NMR supplement
nature structural biology ? NMR supplement ? july 1998 499
Equilibrium NMR studies of unfolded and
partially folded proteins
H. Jane Dyson and Peter E. Wright
Multidimensional NMR studies of proteins in unfolded and partially folded states give unique insights into their
structures and dynamics and provide new understanding of protein folding and function.
Acknowledgments
A.K.D. thanks the Wellcome Trust and The Queen’s
College, Oxford for support. I.D.C. is also supported
by the Wellcome Trust. The authors acknowledge
P. Handford for critical reading of the manuscript.
The Oxford Centre for Molecular Sciences is funded
by the Biology and Biotechnology Sciences Research
Council, Engineering and Physical Sciences Research
Council and Medical Research Council.
Iain D. Campbell and A. Kristina Downing
are at the Department of Biochemistry,
University of Oxford, South Parks Road,
Oxford OX1 3QU, England and the Oxford
Centre for Molecular Sciences, New
Chemistry Laboratory, University of Oxford,
South Parks Road, Oxford OX1 3QT, England.
Correspondence should be addressed to
I.D.C. email: idc@bioch.ox.ac.uk
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In recent years NMR has developed into
one of the two leading technologies,
together with X-ray crystallography, for
determining the three-dimensional struc-
tures of folded proteins at atomic resolu-
tion. However, NMR is unequaled in its
ability to characterize the structure and
dynamics of unfolded and partially folded
states of proteins. Such non-native pro-
tein states do not adopt unique three-
dimensional structures in solution but
fluctuate rapidly over an ensemble of con-
formations. Structural characterization of
non-native states is of great interest
because of their importance in protein
folding, in the transport of proteins across
membranes, in cellular processes such as
signal transduction, and in the develop-
ment of amyloid diseases (Fig. 1).
Knowledge of the structure of protein
folding intermediates is of central impor-
tance for a detailed understanding of pro-
tein folding mechanisms. Likewise,
characterization of the ensemble of con-
formations sampled by denatured pro-
teins can provide insights into the nature
of the free energy landscape at the very
beginning of the folding process. Finally,
it is now recognized that many proteins
and protein domains are only partially
structured or are unstructured under
physiological conditions and only become
structured upon binding to their biologi-
cal targets. Knowledge of the structural
propensities of these domains is essential
to a proper understanding at the molecu-
lar level of their biological functions and
interactions.
Understanding the fundamental molec-
ular mechanisms by which proteins fold
into the complex structures required for
biological activity remains one of the cen-
tral challenges in structural biology. NMR
has emerged as an especially important
tool for studies of protein folding because
of the unique structural insights it can
provide into many aspects of the folding
process
1
. Applications range from direct
or indirect characterization of kinetic
folding events (reviewed in the accompa-
nying article by Dobson and colleagues
2
)
to structural and dynamic characteriza-
tion of equilibrium folding intermediates,
partly folded states, peptide fragments,
and fully denatured states of proteins. For
most proteins, refolding is very rapid and
any intermediates formed are populated
only transiently and are therefore difficult
to study by direct real-time NMR experi-
ments. An especially powerful method for
obtaining site-specific information on the
structure of folding intermediates is
hydrogen exchange pulse labeling com-
bined with 2D NMR detection
3,4
. A typi-
cal experiment involves rapid dilution of
denatured protein in H
2
O buffer to initi-
ate refolding; the protein is allowed to
refold for a short period (typically mil-
liseconds–seconds) before mixing with a
high pH labeling buffer in D
2
O solution.
