similar to other applications in physical
chemistry,
2
〈3 cos
2
(Θ) - 1〉/2 can be interpreted as a local order
parameter S of the internuclear vector relative to an external
director. S adopts a value of 1, if there is perfect alignment of
the bond along the magnetic field, -
1
/
2
if there is perfect
alignment perpendicular to the magnetic field, and 0 if for
example all orientations are equally probable or if there is perfect
alignment along the magic angle Θ) 54.7°. By this geometrical
dependence and because usually many different RDCs can be
determined, RDCs bear particular promise for a detailed,
quantitative characterization of conformations in partially folded
or unfolded proteins.
Thus RDCs have revealed residual structure in urea-denatured
forms of staphylococcal nuclease
3
and natively unfolded alpha-
synuclein,
4
R-helix propensities in the unfolded S-peptide,
5
the
acyl-coenzyme A binding protein
6
and myoglobin,
7
and â-turn
propensities above the melting transition of the T4 fibritin foldon
â-hairpin.
8
For shorter peptides, modest RDCs have been
observed and interpreted as a local stiffness of the backbone.
9
RDCs of unfolded polypeptides have been described theoreti-
cally by polymer random flight models
10-12
and more recently
†
University of Basel.
‡
Institut de Biologie Structurale Jean-Pierre Ebel.
(1) Tjandra, N.; Bax, A. Science 1997, 278, 1111-1114.
(2) Doruker, P.; Mattice, W. L. J. Phys. Chem. B 1999, 103, 178-183.
(3) Shortle, D.; Ackerman, M. S. Science 2001, 293, 487-489.
(4) Bertoncini, C. W.; Jung, Y. S.; Fernandez, C. O.; Hoyer, W.; Griesinger,
C.; Jovin, T. M.; Zweckstetter, M. Proc. Natl. Acad. Sci. U.S.A. 2005, 102,
1430-1435.
(5) Alexandrescu, A. T.; Kammerer, R. A. Protein Sci. 2003, 12, 2132-2140.
(6) Fieber, W.; Kristjansdottir, S.; Poulsen, F. M. J. Mol. Biol. 2004, 339,
1191-1199.
(7) Mohana-Borges, R.; Goto, N. K.; Kroon, G. J.; Dyson, H. J.; Wright, P. E.
J. Mol. Biol. 2004, 340, 1131-1142.
(8) Meier, S.; Guthe, S.; Kiefhaber, T.; Grzesiek, S. J. Mol. Biol. 2004, 344,
1051-1069.
(9) Ohnishi, S.; Shortle, D. Proteins 2003, 50, 546-551.
(10) Louhivuori, M.; Paakkonen, K.; Fredriksson, K.; Permi, P.; Lounila, J.;
Annila, A. J. Am. Chem. Soc. 2003, 125, 15647-15650.
Published on Web 09/26/2006
13508
9
J. AM. CHEM. SOC. 2006, 128, 13508-13514 10.1021/ja063606h CCC: $33.50 2006 American Chemical Society

by statistical models derived from coil subsets of the Protein
Data Bank.
13,14
At present, a systematic experimental charac-
terization of the influence of individual amino acids on the RDC-
derived local and global order of polypeptides is lacking.
Here, we have systematically investigated the effect of various
single and multiple amino acid substitutions on the conformation
of short peptides as monitored by
1
H
Ν
-
15
N and
1
H
R
-
13
C
R
RDCs
detected at natural abundance of
15
N and
13
C in strained
polyacrylamide gels.
15,16
The RDCs show specific dependencies
on the amino acid substitutions that for the investigated cases
correlate to steric or hydrophobic interactions with adjacent
amino acids. The determination of RDC-derived order param-
eters in conjunction with a systematic variation of the amino
sequence opens the possibility for a rigorous experimental
characterization of individual amino acid/amino acid interactions
in polypeptides.
Results
The peptides used in this study were all derived from the
sequence EGAAXAASS where X is the amino acid under
investigation. This sequence was based on the rationale of
providing neutral next neighbor alanine residues for X and
making the peptides water-soluble by hydrophilic residues at
their N- and C-terminal ends. In total, 14 amino acids X were
investigated; they comprise G, V, L, I, P as aliphatic; T, N, Q
as polar; K, D, E as charged; and Y, W, H as aromatic residues.
