Báo cáo khoa học: Characterization of the molten globule state of retinol-binding protein using a molecular dynamics simulation approach ppt

Characterization of the molten globule state of
retinol-binding protein using a molecular dynamics
simulation approach
Emanuele Paci
1
, Lesley H. Greene
2
, Rachel M. Jones
2
and Lorna J. Smith
2
1 Institute of Molecular Biophysics, School of Physics and Astronomy, University of Leeds, UK
2 Department of Chemistry and Oxford Centre for Molecular Sciences, Chemistry Research Laboratory, University of Oxford, UK
The detailed characterization of molten globule states
of proteins continues to be an area of intense research
activity and interest (for reviews see [1,2]). This is in
part because these equilibrium partially folded states
have been found to have many similarities to kinetic
protein folding intermediates. As such, the properties
of these states can therefore give important insights
into the determinants of protein structure and folding
[2]. Molten globule states of proteins are also postula-
ted to be involved in a range of important physiologi-
cal processes, including the insertion of proteins into
membranes, the release of bound ligands and aggrega-
tion [1]. In this latter area, molten globule-like species
are thought in some systems to be precursors of amy-
loid fibril formation [3,4].
Molten globule ensembles are characterized by hav-
ing a pronounced amount of secondary structure, in a
compact state that lacks most of the specific tertiary

interactions coming from tightly packed side chain
groups [1,2]. One of the molten globule states that has
been studied in the most depth is that of a-lactalbumin
[5]. In this case, it has been possible using nuclear
magnetic resonance (NMR) methods to gain a residue
specific picture of the noncooperative unfolding of the
molten globule during denaturation with urea [6–8];
data from these experiments have also been used as
Keywords
lipocalin; molecular dynamics; molten
globule; protein folding; retinol-binding
protein
Correspondence
L. J. Smith, Department of Chemistry,
University of Oxford, Chemistry Research
Laboratory, Mansfield Road, Oxford OX1
3TA, UK
Fax: +44 1865 285002
Tel: +44 1865 275961
E-mail:
E. Paci, Institute of Molecular Biophysics,
School of Physics and Astronomy,
University of Leeds, Leeds LS2 9JT, UK
Tel: +44 113 3433806
E-mail:
(Received 25 May 2005, revised 12 July
2005, accepted 3 August 2005)
doi:10.1111/j.1742-4658.2005.04898.x
Retinol-binding protein transports retinol, and circulates in the plasma as a
macromolecular complex with the protein transthyretin. Under acidic con-

ditions retinol-binding protein undergoes a transition to the molten globule
state, and releases the bound retinol ligand. A biased molecular dynamics
simulation method has been used to generate models for the ensemble of
conformers populated within this molten globule state. Simulation con-
formers, with a radius of gyration at least 1.1 A
˚
greater than that of the
native state, contain on average 37% b-sheet secondary structure. In these
conformers the central regions of the two orthogonal b-sheets that make
up the b-barrel in the native protein are highly persistent. However, there
are sizable fluctuations for residues in the outer regions of the b-sheets,
and large variations in side chain packing even in the protein core. Signifi-
cant conformational changes are seen in the simulation conformers for resi-
dues 85–104 (b-strands E and F and the E-F loop). These changes give an
opening of the retinol-binding site. Comparisons with experimental data
suggest that the unfolding in this region may provide a mechanism by
which the complex of retinol-binding protein and transthyretin dissociates,
and retinol is released at the cell surface.
Abbreviations
ANS, 8-anilino-1-napthalenesulphonate; MD, molecular dynamics; RBP, retinol-binding protein; R
g
, radius of gyration; RMSD, root-mean-
square deviation; TTR, transthyretin.
4826 FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS
restraints in a novel approach to determine the free
energy landscape of this molten globule [9]. In the
majority of the proteins whose molten globule states
have been characterized, both by experimental and the-
oretical methods, predominantly a-helical secondary
structure persists in the molten globule state [10–13].

In contrast, in this paper we concentrate on a protein
which is rich in b-sheet secondary structure in the
native state, and which retains the majority of this
b-sheet secondary structure in the molten globule state,
human serum retinol-binding protein (RBP).
RBP is a member of the lipocalin superfamily [14].
The proteins in this superfamily adopt a similar fold;
an eight-stranded up-and-down b-barrel with a C-ter-
minal helix. However, they have a wide range of func-
tions, and high levels of sequence divergence, with
many members sharing under 20% sequence identity
[14,15]. The lipocalins are widely distributed through-
out the eukaryotic and prokaryotic kingdoms [16,17].
Many of the lipocalins act as transporters for small
nonpolar ligands, such as retinoids, haem, phero-
mones, lipids, prostaglandins and pigments [18]. RBP
transports all-trans-retinol (vitamin A) from its storage
sites in the liver to target tissues [19]. It is postulated
that the local decrease in pH at the surface membrane
of target cells triggers the release of retinol, by a mech-
anism that is dependent on the conversion of RBP to
a molten globule state [20,21]. It is possible that similar
mechanisms may prompt ligand release for other mem-
bers of the lipocalin superfamily. For example, the
release of lipid ligands by human tear lipocalins under
acidic conditions is thought to be associated with a
transition to the molten globule state [22,23].
The molten globule state of RBP, formed under acidic
conditions, has been shown to exhibit the key character-
istics typical of these partially folded states. Stoke’s