Amide protons that become involved in
hydrogen bonded secondary structures
during the refolding period are protected
from exchange, whereas amide protons in
regions of the polypeptide that remain
unfolded are exchanged with deuterium
by the labeling pulse. After quenching of
exchange and completion of folding, 2D
NMR supplement
NMR spectra are acquired to identify the
protected amides and monitor the pro-
gressive stabilization of hydrogen bonded
secondary structure during kinetic refold-
ing. Although its importance should not
be underestimated, the primary limitation
of the pulse labeling method is that it pro-
vides information only on the location of
amide protons that become protected
from exchange during folding; the nature
of the structures that give rise to protec-
tion must be deduced indirectly and ele-
ments of structure that are insufficiently
stable to protect amides from exchange
will go undetected. Fortunately, for some
proteins, partially folded states that corre-
spond closely to kinetic folding interme-
diates can be stabilized at equilibrium,
thereby opening the way to direct NMR
analysis. In addition, direct NMR studies
of fully denatured states provide valuable
insights into the nature of the conforma-
tional ensemble at the starting point of
protein folding, while studies of peptide
fragments reveal the intrinsic conforma-
tional propensities of the polypeptide
chain and identify potential folding initia-
tion sites.
The challenge of assignments
Characterization of unfolded and partial-
ly folded states of proteins by NMR pre-
sents special challenges because the
polypeptide chain in such states is inher-
ently flexible and rapidly interconverts
between multiple conformations.
Consequently, the chemical shift disper-
sion of most resonances is poor and
sequence-specific assignment of reso-
nances is difficult (Fig. 2). Exceptions are
the backbone
15
N and
13
C' (that is, car-
bonyl carbon) resonances, which are
influenced both by residue type and by
the local amino acid sequence and there-
fore remain well-dispersed, even in fully
unfolded states
5,6
. Multi-dimensional
triple resonance NMR experiments
which establish sequential connectivities
through the well-resolved
15
N and
13
C'
resonances provide a robust method for
obtaining unambiguous resonance
assignments
7–9
. The lack of
1
H and
aliphatic
13
C chemical shift dispersion for
unfolded or partially folded proteins
means that it is extremely difficult to
assign unambiguously the NOEs that
could provide key information on sec-
ondary structure and tertiary contacts.
Fortunately, recently developed NMR
experiments help to overcome this prob-
lem by transferring the NOE information
to the relatively well-resolved
15
NH or
13
C' resonances
6
.
One significant advantage in NMR
studies of proteins in highly unfolded
states is that resonances are generally nar-
row due to the rapid fluctuations of the
polypeptide chain. As a result, high quali-
ty 2D and 3D spectra can be obtained at
surprisingly low protein concentrations;
indeed, our own experience is that excel-
lent data can be obtained at concentra-
tions of 0.1 mM or lower. In addition,
sequential assignments can be made using
triple resonance experiments that other-
wise may be unsuitable for a folded
protein of comparable molecular weight.
NMR spectroscopy of partially folded
proteins can be even more challenging in
that resonances are at least as broad as
those of native folded proteins but
with the limited dispersion found in com-
pletely unfolded states; in many cases,
NMR studies are impeded by severe
resonance broadening that results from
conformational fluctuations on a milli-
second–microsecond time scale
8,10
.
Structural characterization
Once resonance assignments have been
completed, detailed information on the
conformational propensities of the
polypeptide chain can be readily derived
from chemical shifts, NOEs or coupling
constants. The patterns and relative inten-
sities of the sequential and medium range
NOEs provide information on the
propensity of the polypeptide to populate
the a and b regions of f,y space or to
form ordered helical structures
11,12
. The
deviations of chemical shifts from random
coil values, especially for
13
Ca and
1
Ha ,
provide a convenient and sensitive probe
of the secondary structural propensities
13
.
Main chain coupling constants also give
insights into the conformational ensemble
populated by an unfolded or partly folded
protein
14
. Careful analysis of NMR data
for unfolded proteins and peptide frag-
ments of proteins has led to a description
of the random coil state as a statistical dis-
tribution of backbone dihedral angles in
f,y space
15
. It is becoming increasingly
clear that many unfolded proteins do not
simply form statistical random coils but
exhibit measurable propensities to popu-
late native-like backbone conformations.
Secondary structure
NMR is particularly useful for determining
secondary structural propensities on a
residue-by-residue basis in unfolded and
partly folded proteins; this is necessary for
an understanding of the local interactions
that are likely to participate in the initiation
of protein folding
1,12
. Information obtained
under non-denaturing or very weakly
denaturing conditions is most relevant
since it more closely relates to the condi-
tions prevailing at the start of a protein
folding reaction. For many proteins, the
unfolded state can only be obtained in
solutions of strong denaturants which will
have a pronounced effect on the popula-
tion of residual structured conformers. The
ensemble of conformations sampled by a
polypeptide can differ significantly
between denaturing and non-denaturing
conditions
16,17
and subtle differences in the
location of residual structure have been
observed for different denaturants
7
.
