Báo cáo khoa học: Conformational stability of neuroglobin helix F – possible effects on the folding pathway within the globin family potx

Conformational stability of neuroglobin helix F – possible
effects on the folding pathway within the globin family
Luca Codutti
1,
*, Paola Picotti
2,
*
,
, Oriano Marin
2
, Sylvia Dewilde
3
, Federico Fogolari
1
, Alessandra
Corazza
1
, Paolo Viglino
1
, Luc Moens
3
, Gennaro Esposito
1
and Angelo Fontana
2
1 Department of Biomedical Sciences and Technologies and MATI Centre of Excellence, University of Udine, Italy
2 CRIBI Biotechnology Centre, University of Padua, Italy
3 Department of Biochemistry, University of Antwerp, Belgium
Introduction
Globins are well-known proteins that share the charac-
teristic of a typical prosthetic group, traditionally

named heme, and corresponding to a protoporphyrin
scaffold carrying a single iron ion, formally in the +2
or +3 oxidation state. The metal ion can coordinate
several ligands other than protein groups or porphyrin
ring atoms. Among the exogenous ligands, molecular
oxygen has a specific relevance for the function of
Keywords
circular dichroism; globin folding; myoglobin;
neuroglobin; NMR
Correspondence
G. Esposito, Dipartimento di Scienze e
Tecnologie Biomediche, University of Udine,
P. le Kolbe 4, 33100 Udine, Italy
Fax: +39 0432 494301
Tel: +39 0432 494321
E-mail:
A. Fontana, CRIBI Biotechnology Centre,
University of Padua, Viale G. Colombo 3,
35121 Padua, Italy
Fax: +39 049 8276159
Tel: +39 049 8276156
E-mail:
†Present address
Institute of Molecular Systems Biology, ETH
Zurich, Switzerland
*These authors contributed equally to this
work
(Received 15 April 2009, revised 17 June
2009, accepted 15 July 2009)
doi:10.1111/j.1742-4658.2009.07214.x

Neuroglobin is a recently discovered member of the globin family, mainly
observed in neurons and retina. Despite the low sequence identity (less
than 20% over the whole sequence for the human proteins), the general
fold of neuroglobin closely resembles that of myoglobin. The latter is a
paradigmatic protein for folding studies, whereas much less is known about
the neuroglobin folding pathway. In this work, we show how the structural
features of helix F in neuroglobin and myoglobin could represent a pivotal
difference in their folding pathways. Former studies widely documented
that myoglobin lacks helix F in the apo form. In this study, limited prote-
olysis experiments on aponeuroglobin showed that helix F does not
undergo proteolytic cleavage, suggesting that, also in the apo form, this
helix maintains a rigid and structured conformation. To understand better
the structural properties of helices F in the two proteins, we analyzed pep-
tides encompassing helix F of neuroglobin and myoglobin in the wild-type
and mutant forms. NMR and CD experiments revealed a helical conforma-
tion for neuroglobin helix F peptide, at both pH 7 and pH 2, absent in the
myoglobin peptide. In particular, NMR data suggest a secondary structure
stabilization effect caused by hydrophobic interactions involving Tyr88,
Leu89 and Leu92. Molecular dynamics simulations performed on the apo
and holo forms of the two proteins reveal the persistence of helix F in neu-
roglobin even in the absence of heme. Conversely myoglobin shows a
higher mobility of the N-terminus of helix F on heme removal, which leads
to the loss of secondary structure.
Abbreviations
Fmoc, 9-fluorenylmethoxycarbonyl; Mb, myoglobin; MbF-P88A, fragment 79–97 of sperm-whale myoglobin with Pro88 replaced by Ala88;
MbF-wt, fragment 79–97 of sperm-whale myoglobin; Ngb, neuroglobin; NgbF-A90P, fragment 79–100 of human neuroglobin with Ala90
replaced by Pro90; NgbF-wt, fragment 79–100 of human neuroglobin; NOESY, nuclear Overhauser enhancement spectroscopy; PME,
particle mesh Ewald; TFA, trifluoroacetic acid; TFE, 2,2,2-trifluoroethanol; TOCSY, total correlation spectroscopy.
FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS 5177
globins, such as myoglobins (Mbs) and hemoglobins,

which are generally considered to be oxygen storage
and transport proteins [1,2], although other views have
been proposed [3,4]. In addition to Mb and hemoglo-
bin, over recent years two additional globins have been
found to occur in a wide variety of vertebrates, namely
neuroglobin (Ngb) [5] and cytoglobin [6]. These two
globins exhibit the interesting feature of endogenous
hexacoordination of iron [7], whatever its oxidation
state. In brief, the metal ion coordinates the four pyr-
role nitrogens of the porphyrin group and two imidaz-
ole nitrogens of two different histidine residues of the
protein, whereas, in Mbs and nearly all hemoglobins,
only one iron coordination site is given by a histidine
imidazole. The two histidines involved in iron coordi-
nation are commonly referred to as proximal and dis-
tal, depending on the relative separation from the
heme metal, and occur at definite locations of the Mb
structural domain. The latter consists of eight helices
(A–H) with intervening loops packed with a character-
istic fold of two triple-helix layers with nearly orthogo-
nal relative rotation (three-over-three) (Fig. 1). The
heme group accommodates between the two parallel
helices E and F, the proximal histidine being provided
by helix F (residue F8) and the distal histidine being
provided by helix E (residue E7), according to consen-
sus numbering [7]. Although the distal histidine bind-
ing to the metal ion is the signature of endogenous
hexacoordination of Ngbs and cytoglobins, only the
proximal histidine coordination occurs invariably in all
globins.