radii, from diffusion coefficient measurements, have
demonstrated that the molten globule retains a compact
fold [20]. The mean molecular dimensions of the parti-
ally folded ensemble are only 13% larger than those
of the native state. Far- and near-UV circular dichroism
(CD) spectra show that the protein contains a significant
level of secondary structure, but has a considerable level
of disorder in side chain packing, respectively [20,24,25].
Obtaining detailed information at an atomic level about
the molten globule state using techniques such as NMR
spectroscopy is challenging. Partially folded states such
as molten globules are ensemble of interconverting con-
formers [26]. Slow interconversion between populated
conformers gives rise to broadened NMR resonances,
while averaging of chemical shifts across the populated
ensemble gives a limited chemical shift dispersion [6,27].
Therefore, to develop a detailed model to further our
understanding of the molten globule state of RBP,
in vitro and in vivo experimental studies have been com-
plemented with a molecular dynamics (MD) simulation
study. The results of this are reported here.
It is not possible to explore adequately the conform-
ational space accessible to partially folded proteins,
within the simulation timescale currently accessible to
conventional MD simulations of proteins in explicit
solvent. The exploration of non-native conformations
is therefore usually achieved, either by using very high
temperatures in the simulations, or by introducing a
suitable perturbation in a biased MD simulation, often
using implicit solvent models (for a review of this topic

see, e.g [28,29]). Explicitly modelling the perturbation
induced by a change in the solution pH would not
prompt the transition from the native to the molten
globule state, on a timescale which can be directly
simulated. In this work therefore we have used three
different perturbations in turn. One perturbation forces
an increase in the protein radius of gyration, the sec-
ond perturbation induces the breaking of native con-
tacts in the structure and the third perturbation is
aimed to speed up the exploration of diverse (in terms
of mutual RMSD) conformations. These various per-
turbations are applied using a particularly ‘soft’ time-
dependent bias [30,31], designed to generate low energy
pathways in the conformational space. The large num-
ber of diverse and moderately non-native conforma-
tions generated with this biased molecular dynamics
approach are then used as initial conformations for
unperturbed, room temperature simulations. This
method allowed us to explore local free energy minima
in a broad region of the conformational space close to
the native state. The approach is designed to provide a
qualitative map of the free energy landscape, in a
region of the conformation space compatible with the
experimental knowledge of the molten globule state.
By applying this sampling approach to RBP, we are
able to identify a broad basin of low energy partially
folded conformers that are compatible with the avail-
able experimental data [20,24,25,32–34]. These con-
formers provide a model for the molten globule state
of RBP that allows us to gain insight into the determi-

nants of protein folding and the mechanism of retinol
delivery and release, an important physiological prob-
lem which remains unresolved.
Results and Discussion
Sampling of conformational space
As described in the Experimental procedures section,
a biased MD simulation method has been used to
E. Paci et al. MD simulations of the RBP molten globule state
FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS 4827
generate 160 diverse configurations of RBP. Each of
these was then used as an initial configuration in an
unbiased MD simulation of 1.2-ns length. Figure 1(A)
shows a plot of the heavy atom RMSD (root-mean-
square deviation) from the X-ray structure as a func-
tion of the radius of gyration (R
g
), for structures taken
every 10 ps through the 160 unbiased MD simulations.
These data demonstrate the broadness of the conform-
ational space explored in the study. Analysis of the
range of RMSD values seen for the MD conformers
shows that there are three distinct peaks in the RMSD
distribution (Fig. 1B). These correspond to different
levels of unfolding. To aid the analysis, the conformers
have been divided into three groups on the basis of
their RMSD values. Conformers in group 1 have an
RMSD less than 4 A
˚
and an R
g

less than 16.5 A
˚
.
Group 2 conformers have an RMSD in the range
4–7 A
˚
and an R
g
in the range 16.5–17.7 A
˚
, while
group 3 conformers have an RMSD above 7 A
˚
and an
R
g
greater than 17 A
˚
. The characteristics of the group
1 conformers are very native-like, in keeping with their
low RMSD values (< 4 A
˚
). For example, 81 of the
residues that are in regions of secondary structure in
the native protein have secondary structure popula-
tions greater than 0.8 in the group 1 ensemble of con-
formers. We therefore focus our attention on the
conformers in groups 2 and 3 that display a greater
level of unfolding. The native state structure of RBP
has an R

