Side-Chain
1 Conformations in Urea-Denatured Ubiquitin and
Protein G from 3J Coupling Constants and
Residual Dipolar Couplings
Navratna Vajpai,† Martin Gentner,† Jie-rong Huang,† Martin Blackledge,‡ and
Stephan Grzesiek*,†
Biozentrum, UniVersity of Basel, Klingelbergstrasse 50/70, 4056 Basel, Switzerland, and
Institut de Biologie Structurale Jean-Pierre Ebel, CEA, CNRS, UJF UMR 5075,
41 Rue Jules Horowitz, Grenoble 38027, France
Received December 7, 2009; E-mail: stephan.grzesiek@unibas.ch
Abstract: Current NMR information on side-chain conformations of unfolded protein states is sparse due
to the poor dispersion particularly of side-chain proton resonances. We present here optimized schemes
for the detection of 3JHRH
, 3JNH
, and 3JC′H
scalar and 1DC
H
residual dipolar couplings (RDCs) in unfolded
proteins. For urea-denatured ubiquitin and protein G, up to six 3J-couplings to 1H
are detected, which
define the 1 angle at very high precision. Interpretation of the 3J couplings by a model of mixed staggered
1 rotamers yields excellent agreement and also provides stereoassignments for 1H
methylene protons.
For all observed amino acids with the exception of leucine, the chemical shift of 1H
3 protons was found
downfield from 1H
2. For most residues, the precision of individual 1 rotamer populations is better than
2%. The experimental 1 rotamer populations are in the vicinity of averages obtained from coil regions in
folded protein structures. However, individual variations from these averages of up to 40% are highly
significant and indicate sequence- and residue-specific interactions. Particularly strong deviations from the
coil average are found for serine and threonine residues, an effect that may be explained by a weakening
of side-chain to backbone hydrogen bonds in the urea-denatured state. The measured 1DC
H
RDCs correlate
well with predicted RDCs that were calculated from a sterically aligned coil model ensemble and the
3J-derived 1 rotamer populations. This agreement supports the coil model as a good first approximation
of the unfolded state. Deviations between measured and predicted values at certain sequence locations
indicate that the description of the local backbone conformations can be improved by incorporation of the
RDC information. The ease of detection of a large number of highly precise side-chain RDCs opens the
possibility for a more rigorous characterization of both side-chain and backbone conformations in unfolded
proteins.
Introduction
A detailed, quantitative description of the unfolded state of
proteins is crucial for understanding protein folding,1 protein
misfolding and aggregation in amyloidogenic diseases such as
Alzheimer’s and Parkinson’s,2 and function of intrinsically
disordered proteins.3,4 Such a description is both experimentally
and theoretically highly challenging, because only a limited
number of measurable parameters are available to describe the
vast space of possible unstructured conformations.
During recent years, the conformations of the backbone of
unfolded proteins have been described in some detail using
paramagnetic relaxation enhancements PREs5,6 and RDCs.7,8
These two parameters report on well-defined ensemble averages
of the long- and short-range backbone geometry and are thus
more amenable to a rigorous quantitative interpretation than
chemical shifts, NOE, or relaxation data. Both PREs and RDCs
have revealed long-range contacts and residual structure in many
denatured proteins showing that such states contain structural
bias that may drive them toward a folded structure. The correct
prediction of such structural propensities of denatured states
from the amino acid sequence may be an important step toward
solving the protein folding problem.
RDCs offer particular advantages for the characterization of
unfolded states, because they do not require additional chemical
labeling and can be detected with ease for a large number of
internuclear vectors; for example, a recent study showed that
up to seven RDCs per peptide unit could be determined for
urea-unfolded ubiquitin.9 For a number of unfolded proteins,
trends of backbone RDCs along the polypeptide sequence could
be reproduced in structural ensembles created according to the
† University of Basel.
‡ Institut de Biologie Structurale Jean-Pierre Ebel.
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Published on Web 02/15/2010
10.1021/ja910331t  2010 American Chemical Society3196 9 J. AM. CHEM. SOC. 2010, 132, 3196–3203

amino-acid-specific phi/psi angle propensities in non-alpha, non-
beta conformations of PDB structures (PDB coil libraries).8,10,11
This indicates that the so-called coil model12,13 is a good, first
approximation of the unfolded state ensemble. In turn, deviations
from the coil model point to residual order within the unfolded
state. Such deviations have revealed highly populated turn
conformations in the natively unfolded Tau protein14 and have
shown that urea binding drives the backbone to more extended
conformations for ubiquitin.9 Additional long-range RDCs
between amide protons have given evidence for a remaining,
significant (10-20%) population of the first
-hairpin (residues
1-18) in (8 M) urea-denatured ubiquitin.15
In contrast to the backbone of unstructured polypeptides,
experimental information on side chains is rather sparse. Such
investigations are severely hampered by the poor dispersion of
side-chain signals resulting from the conformational averaging.