ApoMb, the heme-free Mb, retains the highly helical
fold of native Mb at neutral pH. However, NMR [8,9]
and limited proteolysis [10,11] studies have shown that
helix F in apoMb is disordered and readily cleaved by
proteases. The limited proteolysis pattern also led to
the establishment that no other potential cleavage sites
of apoMb undergo hydrolysis, as a consequence of the
stability of the helical fold of the protein [11]. An
important determinant responsible for the conforma-
tional flexibility of the chain segment encompassing
helix F in apoMb has been recognized in the nature of
residue F3, a proline residue that disrupts the local
a-helical conformation and destabilizes significantly the
whole helix F (Fig. 1). Indeed, substitution of the helix-
breaking Pro88 (F3) residue with the helix-forming ala-
nine residue in sperm-whale apoMb successfully meets
expectations and apparently restores the local helix
geometry, as inferred from CD profiles and limited
proteolysis [11], although none of the techniques can
distinguish partial from full restoration. In these earlier
studies [8,11], it was proposed that helix F in the native
holoprotein is stabilized by interactions with the heme
moiety, counterbalancing the helix-breaking effect of
proline. As a proline residue at location F3 occurs in
more than 90% of Mb sequences and several hemoglo-
bin chains, these globin species in their apo form
should exhibit low, if any, helical propensity in the
corresponding helix F segment. This is in agreement
with the proposed main folding pathway of apoMb at
neutral pH, sketched as U fi AGH fi ABGH fi

ABCDEGH fi N, where U and N are the unfolded
A
B
C
Fig. 1. Three-dimensional structure of
human Ngb (A) and sperm-whale Mb
(B). The models were constructed from
the X-ray structure of the Ngb mutant
C46G ⁄ C55S ⁄ C120S (PDB code 1OJ6, chain
B) and sperm-whale Mb (PDB code 1VXD).
Helix F is highlighted by a white ellipsoid in
both diagrams. (C) Sequences of the pep-
tides that were addressed in the present
study, i.e. helix F encompassing fragments
79–100 of Ngb and 79–97 of sperm-whale
Mb, together with the corresponding vari-
ants. A box highlights the actual extension
of helix F in the parent protein structures.
The wild-type sequences are indicated as
NgbF-wt and MbF-wt; the variant
sequences are identified by the correspond-
ing mutations, i.e. NgbF-A90P and MbF-
P88A, respectively.
Globin helix F conformational stability L. Codutti et al.
5178 FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS
and native conformations, respectively, and A, B, etc.
are the helix segments [12]. The folding scheme confirms
the absence of a stably folded helix F in native apoMb,
in contrast with the N state of the holoprotein [12].
The stability of the globin domain has been

addressed recently by comparing the apo forms of
horse Mb and human Ngb at acidic pH (P. Picotti,
unpublished results). The well-established instability of
apoMb at low pH [13,14] was confirmed. At variance
with the extensive loss of apoMb secondary structure,
the apoNgb chain was observed to preserve most of
the helical fold at acidic pH and limited proteolysis
experiments suggested that only the N-terminal frag-
ments were sufficiently flexible to become susceptible
to proteolytic cleavage. Therefore, among the pre-
served helical segments of apoNgb, there was also
helix F. This finding appears to be consistent with the
absence of a proline residue in the chain segment
encompassing helix F in apoNgb (Fig. 1).
A useful approach to the study of the mechanism
of protein folding entails the analysis of the confor-
mational preferences of isolated peptide fragments.
Short linear peptide fragments cannot exhibit the
tertiary interactions that they establish in the intact
proteins. Therefore, the assessed conformational
trends of isolated fragments are the same as those
occurring in the protein chain during the early stages
of folding, when only local and inherent conforma-
tional propensities drive the folding process. Along
these lines, a direct CD and NMR investigation was
carried out previously in order to establish the folding
propensities of Mb peptide fragments [15]. In addition
to confirming inherent helix propensities for AB and
GH segments, this study also showed that the helix F
fragment has quite a low propensity towards helical

geometry, even in the presence of 2,2,2-trifluoro-
ethanol (TFE).
In order to investigate more deeply the suggested
differences in helix F stability within the globin family,
limited proteolysis of apoNgb at neutral pH and the
intrinsic conformational stability of peptides encom-
passing the different helix F variants (Fig. 1) are
addressed here. Indeed, it is shown that helix F in apo-
Ngb has a strong propensity for a-helical secondary
structure, at variance from helix F in apoMb. These
results allowed us to infer that the folding pathway of
apoNgb is different from that of apoMb, despite the
similarity of their overall fold. The conclusion reached
in this study reinforces the view that the same protein
structural topology does not imply the same folding
pathway. An analogous view was also expressed in a
comparative study of the folding pathway of Mb [12]
and leghemoglobin [16].
Results and discussion
Limited proteolysis
Limited proteolysis experiments were performed on
wild-type human apoNgb at neutral pH with the
enzyme thermolysin (Fig. 2). Compared with the incu-
bation times typically required by horse or sperm-
whale apoMb, i.e. seconds [10,11], apoNgb proteolysis
proved to be much slower. After 4 min of incubation,
apoNgb shows only two sites of preferential cleavage,
i.e. at the level of the N-terminal helix (helix A), pre-
cisely between Ala15 and Val16, and at the interhelical
segment between helix F and helix G, precisely