g
of 15.9 A
˚
. The 13% increase in molecular
dimensions seen experimentally on forming the molten
globule state would correspond to an effective R
g
of
18 A
˚
for the molten globule ensemble. The group 3
conformers, with an R
g
in the range 17–20 A
˚
, show an
appropriate level of expansion on average for the mol-
ten globule state (Fig. 1A). The properties of this
group of conformers have therefore particularly been
compared with experimental data for this state.
Secondary structure persistence
The b-barrel in the native structure of RBP consists of
eight b-strands (A-H) [35]. These are arranged in two
orthogonal b-sheets, with some of the b-strands being
involved in both of the sheets. The first sheet consists
of strands ABCDEF, and the second sheet contains
strands EFGHA (Fig. 2). The native structure also
contains an a-helix (residues 146–158), which packs
onto the b-sheet formed by the second set of strands.
All of the b-strands present in native RBP show a high

level of persistence in the group 2 conformers (Figs 2
and 3). However, in many of the structures some of
these strands are reduced in length, or have irregularit-
ies compared to those in the native state. For example,
for strand F (residues 100–109) the mean b-sheet
populations for the first five residues are only 0.05–
0.15. For strand E (residues 85–92) the mean b-sheet
populations for the terminal residues 85 and 91–92 are
0.05–0.13, while those for residues 86–90 are 0.71–0.84.
The disruption of this b-strand results predominantly
0
5
10
15
20
Å
0
0.1
0.2
0.3
0.4
0.5
normalized probability
RMSD
R
g
B
15 16
17 18 19 20
R

g
(Å)
0
2
4
6
8
10
12
RMSD (Å)
Group2
Group3
Group1
A
Fig. 1. (A) Relationship between the protein radius of gyration and
the RMSD value from the X-ray structure of native RBP, for the
16 000 conformers taken at 10-ps intervals along the unbiased
simulations. The radius of gyration and RMSD values are calculated
using all heavy atoms. The black diamond corresponds to the native
state following the 2 ns equilibration simulation. The definitions of
the three groups of conformers used in the analysis are shown. (B)
The distribution of radius of gyration (filled bars) and RMSD values
(open bars) across the 16 000 simulation conformers.
MD simulations of the RBP molten globule state E. Paci et al.
4828 FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS
from the loss of hydrogen bonds between strands E
and F, although in some structures the hydrogen
bonds between strand D and E are also missing. The
a-helix also shows a high level of persistence in the
group 2 conformers, with a-helical populations in

the range 0.59–0.99 for the residues involved.
In the group 3 conformers the changes are more
pronounced (Figs 2 and 3, and Fig. 1 in the supple-
mentary material). All the b-strands now show reduc-
tions in length compared to those in the native
structure. Even strand H (residues 129–138), one of
the particularly persistent strands, is reduced in length
by three residues in more than half of the structures,
with residues 129 and 130 having b-sheet populations
of less than 0.1. In addition, in group 3 strand E and
the first half of strand F are almost completely lost.
Residues 85–92 and 100–104 show b-sheet populations
of less than 0.3. A significant disorder is seen across
the ensemble of group 3 conformers in the region con-
taining strand E, the first part of strand F and the con-
necting E–F loop (residues 85–104). The changes in
this region correlate with results from crystallographic
studies of bovine RBP. These report a conformational
change in the E–F loop at low pH [34]. In addition,
changes in this region were reported in a simulation of
the apo form of RBP reported previously [36].
In almost all the group 3 conformers, however, a
central region of the b-sheets is preserved. Residues in
the central regions of strands B, C, D, G and H and
part of strand A have b-strand populations greater
than 0.9. A persistent section comprising the central
regions of strands B, C and D together with part of
strand A in b-sheet 1, and the C-terminal region of
strand F with the central parts of strands G and H in
b-sheet 2, have residues with b-strand populations

greater than 0.8 (Figs 2 and 3). Hence in the group 3
conformers the central area of each of the two ortho-
gonal b-sheets, that make up the b-barrel in the native
protein, are retained. This is interesting as the two
parts of the polypeptide chain that form these persist-
ent central regions of the b-sheets have closely similar
amino acid sequences. In particular, the sequence of
RBP contains an internal repeat with residues 36–83
(includes b-strands BCD) and 96–141 (includes
b-strands FGH) having 34% identity [35]. This may
account, at least in part, for the similar behaviour of
these regions in the simulations. The central region
of the a-helix is also very persistent in the group 3
A
B
C
D
E
F
G
H
A
B
C
Fig. 2. (A and B) The X-ray structure of human serum retinol-binding protein [35]. (A) The b-strands are labelled, those in b-sheet 1 are
shown in red and those in b-sheet 2 are shown in cyan. The a-helix is blue and the retinol is magenta. (B) Only the residues that have a sec-
ondary structure persistence greater than 0.8 in the group 3 conformers (Fig. 3) are coloured. The figure was generated using the program
MOLSCRIPT [53]. (C) Backbone trace of representative structures from the three groups of simulation conformers (left, group 1; centre, group
2; right, group 3). In each case the average structure over the cluster centres is shown in red.
E. Paci et al. MD simulations of the RBP molten globule state

FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS 4829
conformers, residues 151–156 having a-helical popula-
tions greater than 0.9.
Experimental estimates of secondary structure con-
tent from far-UV CD spectra give 45 and 40% b-sheet
for the native and molten globule states of RBP,
respectively [26]. The b-sheet estimate for the native-
state is in close accord with that observed in the X-ray
structure (46%) [35]. In the group 3 simulation con-
formers, 53 residues have a b-sheet population of 0.60
or greater, and 11 residues have a b-sheet population
in the range 0.40–0.60. Taken together this corres-
ponds to 37% of residues in b-strand secondary struc-
ture, a value similar to that seen experimentally for the
molten globule state. The experimental CD data show
that there is an increase in a-helical secondary struc-
ture on forming the molten globule state (8% native;
24% molten globule [25]). A large increase in a-helical
secondary structure is not observed, on average, in the
simulation conformers. This difference may reflect
sampling and force field limitations in the simulations,
and the difficulty of interpreting experimental CD data
in a quantitative fashion. The difference may also
reflect the fact that we do not model explicitly the con-
ditions under which the molten globule is stable in the
simulations, but rather identify conformers that are
low in energy under native conditions. However,
although there is not a large increase in a-helical sec-
ondary structure, the native state a-helix for residues
146–158 is essentially retained in all the simulation

conformers. In addition, in some of the conformers,
particularly those in group 2, turns, some of a helical
character, do form for residues 93–96. These residues
are in the loop connecting strands E and F in the
native protein, a region where the native structure is
significantly disrupted in the simulations. It is therefore
possible that this is the part of the RBP sequence that
forms non-native helical secondary structure when the
molten globule state is adopted.
Variations in side chain packing
Despite the high persistence of the central regions of
b-sheet secondary structure even in the conformers
in group 3, significant changes are observed in the
0
0.2
0.4
0.6
0.8
probability
02040
60
80 100 120 140
160
residue number
0
0.2
0.4
0.6
0.8
A

BC D E FGH
Fig. 3. Fraction of the simulation conformers belonging to groups 2 (upper panel) and 3 (lower panel) in which certain secondary structure
elements are present. Secondary structure was calculated using the program DSSPcont [54] which identifies regions of secondary structure
through an analysis of hydrogen bonding patterns. b-sheet secondary structure is shown with open bars, helical (a,3
10
and p) secondary
structure is shown with filled black bars, and turns and bends are shown with grey bars. The secondary structure present in the native state
of RBP is indicated at the top of the figure, with the b-strands labelled A–H (open bars, b-strands; filled bars, a-helices).
MD simulations of the RBP molten globule state E. Paci et al.
4830 FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS
packing of hydrophobic side chains. The distances
between pairs of aromatic side chains and between
aromatic and other hydrophobic side chains, that are
close in the native state, have been analyzed in the
simulation conformers. Some representative examples
of the distance distributions for conformers in groups
2 and 3 are shown in Fig. 4. A very broad distribution
of distances across the simulation conformers is seen
when one of the side chains involved is from residues
85–104. This is the region that forms strand E and
part of strand F in the native structure, and is disor-
dered in many of the simulation conformers. For
example, the distance Tyr90–Met73 ranges from 3.3 to
46.6 A
˚
, while the distance Phe36–Tyr90 varies from
5.9 to 52.0 A
˚
in the group 3 conformers (Fig. 4).
Fluctuations in the distances are seen even for resi-

dues in regions where the native structure is retained
to a significant extent. Here though, the variations are
more limited. Thus for Phe20–Phe137 and for Phe36–
Tyr133 the distance ranges are 3.2–15.3 A
˚
and
2.9–13.8 A
˚
, respectively, in the group 3 conformers
(Fig. 4). These changes in the packing of aromatic side
chains in the group 3 conformers are in accord with
the experimental loss of a near-UV CD spectrum on
forming the molten globule state of RBP [20,24,25].
These experimental data reflect the absence of fixed
asymmetric environments for aromatic and cysteine
residues in the molten globule.
It is interesting that relatively short distances are
retained for some of the side chains that are in contact
with Trp24 in the native structure. Thus the distances
Trp24–Phe137 and Trp24–Arg139 are 3.0–6.8 A
˚
and
2.7–8.4 A
˚
, respectively, in the group 3 conformers
(Fig. 4). Mutational studies have shown that Trp24,
and its side chain interactions, play an important role
in stabilizing the RBP structure, and potentially in pre-
venting misfolding [25]. Trp24 and Phe137 are in a
hydrophobic cluster in the native structure, while the