Because their tendency towards structure
formation is governed by local rather than
long-range interactions, short linear pep-
tide fragments of proteins are an ideal vehi-
Fig. 1 Schematic diagram summarizing the roles of unfolded, partially folded proteins, and mis-
folded proteins in biology.
500 nature structural biology ? NMR supplement ? july 1998
NMR supplement
nature structural biology ? NMR supplement ? july 1998 501
cle for elucidation of the intrinsic propen-
sities of sequences to fold under non-dena-
turing conditions
12
. Studies of peptide
fragments of proteins and of proteins that
are unfolded under non-denaturing or
weakly denaturing conditions show that
the intrinsic conformational propensities
of the polypeptide backbone frequently
reflect the secondary structure found in
the native folded protein
8,9,18–20
. In other
words, the conformations populated by
the unfolded polypeptide are not distrib-
uted randomly over the low energy regions
of f,y space but are biased in a way that
reflects the secondary structural propensi-
ties of the local amino acid sequence. In
addition, turn-like structures are frequent-
ly populated in unfolded states of proteins
and in peptide fragments. The observation
of conformational preferences for forma-
tion of secondary structure or hydropho-
bic clusters in short peptides shows that
local interactions determined by the
amino acid sequence bias the conforma-
tional search toward specific structured
forms, even in the absence of stabilization
by long-range interactions with the
remainder of the protein.
The molten globule
Molten globules are compact states that
contain native-like secondary structure
but which lack the unique side chain
interactions that characterize the tertiary
structure of the native protein.
Equilibrium molten globules are formed
by many proteins under partially denatur-
ing conditions. Unfortunately, the confor-
mational heterogeneity and complex
dynamics of these species frequently result
in extremely broad and featureless NMR
spectra which make direct NMR structur-
al analysis difficult. Nevertheless, numer-
ous NMR experiments have been devised
to provide structural information on
molten globule states, including hydrogen
exchange measurements
21
, magnetization
transfer experiments
10
, and denaturant
titrations to allow residue-specific charac-
terization of the hydrophobic core
22
. The
molten globule state formed by apomyo-
globin at pH 4 is exceptional in the quality
of the NMR spectra that it yields; this
species is stable at relatively high tempera-
ture where there is sufficient internal
motion to give rise to narrow resonances
and permit use of multidimensional NMR
experiments
9
. As a consequence, it has
been possible to make complete backbone
NMR assignments and obtain highly
detailed insights into secondary structure
and backbone dynamics. The apomyoglo-
bin molten globule is of particular interest
and importance because it corresponds
closely to an intermediate formed during
kinetic refolding of the protein
23
. High
quality NMR spectra can often be
obtained from partially folded compact
species formed by denaturation of pro-
teins with alcohols
24
; however, the rele-
vance of such states to protein folding
remains to be established.
Tertiary structure
Characterization of residual tertiary struc-
ture in unfolded and partially folded pro-
teins is extremely challenging given their
intrinsic flexibility. While observation of a
long-range NOE between two protons
definitively indicates that they must be in
close proximity in at least some structures
in the conformational ensemble, determi-
nation of the nature of the folded struc-
ture is difficult unless an extensive
network of NOEs can be observed. Newly
developed methods for assigning NOE
peaks in partly folded states may eventual-
ly provide sufficient data in favorable cases
to allow a detailed description of highly
populated structures
20
. For the 434 repres-
sor, for example, enough NOEs were
observed to permit distance geometry cal-
culations of the three-dimensional struc-
a b c
Fig. 2
1
H-
15
N HSQC spectra of apomyoglobin at three pHs, illustrating the decrease in resonance dispersion in the
1
H dimension as the protein
unfolds. Note that the
15
N dimension remains relatively well-dispersed, an important factor in successful assignment of resonances of unfolded pro-
teins. a, pH 2.0 (acid-unfolded state); b, pH 4.0 (equilibrium molten globule intermediate state); c, pH 6.0 ( folded native apoprotein). (Reproduced
from ref. 9 with permission).