between Ala98 and Val99. The proteolysis pattern of
the latter region also involves hydrolysis between Ser91
and Leu92, with the formation of fragment 16–91,
which becomes the predominant species at longer
protease incubation times. The later onset of fragment
16–91 demonstrates that it derives from further proteo-
lytic digestion of the initially formed species 16–98 at
the level of the newly exposed C-terminus. However, a
Fig. 2. Limited proteolysis of human apoNgb at neutral pH. Proteol-
ysis of apoNgb by thermolysin (enzyme to substrate ratio 1 : 100
by weight) was conducted at 25 °Cin50m
M Tris-HCl, 0.15 M NaCl,
pH 7.0. The proteolysis mixture was analyzed by reverse-phase
HPLC after 4 and 30 min of incubation. The identities of the protein
fragments were established by electrospray mass ionization mass
spectrometry and are indicated by the labels near the chromato-
graphic peaks.
L. Codutti et al. Globin helix F conformational stability
FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS 5179
significant amount of undigested full-length protein is
still present at either proteolysis intervals. Therefore,
the cleavage location is partially dependent on the
incubation time of the substrate with the protease, at
least within the limits of the experimental protocol.
Cleavage at the level of the turn-like fragment, joining
helix F to the rest of the C-terminal region, suggests
that this region is highly flexible in Ngb at neutral pH,
and thus it is a proper protease substrate. The addi-
tional cleavage site at the N-terminal region of Ngb
(at the end of helix A) recalls the previously observed

limited proteolysis pattern of apoMb at acidic pH,
with a cleavage extended to most of helix B at low pH
[13]. Strikingly, at both pH conditions, helix F of
human apoNgb does not undergo proteolytic cleavage
as instead observed in apoMbs, under either neutral
[10,11] or acidic conditions [13], suggesting that the
apoNgb helix F maintains a sufficiently rigid structure
to prevent proteolysis. These results are perfectly in
line with the agadir [17] secondary structure predic-
tions reported in Fig. 3, which illustrates the helical
propensity for the whole Ngb sequence, at neutral
and acidic pH, and for wild-type or mutated helix F
peptides of Ngb and Mb.
CD analysis
Far-UV CD measurements (Fig. 4) of the peptides
under investigation were conducted in different experi-
mental conditions in order to analyze their content of
secondary structure. The investigated peptides encom-
pass the native sequences of helix F in sperm-whale
Mb (MbF-wt) and human Ngb (NgbF-wt) and the
corresponding mutants obtained by the replacement of
Pro88 (ProF3, in the globin consensus map [7]) with
an alanine in Mb (MbF-P88A), and of Ala90 (AlaF2)
with a proline in Ngb (NgbF-A90P) (see Fig. 1). Fig-
ures 4A,B depict the spectra obtained for NgbF-wt at
neutral pH conditions with increasing amounts of TFE
and in aqueous solution at decreasing pH, respectively.
At neutral pH, the CD spectrum of NgbF-wt displays
two prominent minima at 208 and 222 nm, typical of
a-helical polypeptides. The helix content of NgbF-wt

steadily increases with TFE, from 35% without
organic solvent to 56% at 20% TFE. On lowering the
pH (Fig. 4B), instead, the helix content decreases from
the same initial value as in Fig. 4A to 30% at pH 2.2.
The mutation of Ala90 into proline destroys the helix
content of the parent sequence, as evident from the
corresponding CD spectrum typical of random coil
peptides (Fig. 4C). The addition of TFE restores some
helix content (17%) in NgbF-A90P, to an extent, how-
ever, much below that observed for NgbF-wt. Far-UV
CD spectra collected on the peptides encompassing the
sequence of helix F in Mb are reported in Fig. 4D. As
expected from previous results on the whole protein
and mutants thereof [11], as well as on isolated frag-
ments [15], the peptide MbF-wt, with the natural
sequence bearing a proline in position F3, displays
very little helical content at neutral pH, whereas the
peptide MbF-P88A, where the proline is replaced by
alanine, exhibits a slightly higher helix content (16%).
It is worth noting that the experimental helix content
obtained from CD data for the isolated peptides paral-
lels the expectations obtained using agadir semi-
empirical predictions on the corresponding native and
mutant full-length proteins (Fig. 3, right).
NMR analysis
1
H NMR spectra were collected only for the NgbF-wt
fragment at two different pH values, i.e. pH 6.3 and
Ngb, pH 2.0
Ngb, pH 7.0