side chains of Trp24 and Arg139 form an amine–
aromatic interaction that closes the base of the b-barrel
structure. These residues are part of an evolutionary
conserved set of residues in the lipocalin superfamily,
which it is suggested may fold on a faster timescale
than nonconserved regions [15]. In accord with this,
stopped flow fluorescence studies have shown that
Trp24 is in a near native-like hydrophobic environment
Fig. 4. Normalized distributions of the distances between selected residues for the simulation conformers in groups 2 (top panels) and 3
(bottom panels). The shortest distances between atoms in these side chains in the native state structure of RBP are indicated in paren-
theses. (A) Met73–Tyr90 (native state 3.8 A
˚
); (B) Phe36–Tyr90 (native state 6.9 A
˚
); (C) Phe20–Phe137 (native state 5.0 A
˚
); (D) Phe36–Tyr133
(native state 3.4 A
˚
); (E) Trp24–Phe137 (native 3.2 A
˚
); (F) Trp24–Arg139 (native state 3.7 A
˚
).The scale on the y -axis is arbitrary but the same
for each pair of residues.
E. Paci et al. MD simulations of the RBP molten globule state
FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS 4831
during the early stages of folding [15]. The persist-
ence of native-like contacts for Trp24 in the simula-
tion group 3 conformers suggests that these contacts

may be retained in the molten globule state. This would
be consistent with the significance of this side chain,
and its contacts, for the folding and stability of the
protein.
Common interactions may be topological
constraints in the molten globule
The simulation conformers in groups 1–3 all share the
same topology, while varying to a large degree in sec-
ondary structure persistence and side chain disorder.
This study provides an ideal model system to investi-
gate whether there is a common set of native-like inter-
actions maintained within low energy partly unfolded
structures, which provide a putative model for the
molten globule. For this purpose, we computed the
pairwise effective energy between pairs of residues,
averaged over all the structures belonging to the differ-
ent groups defined above. A network of the interac-
tions was built, by linking together residues whose
pairwise interaction is larger than a threshold (2.5 kcalÆ
mol
)1
), and removing isolated residues. The resulting
network is shown in Fig. 5 for the very native-like
group 1 and for the molten globule-like group 3 struc-
tures. The networks were analyzed using a network
principle termed ‘betweenness-centrality’ [37,38]. This
measure allows the identification of key nodes govern-
ing the network of interactions in proteins [39,40]. The
light grey residues in Fig. 5 have the highest between-
ness score, and are therefore the most influential to the

network. The interaction network representing group 3
is simpler, and the set of key nodes is smaller than for
group 1.
Interestingly, the structure of the networks of the
group 1 and group 3 conformers is similar, and the set
of residues that have a high betweenness in group 3
have also high betweenness in group 1 conformers.
These are Arg10, Lys12, Asn14, Val107, Glu108,
Thr109, Tyr111, Val116, Arg139 and Arg155. All these
10 residues are clustered together on the second face of
the
1
b-barrel, in the same overall region of the tertiary
structure. However, they reside in six different struc-
tural elements, being located in the N-terminal region,
on
2
b-strands F, G, H, in the F–G loop and in the
C-terminal helix in the native RBP structure. There are
wide variations in the characteristics of the contacts
between these residues. For example, in some cases
there are interactions between charged or polar side
chain groups (e.g. Glu108 has contacts to Lys12 and
R2
F45
S46
V42
A43
L35
A26

I41
E44
E16
G22
A130
S134
F135
E131
C174
A71
S21
R19
T128
A28
T56
R62
A55
A57
M53
S54
E33
G51
E39
G92
L37
S7
V6
V61
G59
L125

E49
L122
K85
M73
Y114
E72
E102
V74
G75
C4
E103
N124
W91
V93
K99
G100
W105
F86
T76
A84
T80
Y173
H170
G172
Q149
F77
C120
F137
L144
R166

R163
I106
Y165
A115
V69
E158
R153
K58
K17
Q156
Y118
K30
S132
K89
M88
T78
N101
K87
Q98
Q117
E68
K29
N40
N171
W24
V136
G127
Y90
H104
S119

Q38
R121
Y133
R60
M27
I168
R5
Y25
S138
T113
R10
R155
E140
E112
V107
V116
E108
K12
T109
N14
R139
Y111
R2
Q98
E102
N101
I106
C4
A115
Y114

Y90
K85
N124
V69
M73
A71
G75
E82
F77
S119
L125
G59
E158
N171
Q164
G172
Q149
Y173
R153
L144
T128
C129
F135
S134
V136
E140
F137
N66
C174
M27

E16
K17
E13
F36
S7
R19
E33
V6
E31
Y25
W24
S21
G22
Q38
E39
M53
S54
A55
A57
S46
F45
E44
V42
A43
T56
K58
K87
E68
V61
N40

K29
R60
R121
Y165
Q117
Y133
R166
K30
W105
S138
G127
E131
Y118
L122
A130
R163
S132
T113
R5
R139
R10
Y111
R155
V107
N14
V116
E108
T109
K12
A