A small number of 3JHRH
couplings have been determined in
shorter unfolded peptides without 13C labeling,16,17 which
correlated with predictions from 1 coil distributions. However,
no stereoassignments of methylene H
2 and H
3 resonances were
obtained. A more advanced study18 determined heteronuclear
3JNCγ and 3JC′Cγ couplings in urea-denatured lysozyme. Assuming
staggered 1 rotamers, estimates for their populations were
derived for approximately 50 amino acids, which also showed
correlations to coil model predictions for most amino acids with
the exception of aromatics. Precision in this analysis was limited
by the lack of precise Karplus coefficients for 3JNCγ and 3JC′Cγ
and the fact that the three populations p-60°,60°,180° (two inde-
pendent parameters because p-60° + p60° + p180° ) 1) were
determined from only two experimental values.
In the present study, we have improved the description of 1
conformations in unfolded proteins by optimized heteronuclear
experiments involving
-protons, which are able to resolve most
methylene H
2 and H
3 pairs. Stereoassignments and 1 angle
information could be obtained for the predominant part of
residues in urea-denatured ubiquitin and protein G from an
extensive set of up to six three-bond scalar couplings (3JNH
2,3,
3JC′H
2,3, and 3JHRH
2,3). A combined analysis of all 3J couplings
according to the staggered conformer model yields individual
populations with a maximal error of 2%. This analysis is
corroborated by independent 1DC
H
2,3 RDC data detected in
strained polyacrylamide gels.19,20 These side-chain RDCs agree
well with theoretical RDCs calculated from the 3J-derived 1
conformer distribution and a coil model ensemble of backbone
conformations generated by the program Flexible-Meccano.11
The obtained 1 conformer populations cluster around coil model
averages, but individual variations in particular for serines and
threonines of up to 40% are significant and indicate sequence-
and residue-specific preferences.
Materials and Methods
Sample Preparation and NMR Spectroscopy. 15N/13C-labeled
human ubiquitin and protein G (GB1 sequence 1MQYKLILNGK
11TLKGETTTEA 21VDAATAEKVF 31KQYANDNGVD 41GEW-
TYDDATK 51TFTVTE) were prepared according to standard
protocols.21 Ubiquitin NMR samples contained 1.0 (0.6) mM 15N/
13C-labeled protein in 10 mM glycine, 8 M urea, pH 2.5, 95/5%
H2O/D2O for measurement under isotropic (anisotropic) conditions.
Protein G samples contained 0.6 mM 15N/13C-labeled protein in
10 mM glycine, 7.4 M urea, pH 2.0, 95/5% H2O/D2O. Residual
alignment of urea-denatured proteins was achieved by introducing
the protein solutions into 7% (w/v) polyacrylamide gels and
horizontal compression (aspect ratio 2.9:1) in NEW-ERA sample
tubes22 yielding maximal |1DNH| RDCs of about 13 Hz for both
proteins.
All NMR experiments were carried out at 298 K on a Bruker
Avance DRX 800 spectrometer equipped with a TCI cryoprobe.
Spectra were processed with NMRPipe23 and evaluated with
NMRView24 and PIPP.25
Assignments. Assignments of urea-denatured ubiquitin (BMRB
entry 4375)26 and protein G27 were transferred to our sample
preparations and extended by a combination of CBCA(CO)NH,28
HNCO,29 HBHA(CO)NH,28 and HNHB30 experiments. To obtain
higher resolution, the constant time 15N acquisition period was
increased in these experiments to about 40 ms. Note that for
CBCA(CO)NH, HNCO, and HBHA(CO)NH, this still achieves a
transfer of about 95% via the 1JNC′ (∼15 Hz) coupling. Almost
complete assignments of all 1HN, 15N, 13C′, 13CR, 13C
, 1HR, and
1H
resonances were obtained from this procedure. Missing
assignments mainly comprise amino acids preceding proline or were
due to signal degeneracy of some geminal protons. The obtained
chemical shifts are close to the published data with the exception
of residues in the vicinity of the mutated T2Q site in protein G.
They also extend the previous data by the stereoassignments of

-methylene protons and the 13C′ chemical shifts (protein G). The
assignments are deposited in the BMRB data bank under accession
numbers 16626 (ubiquitin) and 16627 (protein G).
Determination of Scalar and Residual Dipolar Coupling
Constants. 3J scalar couplings carrying information on the 1 angle
of the denatured proteins were obtained from modified versions of
quantitative 3JNH
-HNHB,30 3JC′H
-HN(CO)HB,31 and 3JHRH
-HAHB-
(CACO)NH32 experiments. 1DC
H
RDCs for 1 angle information
were determined from an HBHA(CO)NH28 experiment, where an
IPAP detection scheme33 was introduced into the mixed constant
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J. AM. CHEM. SOC. 9 VOL. 132, NO. 9, 2010 3197
Side-Chain 1 Conformations in Urea-Denatured Proteins A R T I C L E S