Ngb A90P
Mb WT
Mb P88A
Ngb WT
Fig. 3. Left: helical propensity of the poly-
peptide sequences of Ngb at neutral and
acidic pH calculated using the
AGADIR algo-
rithm [17]. The locations of the eight helices
(A–H) along the polypeptide chain of the
protein are also indicated by boxes, accord-
ing to the structural features obtained from
the PDB record. Right:
AGADIR-predicted heli-
cal propensities for helix F of wild-type
human Ngb, A90P human Ngb, wild-type
sperm-whale Mb and P88A sperm-whale
Mb.
Globin helix F conformational stability L. Codutti et al.
5180 FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS
pH 2.1 in water, and pH 6.3 in 10% aqueous TFE.
Detailed analysis was performed, however, only for the
datasets obtained in water to which we will refer,
unless otherwise indicated. Spin systems were first
identified, for both pH conditions, in the total correla-
tion spectra, and then assigned on the basis of nuclear
Overhauser enhancement spectroscopy (NOESY) map
sequential connectivity patterns [18]. A general over-
view of the NMR information shows that, for both
series of experiments, no long-range restraints (interac-

tions between nuclei more than five residues apart)
were detected. Figure 5 displays the distribution along
the sequence of all collected restraints under both
experimental conditions. Secondary structure meaning-
ful cross-peaks have been found for both pH condi-
tions and are shown in Fig. 6. Even a cursory
examination suggests that typical helix conformational
patterns occur in the central region of the investigated
peptide at both pH conditions, whereas the N-terminal
and C-terminal segments appear to be poorly struc-
tured. An additional interesting feature that emerges
from nuclear Overhauser enhancement restraints is the
occurrence, at both pH conditions, of medium-range
hydrophobic interactions between Tyr88 H
d
or H
e
and
Leu85, Leu89, Leu92 H
d
.
After removing all redundancies, the experimental
restraint sets consisted of 211 meaningful interatomic
distances for experiments made at pH 6.3, and 236
meaningful interatomic distances for experiments made
at pH 2.1. The two series formed the experimental
databases for the subsequent restrained modeling.
Table 1 summarizes the final output of restrained
modeling.
Structural validation performed using the software

aqua and procheck-nmr [19] confirmed the presence
of a regular a-helix secondary structure at both pH
conditions, with slightly different lengths. At pH 6.3,
the a-helix involves residues from Glu86 to Leu92,
whereas, at pH 2.1, the a-helix extends from Glu87 to
Ser91, in qualitative agreement with the estimates
10
AB
CD
NgbF, pH 7.2
–10
0
0% TFE
10% TFE
–20
20% TFE
[θ] × 10
–3
(deg·cm
2
·dmol
–1
)
Wavelength (nm)
200 210 220 230 240 250
0
NgbF
–10
–5
pH 2.2

pH 7.2
pH 4.1
–15
[θ] × 10
–3
(deg·cm
2
·dmol
–1
)
Wavelength (nm)
200 210 220 230 240 250
NgbF, pH 7.2
–5
0
–15
–10
NgbF-A90P
NgbF-A90P, 20% TFE
[θ] × 10
–3
(deg·cm
2
·dmol
–1
)
Wavelen
g
th (nm)
200 210 220 230 240 250

MbF, pH 7.2
MbF-P88A
[θ] × 10
–3
(deg·cm
2
·dmol
–1
)
–5
0
–20
–15
–10
MbF
Wavelen
g
th (nm)
200 210 220 230 240 250
Fig. 4. CD characterization of peptides
encompassing helix F of human Ngb and
sperm-whale Mb (see Fig. 1). (A) Far-UV CD
spectra of NgbF-wt peptide dissolved in
50 m
M Tris-HCl ⁄ 0.15 M NaCl, pH 7.0, in the
presence of different amounts of TFE. (B)
Far-UV CD spectra of NgbF-wt peptide dis-
solved in 10 m
M HCl, pH 2.2 or pH 4.1. The
spectrum at pH 7.2 is redrawn for compari-

son. (C) Far-UV CD spectra of NgbF-A90P
peptide dissolved in 50 m
M Tris-HCl ⁄ 0.15 M
NaCl, pH 7.0, in the presence of 20% TFE.
(D) Far-UV CD spectra of MbF-wt and
MbF-P88A peptides dissolved in 50 m
M
Tris-HCl ⁄ 0.15 M NaCl, pH 7.0. All spectra
were recorded at 25 °C.
L. Codutti et al. Globin helix F conformational stability
FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS 5181
obtained from CD data. Over these fragments, the
average upfield deviations of H
a
chemical shifts from
the values of statistically disordered structures [20] are
0.21 ± 0.11 and 0.19 ± 0.13 p.p.m. under neutral and
acidic conditions, respectively (the corresponding value
in TFE is 0.25 ± 0.12 p.p.m.). Such deviation extents
are above the chemical shift index threshold to validate
helical tracts [21], which, for a linear peptide in water,
suggests that a helical geometry is locally significantly
populated. Validation of the secondary structure for
the remaining residues in both pH conditions con-
firmed a statistically disordered state. According to
procheck-nmr, at both pH conditions, refined struc-
tures showed no dihedral angle of the fragment 86–92
in disallowed Ramachandran regions. The helical seg-
ments also revealed low accessibility because of a
Number of constraints Number of constraints