B
Fig. 5. Network of effective interactions averaged over the structures of group 1 (A) and group 3 (B). The average effective energy between
pairs of residues including solvation free energy was computed as described in Paci et al. [55]. The network was built connecting pairs of
residues, whose absolute value of the interaction is larger than 2.5 kcalÆmol
)1
, and that are more than four residues apart in the sequence.
The threshold of 2.5 kcalÆmol
)1
is somewhat arbitrary and is chosen so that the representation of the network of interactions is shown most
clearly. Residues represented as light grey ovals are those with a betweenness larger than 5% (graphical representation and betweenness
obtained with the program
VISONE [56]).
MD simulations of the RBP molten globule state E. Paci et al.
4832 FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS
Arg155) while in other cases there are interactions
between pairs of hydrophobic groups (e.g. Val107 has
contacts to Val116). In addition, six of these 10 resi-
dues are also among the most highly conserved resi-
dues and interactions in the lipocalin superfamily
(N14, V107, E108, T109, Y111, R139) [15]. The simi-
larity of the results for the group 1 and group 3 con-
formers suggests that many, but not all, of the
interactions that are crucial for determining the native
state fold of the protein are retained in the RBP mol-
ten globule. Furthermore, the conservation of the resi-
dues with the high betweenness in RBP across the
lipocalin superfamily indicates the important role that
folding to a specific native state plays in determining
evolutionary selection. The results for RBP are partic-
ularly interesting in the light of a recently reported and

complementary analysis of networks of conserved
interactions in the intracellular lipid-binding protein
family [41]. Proteins in this family are similar to the
lipocalins having a b-barrel structure. However, the
proteins are in general smaller than the lipocalins and
their fold is different, consisting of a 10-stranded
b-barrel with a helix-turn-helix motif near the N-termi-
nus. From this work on the intracellular lipid-binding
protein family it was suggested that a network of con-
served hydrophobic side chain interactions in the
b-sheet region of the protein could provide a potential
folding nucleation site. This has interesting parallels
with an earlier reported study of the lipocalins [15].
Retinol binding site
Retinol binds to RBP in the core of the b-barrel.
When bound, retinol is almost totally encapsulated by
the protein, the b-ionone ring lying in the centre of the
protein with the isoprene chromophore stretching
along the barrel axis [35]. RBP releases retinol under
low pH conditions, undergoing the transition to the
molten globule state. The changes to the structure seen
in the group 3 conformers, especially the disruption in
the strand E to F region, lead to a significant opening
of the retinol binding site to solvent, and so would
prompt loss of bound retinol in the molten globule
state. The simulation analysis therefore proposes that
changes to the E to F region play an important role in
allowing retinol release from RBP. This proposal is
supported by data from studies of ligand binding to a
number of b-lactoglobulins, other members of the

lipocalin superfamily [42,43]. Here, an opening and
closing of the E-F loop region has been shown to pro-
vide a mechanism for the binding and release of lig-
ands. In the b-lactoglobulins the changes are localized
in the E–F loop, and are prompted by the protonation
of a glutamate side chain within the loop. In RBP, the
simulation results suggest that the disorder in the E to
F region is part of much more wide spread conforma-
tional changes, as the protein undergoes the transition
into the molten globule state.
Prior to the simulations reported here, retinol was
removed from the X-ray structure coordinates. This
gave an increase of 251 A
˚
2
in the solvent accessible
surface area of the protein. Comparisons of the side
chain solvent accessibility of the X-ray structure of
RBP (without retinol bound) with that of the group 3
conformers show that, on average, there is a further
increase in mean accessibility of 6.8 A
˚
2
per residue for
the group 3 conformers. Many of the side chains that
make contacts with retinol when it is bound to the
native state have, however, much larger increases in
mean side chain accessibility in the group 3 conform-
ers. For example, for Leu63, Met73 and Phe77 the
mean side chain accessibility increases by 59.3, 85.6

and 77.9 A
˚
2
, respectively (Fig. 6). Experimentally the
molten globule state of RBP has been shown to bind
strongly the hydrophobic dye 8-anilino-1-napthalene-
sulphonate (ANS), giving an intense fluorescence spec-
trum [24]. This property, characteristic of molten
globules, is generally recognized to reflect the presence
of exposed hydrophobic surfaces. The simulation
group 3 conformers show that in the case of RBP the
exposed hydrophobic side chain groups from the reti-
nol binding site are likely to be responsible, at least in
part, for the observed ANS binding.
Transthyretin binding site
RBP circulates in the blood stream in a one-to-one
complex with a second serum protein, transthyretin
(TTR) [32]. This large complex prevents the smaller
RBP from being filtered through the renal glomeruli
[19]. Upon reaching target tissues, RBP and trans-
thyretin dissociate, and retinol is released for delivery
to the cells, in a yet undetermined mechanism. Four
regions of the sequence of RBP at the opening of the
barrel make contacts with TTR [32]. The residues in
RBP in three of the regions involved in these contacts
are identified in Fig. 6. The protein C-terminus is the
fourth region of RBP which makes contacts with
TTR. These residues were missing from the X-ray
structure of RBP [35], and so were excluded from the
simulations reported here. The binding of TTR to