NMR supplement
502 nature structural biology ? NMR supplement ? july 1998
ture of a local hydrophobic cluster
25
.
However, it may often prove to be the case
that backbone or side chain conforma-
tional averaging is so extensive in partially
folded states that observation of long-
range NOEs is difficult, precluding deter-
mination of the folding topology by
conventional NOE-based methods.
The paucity of long-range NOEs in a
denatured fragment of staphylococcal
nuclease (termed D 131D ) led Gillespie and
Shortle to develop an innovative method to
obtain long-range distance constraints by
measuring the enhancement of amide pro-
ton relaxation induced by paramagnetic
nitroxide spin labels
26,27
. Spin labels were
coupled to unique cysteine residues intro-
duced at 14 different sites on the polypep-
tide chain and ~700 long-range distance
constraints were derived from measure-
ments of T
2
relaxation enhancement (that
is, broadening of the resonances of protons
close in space to the spin label). The calcu-
lated ensemble of structures of this dena-
tured state has a global topology that is very
similar to that of the native folded pro-
tein
26,27
(Fig. 3). These results suggest that
the correct folding topology can be estab-
lished in denatured states even in the
absence of cooperative interactions and a
tightly packed hydrophobic core. This spin
labeling approach is highly promising and
should be generally applicable to the eluci-
dation of the folding topologies of other
partially folded proteins.
Dynamics
Unfolded and partially folded proteins are
highly flexible.
15
N spin relaxation mea-
surements can be used to probe the
dynamics of the polypeptide backbone in
these species. Interpretation of
15
N relax-
ation rates and {
1
H}-
15
N heteronuclear
NOEs is not straightforward because the
motions are complex and the common
assumption of isotropic tumbling with a
single correlation time is unlikely to be
valid. Nevertheless, valuable insights into
the backbone motions can be obtained,
using either an extended model-free
analysis or reduced spectral density map-
ping
28,29
. On the basis of the relaxation
measurements reported to date, it is clear
that unfolded states of proteins vary con-
siderably in their dynamical properties. At
one extreme, the backbone fluctuations
show little variation as a function of
sequence
30
while for other proteins there
are clear indications of local interactions
that lead to motional restriction
29,31
. For
molten globule states and other partially
folded species, the molecular motions are
highly heterogeneous and relaxation mea-
Fig. 3 Ca backbone superposition of residues 56–140 of folded staphylococcal nuclease (thick
tube) and five structures calculated for the fragment D 131D (thin line). The three helices are col-
ored red and the three b -strands are shown in yellow, yellow-green, and orange. (Reproduced
from ref. 27 with permission).
Fig. 4 Schematic diagram illustrating the increas-
ing restriction of backbone flexibility as myoglo-
bin folds to increasingly structured and
increasingly compact states, from the acid-unfold-
ed state (U
acid
), to the pH 4.1 molten globule state
(I
MG
), to native apomyoglobin (N
apo
), and finally to
fully folded holomyoglobin (N
holo
). Except for
holomyoglobin, the structures are purely
schematic, shown only to indicate the location of
secondary structure in the various partly folded
states of apomyoglobin, as indicated by NMR
data
9
. The polypeptide fluctuates over an ensem-
ble of conformations in all of these states, and no
single structure suffices to describe its behavior.
The smoothed {
1
H-
15
N} heteronuclear NOE at each
residue is shown, on a color scale from dark blue
(least flexible) to red (most flexible). The F helix
region of apomyoglobin is colored gray, since no
NMR information is available for it
9
. (Adapted
from ref. 9 with permission).
NMR supplement
nature structural biology ? NMR supplement ? july 1998 503
surements can provide key insights into
the structural organization of such states.