Residue number
Residue number
A
B
Fig. 5. Restraint distribution along the sequence of the NgbF-wt
peptide. The restraints obtained at pH 6.3 (A) and pH 2.1 (B) and
subsequently used for simulated annealing calculations are given.
In the histograms, white represents intraresidue restraints, light
grey sequential restraints and dark grey medium-range restraints.
Fig. 6. Secondary structure diagnostic restraints obtained at pH 6.3
(A) and pH 2.1 (B). The bar thickness is proportional to the corre-
sponding nuclear Overhauser enhancement intensity.
Table 1. CYANA 2.1 and DISCOVER output parameters for NgbF-wt
restrained molecular dynamics calculations and subsequent
refinement.
Structure family at pH 6.3 Average Range
CYANA
Average backbone
rmsd to mean ⁄ 10
)1
nm
0.56 ± 0.28 0.33–1.39
Average heavy atom rmsd
to mean ⁄ 10
)1
nm
0.89 ± 0.28 0.57–1.59
Target function ⁄ 10
)2
nm

2
(6.23 · 10
)2
)±
(1.85 · 10
)2
)
Violated distance
constraints
0
Violated van der Waals’
constraints
0
DISCOVER
Average backbone rmsd
to mean ⁄ 10
)1
nm
0.37 ± 0.21 0.07–1.03
Average heavy atom rmsd
to mean ⁄ 10
)1
nm
1.33 ± 0.32 0.68–2.45
Structure family at pH 2.1 Average Range
CYANA
Average backbone rmsd
to mean ⁄ 10
)1
nm

0.36 ± 0.13 0.27–0.65
Average heavy atom rmsd
to mean ⁄ 10
)1
nm
0.59 ± 0.15 0.43–0.94
Target function ⁄ 10
)2
nm
2
(0.28 ± 4.53) · 10
)2
Violated distance
constraints
0
Violated van der Waals’
constraints
0
DISCOVER
Average backbone rmsd to
mean ⁄ 10
)1
nm
0.26 ± 0.13 0.04–0.56
Average heavy atom rmsd
to mean ⁄ 10
)1
nm
1.06 ± 0.38 0.14–1.78
Globin helix F conformational stability L. Codutti et al.

5182 FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS
back-fold trend of the disordered flanking regions
(Fig. 7). Additional validation parameters are reported
in Table S1 (see Supporting information).
A fitting of the 20 final conformers (at both pH con-
ditions) over the a-helix validated zone led to mean
backbone rmsd deviation values of 0.037 ± 0.021 nm
for structures calculated at pH 6.3 and
0.026 ± 0.013 nm for structures calculated at pH 2.1.
Additional details are given in Table 1.
Figure 8 shows a diagrammatic representation of the
superposition of the structure of family conformers at
both pH conditions. The structures were superimposed
to minimize rmsd within the regular helical fragments
in both cases. As apparent from the side-chain distri-
bution, it is likely that hydrophobic interactions
between Tyr88 and both Leu89 and Leu92 side-chains
create a scaffold capable of stabilizing the local helical
fold in either pH conditions. Indeed, the chemical
shifts of the Leu92 side-chain isopropyl moiety are
shifted upfield by 0.13–0.18 p.p.m., whereas Leu89 H
b
resonances occur downfield with respect to the basic
aqueous shift value [20] by 0.18–0.20 p.p.m. at both
pH conditions.
Molecular dynamics simulations
Snapshots have been taken at 100 ps intervals in order
to obtain a statistical ensemble for the three systems
studied. We consider first the molecular dynamics sim-
ulations of the apo forms of Mb and Ngb in order to

check whether any difference in dynamics could be
highlighted even in a simulation time as short as 3 ns.
Although the loss of secondary structure for apoMb
is expected, it is not obvious how fast this process may
be. Molecular dynamics simulation shows that the
N-terminal part of helix F (entailing residues Glu83 to
Leu86) loses its helical conformation in the first 200 ps
of simulation. In particular, the u and w angles formed
by these residues are quite different from those of stan-
dard a-helices and exhibit very large fluctuations.
There is no clear conformational transition towards
completely different conformations, but overall the
backbone is very flexible. The results concerning helix
F are in agreement with earlier simulation studies
[22,23]. This picture is further confirmed by the analy-
A
B
Fig. 7. Overlay of the 20-membered conformer families of the
NgbF-wt peptide. Superpositions were obtained by fitting the struc-
tured regions observed at different pH conditions: (A) pH 6.3; (B)
pH 2.1.
A
B
Fig. 8. A diagrammatic view of the structured regions of the 20
peptide family members at both pH conditions: (A) pH 6.3; (B) pH
2.1. The side-chains involved in the a-helical secondary structure
are highlighted in red.
L. Codutti et al. Globin helix F conformational stability
FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS 5183
sis of backbone rmsds, which are larger than 0.2 nm at