RBP has been shown to stabilize the binding of retinol
to RBP. Studies of the interactions of chimaera
of RBP and epididymal retinoic acid binding pro-
tein (ERABP) with TTR have identified the region
of the RBP sequence that is key to providing this
E. Paci et al. MD simulations of the RBP molten globule state
FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS 4833
stabilization [33]. The residues concerned are those in
the E-F loop, where the most major changes in struc-
ture are observed in the group 3 simulation conform-
ers. This suggests that the dissociation of the RBP–
TTR complex may be linked to release of retinol
bound to RBP, via conformational changes in the E–F
region of the protein. In this respect it is particularly
interesting that, in some of the simulation conformers
analyzed, residues in the E-F region adopted helical
turns. It is therefore possible that a conversion in sec-
ondary structure could be involved in the RBP–TTR
dissociation mechanism. Further work is needed, how-
ever, to investigate this mechanism in more detail,
including a study of the role of the C-terminal region
of the RBP sequence which was excluded from the
simulations reported here.
Comparison with other molten globule states
Overall, analysis of the partially folded conformers
generated for RBP, and comparison with experimental
data, indicates that the structures in group 3 can pro-
vide a model for the ensemble of conformers popu-
lated in the molten globule state of the protein. These
conformers retain considerable secondary structure,

but show disorder in side chain packing, and have
exposed hydrophobic groups. In the group 3 conform-
ational ensemble the central strands of the two ortho-
gonal b-sheets show a high persistence. The structure
in the outer strands is more fluctuating in nature. It is
interesting to compare these secondary structure char-
acteristics with those seen for the molten globule states
of other proteins that have been studied in detail
experimentally. Many of the proteins whose molten
globules have been characterized have predominantly
a-helical secondary structure in their native state, and
this is largely retained in the molten globule state.
Moreover for proteins that contain both a-helices and
b-strands in the native state, it is the a-helical part of
the structure that particularly persists in the molten
globule. The b-sheet regions are more unstructured, in
contrast to the results for RBP. This is seen, for exam-
ple, in human a-lactalbumin [12] and Escherichia coli
ribonuclease HI [13]. Comparison with data for the
molten globule state of two proteins which have a
native state fold that is rich in b-sheet secondary struc-
ture, carbonic anhydrase and b-lactoglobulin, is there-
fore particularly relevant.
Human carbonic anhydrase II contains 10 b-strands
in the native state, but differs topologically from RBP,
having an ab roll fold [44]. In the partially folded state
the central strands 3–7 have native-like structure, but
less ordered structure is found in the peripheral strands
[45,46]. Hence, as in the case of RBP, the core part of
-100

-50
0
50
Surf-Surf
X
TTR binding residues
0 10203040
50 60
70 80 90 100 110 120 130 140
150 160
170
residue number
-100
-50
0
50
Group2
Group3
Fig. 6. Difference between the average side chain accessibility of each residue in the group 2 (upper panel) or group 3 (lower panel) simula-
tion conformers and the side chain accessibility in the X-ray structure of native RBP (with retinol removed). Black bars indicate the residues
that make contacts to bound retinol in the native state, and triangles show the residues that are involved in binding to TTR. The solvent
accessible surface area of the side chains has been calculated using the program
NACCESS [57]. The average increase in surface accessible
area per residues is 6 and 7 A
˚
2
in the structures belonging to group 2 and group 3, respectively, compared to a change of )0.5 A
˚
2
in the

equilibration (control) simulation.
MD simulations of the RBP molten globule state E. Paci et al.
4834 FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS
the b-sheet secondary structure is retained in the mol-
ten globule state, while external regions of the struc-
ture are more disordered. b-lactoglobulin is a member
of the lipocalin superfamily, and like RBP its native
structure contains an up-and-down b-barrel and one
C-terminal a-helix [42]. Hydrogen exchange studies of
the molten globule state of equine b-lactoglobulin
show that the high protection factors are seen for resi-
dues in strands G and H (values > 100 for five resi-
dues). These strands are linked by a disulphide bridge
between Cys106 and Cys119. Moderate protection fac-
tors (10–20) are seen for some residues in the regions
corresponding to strands A, D and F in the native
state, and also the a-helix. Residues out of these
regions have protection factors less than 10 (with the
exception of Asn53 in the B–C loop which has a pro-
tection factor of 11) [47]. These data are consistent
with a high persistence for native-like secondary struc-
ture in the central parts of the second b-sheet (residues
in strands F, G and H together with part of strand A),
while some secondary structure of a less persistent nat-
ure in the centre of the first b-sheet (particularly for
strand D) and in the a-helix [47]. Results for bovine
b-lactoglobulin are very similar, with a high level of
hydrogen exchange protection being seen in the central
parts of the second b-sheet (residues in strands F, G,
H and A) in the partially folded state formed at pH 2