1
D
NH
and
1
D
CAHA
RDCs of amide
1
H-
15
N and
1
H
R
-
13
C
R
internuclear vectors were determined as the difference of the
respective doublet splittings in nondecoupled
1
H-
15
N and
1
H-
13
C HSQCs of anisotropic and isotropic samples. Figure 1 shows
typical examples for the peptides X ) I, G, W. Despite a very
similar overall alignment (see below), the RDCs of the three
peptides vary significantly in the vicinity of amino acid X, e.g.,
1
D
CAHA
equals 16 Hz for I5 but -11 Hz for W5.
Sequential RDCs. Sequential
1
D
NH
and
1
D
CAHA
RDCs for
the 14 investigated peptides EGAAXAASS are shown in Figure
2. It is evident that the aliphatic side chains of the amino acids
I, V, and L at position X5 (Figure 2A) all lead to very similar
RDC profiles and thus indicate similar average orientations of
the
1
H
R
-
13
C
R
and
1
H
Ν
-
15
N internuclear vectors. The absolute
values of
1
D
NH
show a bell-shaped, almost 2-fold increase in
the center of the peptide (A4, X5, A6). This increase is
equivalent to an increase in the average 〈3 cos
2
(Θ) - 1〉/2 for
the respective N-H bond vectors, which may be caused by a
stiffening and/or a kink of the backbone due to the larger side
chain at position X5. Toward both peptide termini the
1
D
NH
RDCs decrease. However, the penultimate residues G2 and S8
show increased
1
D
NH
RDCs, which are analyzed in more detail
below. The increase for these residues deviates from the
expected profile for a random chain polymer,
11
where terminal
fraying causes a continuous bell-shaped decrease of RDCs from
the peptide center toward the termini.
In comparison to the
1
D
NH
RDCs, the variation of
1
D
CAHA
RDCs along the peptide chain is less pronounced. A weak (about
20%) increase in
1
D
CAHA
is also observed at position X5 for
the case of X ) I and L, but it is undetectable for X ) V.
It is remarkable that in all investigated cases the two G2
1
H
R
nuclei showed distinguishable resonances and strongly differing
1
D
CAHA
values. This indicates that the averages of the two
1
H
R
-
(11) Louhivuori, M.; Fredriksson, K.; Paakkonen, K.; Permi, P.; Annila, A. J.
Biomol. NMR 2004, 29, 517-524.
(12) Fredriksson, K.; Louhivuori, M.; Permi, P.; Annila, A. J. Am. Chem. Soc.
2004, 126, 12646-12650.
(13) Jha, A. K.; Colubri, A.; Freed, K. F.; Sosnick, T. R. Proc. Natl. Acad. Sci.
U.S.A. 2005, 102, 13099-13104.
(14) Bernado, P.; Blanchard, L.; Timmins, P.; Marion, D.; Ruigrok, R. W.;
Blackledge, M. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 17002-17007.
Epub 12005 Nov 17011.
(15) Tycko, R.; Blanco, F. J.; Ishii, Y. J. Am. Chem. Soc. 2000, 122, 9340-
9341.
(16) Sass, H. J.; Musco, G.; Stahl, S. J.; Wingfield, P. T.; Grzesiek, S. J. Biomol.
NMR 2000, 18, 303-309.
Figure 1. Detection of amino acid specific order in peptides EGAAXAASS
from RDCs.
1
D
NH
and
1
D
CAHA
RDCs are obtained from the difference in
doublet splittings of nondecoupled natural abundance
1
H-
15
N (left) and
1
H-
13
C HSQCs (right) of peptides EGAAXAASS (X ) I, G, W) under
isotropic (black) and anisotropic (red) conditions in mechanically strained
polyacrylamide gels.
1
H(
13
C) decoupling was omitted during the
15
N(
1
H
R
)
evolution period.
Figure 2. Sequential
1
D
NH
(left) and
1
D
CAHA
(right) RDCs in oriented
peptides EGAAXAASS. Experimental RDCs are given for aliphatic (A),
hydrophilic (B), charged (C), G and P (D), and aromatic (E) substitutions
of the residue X. The specific amino acid substitutions are marked. For
comparison, the behavior of the X ) I substitution is shown in all panels.
For G2, the separately observable
1
D
CAHA
RDCs of both
1
H
R
protons are
shown. For the X ) G substitution (D) only one
1
H
R
resonance is observable.
The corresponding average
1
D
CAHA
is shown (see text). Error bars indicate
statistical errors from repeated experiments.
Residual Dipolar Couplings in Short Peptides ARTICLES
J. AM. CHEM. SOC. 9 VOL. 128, NO. 41, 2006 13509