residues 83 and 84.
In contrast, apoNgb maintains a standard a-helical
conformation at the corresponding residues (Glu86 to
Leu89). The a-helix does, however, start for most of
the time at residue Ser83. The hypothesis put forward
in this work is that Tyr88 plays a crucial role in
scaffolding residues 85, 89 and 92. Indeed, when these
residues are superimposed, the average heavy atom
rmsd between any two pairs of snapshots is 0.088 ±
0.026 nm, therefore showing a rather stable arrange-
ment of these amino acids. This rmsd value can be
compared with that obtained for the corresponding
residues in apoMb, which is 0.126 ± 0.042 nm. In
order to check that the loss of ordered conformation in
apoMb was not a simulation artifact, a 3 ns molecular
dynamics simulation was performed on the holo form
of the same protein. In this simulation, the helical con-
formation is preserved, as expected during all simula-
tions, because of the additional constraint on the helix
provided by the covalently bonded heme group.
In order to further validate the NMR results, 1.2 ns
molecular dynamics simulations were run on the stud-
ied peptides. The results further confirmed all the
available evidence. For the MbF-wt fragment, the heli-
cal conformation is lost after 500 ps at the N-terminal
residues and the helix is found mostly between residues
Leu86 and Ala94. The missing backbone amide proton
of proline seems to be the determinant of secondary
structure loss at the N-terminus, according to the
typical CO(i)–HN(i + 3), CO(i)–HN(i + 4) hydrogen-

bonding pattern of helical conformations. In contrast,
for the NgbF-wt peptide, the regular helical conforma-
tion is maintained after 500 ps, from Ser83 to Val99.
Interactions among hydrophobic moieties of Leu85,
Leu89, Tyr88 and Leu92 appear to be particularly rele-
vant in conferring stability to the helix.
Ngb helix F
The collection and interpretation of structural data at
neutral and acidic pH conditions highlight the interest-
ing features of human Ngb helix F structuring. Previ-
ous evidence has shown that helix F of apoNgb is
preserved from proteolysis at pH 2 (P. Picotti, unpub-
lished results), suggesting the conservation of its sec-
ondary structure in spite of the extreme conditions. By
contrast, at the same pH value, apoMb underwent
extensive proteolysis [13], whereas, at neutral pH,
proteolytic cleavage occurred only at helix F [10,11]. In
the present work, a clear persistence of the a-helical
structure in strong acidic conditions has also been con-
firmed for the isolated NgbF-wt peptide. A first glance
at the amino acid charge position over this peptide
sequence led us to postulate initially a secondary struc-
ture stabilization as a result of favorable charge inter-
action with the a-helix macrodipole [24,25] of Glu86
and Glu87 side-chains. Hence, decreasing the pH to a
value of 2.1 should have affected the whole helix
stability because of a loss of the side-chain-mediated
electric shielding from Glu86 and Glu87 carboxylates.
As mentioned previously, a decrease in pH decreases
the a-helix extension, from residues 86–92 to 87–91,

but does not totally disrupt it. This means that, in the
addressed sequence, the main helix-nucleating driving
forces are likely to arise from other structuring energy
contributions. One such contribution may arise from
the interactions that could be established in NgbF-wt
between Glu86 and Glu87 amides and the carbonyl
and side-chain oxydryl acceptors of the preceding
serine pair, in particular Ser84 occurring in the ideal
position for N-capping [26] (no similar N-capping
potentiality is present in MbF-wt). However, our exper-
imental evidence does not support this N-capping
occurrence in NgbF-wt, but rather hydrophobic inter-
actions. Medium-range interactions revealed by NMR
NOESY spectra at both pH conditions involve princi-
pally Tyr88, Leu89 and Leu92, arranged in a helical
geometry with an ideally suited separation between
Tyr88 and Leu92. This experimental evidence is
compatible with the hydrophobic scaffold-mediated
hypothesis advanced above.
The limited proteolysis pattern observed for apoNgb
after 30 min of incubation with thermolysin is in line
with this interpretation. Indeed, the proteolytic cleav-
age affecting helix F, which is unprotected as a result
of the loss of segment 99–151, occurs between residues
Ser91 and Leu92, i.e. at the C-end of the proposed
hydrophobic scaffold, despite the fact that an even
more favorable thermolysin proteolytic site can be rec-
ognized between Tyr88 and Leu89. Interestingly,
equivalent results were also observed when thermolysin
hydrolysis was performed on the isolated NgbF-wt

peptide (Fig. S3, see Supporting information). Indeed,
by aligning the known Ngb sequences obtained from
the UniProtKB ⁄ Swiss-Prot database (http://www.
expasy.org/cgi-bin/get-similar?name=globin%20family),
a general motif can be recognized to occur in all
F-helices: {L
82
-[SH]-[ST]-L-E-[ED]-[YF]-L-X-X-L-G-
[R,K]-K-H-[R,Q]-A
98
}. In addition to the invariant
His96 (proximal HisF8), which is expected because of
its essential role in heme coordination, Leu92 (LeuF4)
is also well conserved in the globin family [27] and,
indeed, it has structural relevance in maintaining the
position of the His96 imidazole ring [7]. In addition,
the Ngb subfamily is specifically characterized by the
Globin helix F conformational stability L. Codutti et al.
5184 FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS
occurrence of a conserved aromatic residue at position
88 (tyrosine or phenylalanine). The recognized motif
also presents phylogenetic persistence of negatively and
positively charged (at physiological pH) clusters close
to the N- and C-termini of the helix, respectively.
Finally, the presence of conserved leucines, three to
four residues apart, is most noticeable, an arrangement
that creates, with the mentioned aromatic residue at
position 88, the hydrophobic face of an amphipathic
helix (see Fig. 9). The regularly spaced leucine residues
are likely to contribute to the extension of the helix F