[48]. In addition, for bovine b-lactoglobulin significant
hydrogen exchange protection persists for residues in
b-strands G and H in the cold denatured state [49].
These data for b-lactoglobulin are similar to the results
reported here, although in the RBP simulation con-
formers there is an equivalent level of persistence in
the central regions of both of the two b-sheets, while
in b-lactoglobulin the second sheet predominates. The
proposed similarity between the molten globule states
of RBP and b-lactoglobulin suggests that the partially
folded protein characteristics identified here may be
typical of those adopted by other proteins in the lipo-
calin superfamily.
Conclusions
In the model for the molten globule state of RBP
reported here the protein retains a persistent b-sheet
core, although even in these regions there is consider-
able variation in the side chain packing. Out of the
b-sheet core there is much more disorder across the
conformational ensemble. The network analysis of
the simulation conformers shows that there is a small
subset of persistent key interactions. The most signifi-
cant changes in the structure are seen in the region
extending from the start of b-strand E to the middle of
b-strand F (residues 85–104). It is possible that the
conformational changes in this region of the protein in
the simulation conformers, including the presence of
some a-helical character, could play an important role
in the mechanism for the dissociation of the TTR–
RBP complex and retinol release at the cell surface.

Moreover, similarities between the simulation con-
formers of RBP and experimental data for b-lactoglob-
ulin, suggest that the model for the molten globule of
RBP reported here may have relevance to our under-
standing of the properties and ligand binding of other
members of the lipocalin superfamily. This is of partic-
ular interest in the light of the development of
engineered lipocalins for carrying novel ligands in
therapeutic approaches [23,50,51].
Experimental procedures
The initial structure used in the simulations was the crystal
structure of human serum retinol-binding protein at 2 A
˚
resolution (PDB entry 1RBP [35], Fig. 2), with the bound
retinol ligand removed. The C-terminal region of the RBP
sequence is missing from the X-ray structure, as these resi-
dues could not be located in the electron density map (resi-
dues 175–182). We therefore also chose to exclude these
eight C-terminal residues from the simulations reported
here, although it is possible that this truncation of the
sequence could affect the stability of non-native states of
the protein. An equilibration (control) simulation was run
for 2 ns at 300 K. The exploration of the conformational
space outside the native state at room temperature was car-
ried out by a biased molecular dynamics (BMD) scheme,
similar to that used by Paci et al. [30]. All simulations were
performed in implicit solvent (EEF1 [52]). The system was
initially energy minimized to remove bad contacts, heated
up to 300 K in 0.6 ns and then equilibrated for 2 ns. Dur-
ing the last 1 ns of this equilibration simulation the average

RMSD from the native state crystal structure was
2.4 (3.2) A
˚
for Ca (all atoms): i.e. the native state is relat-
ively stable with the implicit solvation method employed.
Following this, a 6 ns simulation was performed using
biased molecular dynamics, with a perturbation increasing
the protein radius of gyration. During this simulation, the
radius of gyration and solvent accessible surface area of the
protein increased up to 30% relative to that of the native
structure, while the heavy atom RMSD from the native
structure reached 13 A
˚
.
Five conformations were picked out from along the tra-
jectory, at approximately equal intervals in radius of gyra-
tion value, ranging from one with an almost native-like
radius of gyration to a maximally denatured conformer.
Five 1.6-ns simulations, one for each initial configuration,
were started with a perturbation favouring the loss of native
contacts between side-chain heavy atoms. A configuration
E. Paci et al. MD simulations of the RBP molten globule state
FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS 4835
every 0.4 ns was saved, and used as an initial conformation
to perform eight sequential 0.4 ns simulations with a pertur-
bation increasing the RMSD from the initial structure. This
procedure generated, in a relatively short simulation time
(6+72 ns), 160 distinct conformations (coordinates and
velocities) that are independent and close to equilibrium
(due to the adiabatic bias used to induce the loss of native

features, the energy of the conformers is no more than 20
kcalÆmol
)1
larger than the average energy in the native
state). These conformations span a range between 2.1 and
11.9 A
˚
in heavy atom RMSD from the native X-ray struc-
ture and between 15.7 and 19.1 A
˚
in protein radius of gyra-
tion. From each of these 160 conformations a 1.2 ns
simulation was started without any bias (the first 0.2 ns
were disregarded in the following analysis). We thus pro-
duced 160 ns of unperturbed equilibrium trajectories span-
ning the conformational space starting from 160 diverse
initial configurations. The total length of the simulation was
more than 300 ns. The present approach, while not suitable
to determine the free energy surface of the protein, which
would be computationally very challenging, enables explora-
tion of many regions of the conformational space corres-
ponding to low-energy non-native structures [30].
Acknowledgements
The work was supported in part by the UK Biotech-
nology and Biological Sciences Research Council
(Grant 43 ⁄ B17889). LHG thanks the NSF for an
International Research Fellows Award (#0000597).
LJS thanks the Oxford University Supercomputing
Centre for access to its facilities. Some of the simula-
tions reported in this paper were performed while EP

was at the Department of Biochemistry, University of
Zurich.
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Supplementary material available
The following material is available for this article
online:
Fig. S1. Contact map for the group 1 and group 3 con-
formers.
4838 FEBS Journal 272 (2005) 4826–4838 ª 2005 FEBS
MD simulations of the RBP molten globule state E. Paci et al.