For example,
15
N relaxation studies of the
pH 4 molten globule state of apomyoglo-
bin show that backbone motions are
highly restricted within a compact
hydrophobic core formed by packing of
three helices whilst other parts of the chain
remain highly fluctional
9
. Similarly,
nuclear spin relaxation studies of a partial-
ly folded state of ubiquitin formed in 60%
methanol reveal the presence of three
loosely coupled secondary structural ele-
ments with enhanced mobility relative to
the native protein
28
.
Intrinsically unstructured proteins
It is now recognized that many proteins
are intrinsically unstructured under phys-
iological conditions
32
. While this has long
been known for certain polypeptide hor-
mones such as glucagon, there is an
increasing awareness that many eukaryot-
ic proteins or protein domains involved in
signal transduction, transcriptional acti-
vation, nucleic acid recognition or cell
cycle regulation adopt stable folded struc-
tures only upon binding to their molecu-
lar targets. Indeed, many genes in
eukaryotic genomes contain regions of
low sequence complexity that encode bio-
logically functional domains which would
not be expected to fold spontaneously into
ordered structures in the absence of addi-
tional stabilizing interactions. NMR is the
method of choice for characterization of
such domains, many of which probably do
not exist as statistical random coils but
will exhibit intrinsic conformational
propensities that may presage the confor-
mation stabilized upon binding. Recent
examples of functional yet unfolded pro-
tein domains include the anti-sigma factor
FlgM
33
, the SH3 domain of the Drosophila
signal transduction protein Drk
16
, a
fibronectin-binding protein from
Staphylococcus aureus
34
, and the kinase
inducible transactivation domain (KID)
from the transcription factor CREB
35
. In
the latter example, NMR analysis shows
that the phosphorylated KID domain is
intrinsically unstructured but undergoes a
folding transition to form a pair of a -
helices upon binding its target domain
from the CREB binding protein (CBP)
35
.
Future perspectives
There can be little doubt that NMR will
continue to make major contributions to
the understanding of the molecular
mechanisms of protein folding and mis-
folding, provide insights into the subtle
relationship between amino acid
sequence and protein structure, and lead
to new understanding of the behavior
and biological function of intrinsically
unstructured protein domains. Clearly it
is of vital importance to understand the
kinetics of collapse and structure forma-
tion that accompany the folding process,
and NMR has important contributions
to make in this field
2
. However, the
intrinsically long time scale of the NMR
experiment makes real-time kinetic
observations problematic. For many pro-
teins, equilibrium NMR studies can pro-
vide valuable and extensive information
on the conformational propensities of
unfolded or partly folded states, informa-
tion that is directly applicable to an
understanding of the folding process.
Recent studies of apomyoglobin pro-
vide an illustrative example of the funda-
mental insights into protein folding
mechanisms that can potentially be
obtained from equilibrium NMR experi-
ments. By careful manipulation of the
solution conditions, several states of
apomyoglobin that differ in structural
content and degree of compaction can be
stabilized for NMR analysis
9
. In this way,
detailed insights can be obtained, at the
level of individual residues, into the pro-
gressive accumulation of secondary struc-
ture and increasing restriction of
backbone dynamics as the chain collapses
during folding to form more compact
states (Fig. 4). Studies such as this are only
a beginning, and the prospects for obtain-
ing even deeper insights into the folding
topology, hydration, and dynamics of the
compact, partially folded states formed by
apomyoglobin and other proteins are
excellent. Future work in the area will be
aided in no small part by the continued
development of novel NMR methodolo-
gies and improvements in NMR instru-
mentation, especially the anticipated
development of spectrometers operating
at or above 900 MHz which will provide
greater sensitivity and dispersion and, as a
consequence, more detailed insights into
the nature of unfolded and partially fold-
ed states.
Acknowledgments
We thank D. Eliezer, P. Jennings and S. Cavagnero for
assistance with preparation of the figures. This work
was supported by grants from the National Institutes
of Health.
H. Jane Dyson and Peter E. Wright are at
the Department of Molecular Biology and
Skaggs Institute for Chemical Biology, The
Scripps Research Institute, 10550 North
Torrey Pines Road, La Jolla, California
92037, USA.
Correspondence should be addressed to
P.E.W. email: wright@scripps.edu or H.J.D.
email: dyson@scripps.edu
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