N-terminal side via hydrophobic stacking. This is con-
sistent with the NMR evidence obtained for NgbF-wt
in aqueous TFE (10%), which suggests some propen-
sity to helix elongation, namely to a helix also involv-
ing the N-terminal fragment 79–85 (Figs. S1 and S2,
see Supporting information), in agreement with the
conspicuous helix content increase also observed by
CD under similar conditions. Inspection of the crystal
structure of human Ngb reveals the relevance of the
helix F amphipathicity. Although the heme surface
contacts the upper side of the helix F hydrophobic
face, a crucial contact involves Leu89, i.e. the first leu-
cine of the helix F hydrophobic scaffold, and Met144
of helix H (Cc
89
–Cc
144
= 0.441 nm). As Met144 is
invariant within Ngb sequences, it can be proposed
that the positioning of the helix F hydrophobic scaf-
fold may be dictated by helix H, i.e. a strongly persis-
tent secondary structure element that has always been
recognized to be involved in the early folding events,
at least in Mb [12] and leghemoglobin [16].
Conclusions
The inherent conformational properties of isolated
protein fragments have often been used to analyse pro-
tein folding pathways [28]. As fragments cannot
develop the long-range interactions of native proteins
that usually form along the folding pathway of the

whole protein chain, the propensity of a protein frag-
ment to adopt a precise secondary structure appears to
be relevant to the early protein folding events. The
results of this study indicate that a peptide encompass-
ing helix F of Ngb has a strong propensity to adopt
an a-helical secondary structure in water solution, as
given by far-UV CD and NMR measurements. As the
isolated Ngb helix F autonomously forms a hydropho-
bic helical scaffold, we advance the hypothesis that
hydrophobic interactions within the core segment
88–92 of Ngb helix F could represent the primary
helix-forming driving force that contributes to the ini-
tial helix core during the folding of apoNgb. In partic-
ular, the presence of a conserved tyrosine residue at
position 88 appears to provide a very stable arrange-
ment of nearby residue side-chains in helix F, thus
making the helical geometry quite stable. In addition,
the hydrophobic scaffold 88–92 appears to be suitably
located to establish a favourable hydrophobic contact
with a conserved residue of helix H, probably an early-
folding one by analogy with previous evidence [12,16].
Although helix F formation seems to occur early in
the folding pathway of Ngb, all models of apoMb
folding pathways so far developed do not include the
structuring of helix F [16,23,29–31]. Molecular dynam-
ics simulations also support this difference. Therefore,
we suggest a different folding pathway for Ngb and
Mb, with helix F being an early nucleating folding
core in Ngb, rather than the last folding step as in
Mb, where helix F is formed only on addition of the

heme moiety.
The globin family has been used previously as an
excellent experimental system for analysing protein
folding mechanisms, as the helical globin fold is highly
conserved between proteins with widely differing
amino acid sequences [32]. It has been proposed that
the folding pathways of evolutionarily related proteins
with similar three-dimensional structure, but different
sequences, should be similar [33,34]. Mb and Ngb
share an almost superimposable three-dimensional fold
and show a low degree of sequence identity (less than
20% over the whole sequence for the human proteins).
In this study, we conclude instead that, despite the
Fig. 9. A ball-and-stick view of the crystallographic structure of
human Ngb helix F with a heme ring. Hydrogen bonds are high-
lighted in green with the corresponding distances calculated by
Swiss PDB viewer.
L. Codutti et al. Globin helix F conformational stability
FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS 5185
strong similarity of the overall fold of Mb and Ngb,
these two proteins display different folding pathways.
A similar scenario has emerged already from the com-
parison of Mb and leghemoglobin, two proteins with
the same type of folded structure, but adopting differ-
ent folding pathways [16]. These two proteins form
rapidly compact helical folding intermediates that
direct the overall folding pathway of the whole poly-
peptide chain, but the details of the pathways are dif-
ferent and depend on the local amino acid sequences.
Although apoMb forms an A(B)GH helical intermedi-

ate [29], leghemoglobin initially forms an intermediate
given by helices G and H and part of helix E [16].
Moreover, recently, it has been shown that the molecu-
lar details of the intermediate formed by leghemoglo-
bin in kinetic experiments differ from those of the
equilibrium molten globule intermediate [35]. There-
fore, individual proteins, such as Ngb, Mb or leghemo-
globin, despite their overall fold similarity, can follow
different folding pathways dictated by the solution
conditions and differences in amino acid sequences
[36].
Experimental procedures
Materials
Thermolysin from Bacillus thermoproteolyticus was pur-
chased from Sigma (St. Louis, MO, USA). Solvents, resin
and coupling reagents for peptide synthesis were obtained
from Applied Biosystems (Foster City, CA, USA). All pro-
tected amino acids were purchased from Novabiochem
(Laufelfingen, Switzerland). HPLC-grade solvents were
obtained from Merck (Darmstadt, Germany).
The expression and purification of the Ngb mutant
C120S was performed as described previously [37]. The
Cys120 to serine replacement in Ngb was made in order to
avoid protein aggregation processes of the apo form of the
protein (apoNgb) as a result of the formation of an inter-
molecular disulfide bond. The preparation of apoNgb was
obtained from the corresponding holoprotein by the
removal of heme by reverse-phase HPLC separation.
Briefly, the holoprotein was loaded onto a C
18

Vydac col-
umn (4.6 · 250 mm; The Separations Group, Oak Ridge,
TN, USA), eluted with a linear gradient of water–acetoni-
trile, both containing 0.05% (v⁄ v) trifluoroacetic acid
(TFA), from 5 to 40% in 5 min and from 40 to 60% in
25 min, at a flow rate of 0.8 mLÆmin
)1
. The effluent was
monitored by absorption measurements at 226 nm and
fractions containing the protein were pooled and then con-
centrated in a SpeedVac system. The possible contamina-
tion of the apoprotein preparation by the holoprotein was
assessed spectrophotometrically, and no significant absorp-
tion was observed in the Soret region.
Peptide synthesis
The peptides used in this study were designed to reproduce
chain segments 79–100 of human Ngb and 79–97 of sperm-
whale Mb and were produced as N-acetylated and C-ami-
dated species. In addition to the wild-type peptides, two
variants were also studied bearing a single residue replace-
ment. The amino acid sequences of the peptides used herein
are shown in Fig. 1B. The peptides were synthesized by
solid-phase peptide synthesis using an automated peptide
synthesizer (model 431-A; Applied Biosystems). The 9-flu-
orenylmethoxycarbonyl (Fmoc) strategy was used through-
out the peptide chain assembly [38]. As solid support the
4-(2¢,4¢-dimethoxyphenyl-Fmoc-aminomethyl)-phenoxyace-
tamido-norleucylaminomethyl resin (Rink amide AM resin)
(Novabiochem) (loading of 0.74 mmmolÆg
)1

) was used. The
side-chain-protected amino acids used were as follows:
Fmoc-Asp(tert-butyl), Fmoc-Glu(tert-butyl), Fmoc-Ser
(tert-butyl), Fmoc-Thr(tert-butyl), Fmoc-Tyr(tert-butyl),
Fmoc-Gln(trityl), Fmoc-His(trityl), Fmoc-Lys(tert-butyloxy-
carbonyl) and Fmoc-Arg(2,2,4,6,7-pentamethyldihydroben
zofuran-5-sulfonyl). Coupling was performed with a single
reaction for 45 min by a 0.45 m solution in N,N¢-dimethyl-
formamide of 2-(1-benzotriazol-1-yl)-1,1,3,3-tetramethyluro-
nium hexafluorophosphate and N-hydroxybenzotriazole in
the presence of N-ethyldiisopropylamine, following the man-
ufacturer’s protocols. At the end of the solid-phase synthe-
sis, the peptidyl-resins were acetylated by treatment with
10% acetic anhydride in N,N¢-dimethylformamide to yield
an N-acetylated peptide. Cleavage of the crude peptides was
performed by reacting the acetylated peptidyl-resins with
TFA–H
2
O–thioanisole–eth anedithiol–p henol ( 10 mL : 0 .5 mL :
0.5 mL : 0.250 mL : 750 mg) for 2.5 h. The peptides were pre-
cipitated with ice-cold ethyl ether and isolated by centrifuga-
tion. The pellets were washed several times with ether,
dissolved in water and lyophilized. Crude peptides were
purified by a preparative reverse-phase HPLC column
(PrepNova-Pak HR C
18
, 250 mm · 10 cm, 6 lm bead size;
Waters, Milford, MA, USA) at 12 mLÆmin
)1
using a linear

gradient of 5–50% acetonitrile in 0.08% TFA. The molecu-
lar masses of the peptides were confirmed by electrospray
mass ionization mass spectrometry using a Micro Q-Tof
mass spectrometer (Waters, Manchester, UK). The purities
of the purified peptides were 98% as evaluated by analytical
reverse-phase HPLC.
Proteolysis experiments
Limited proteolysis experiments with thermolysin were con-
ducted on apoNgb at 25 °C with the proteins dissolved
(0.5 mgÆmL
)1
)in50mm Tris-HCl, 0.1 m NaCl, 1 mm
CaCl
2
, pH 7.0, using an enzyme to substrate ratio of
1 : 100 (by weight). At time intervals, aliquots were taken
from the reaction mixture and proteolysis was stopped by
the acidification of the solutions by adding TFA (final con-
Globin helix F conformational stability L. Codutti et al.
5186 FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS
centration, 0.1%). The proteolysis mixtures were then sepa-
rated by reverse-phase HPLC utilizing a C
18
Vydac column
in the experimental conditions described previously. The
identity of the fragments was established by electrospray
mass ionization mass spectrometry using a Micro Q-Tof
mass spectrometer (Waters).
CD spectroscopy
The CD spectra were recorded at 25 °C with a J-710 spectro-

polarimeter (Jasco Corporation, Tokyo, Japan) equipped
with a thermostatically controlled cell holder. The instru-
ment was calibrated with d-(+)10-camphorsulfonic acid.
CD measurements in the far-UV region were performed at a
peptide concentration of 0.15 mgÆmL
)1
in a quartz cell (path
length, 1 mm) at 25 °C. CD spectra were recorded in 50 mm
Tris-HCl, 0.1 m NaCl, pH 7.3, or in 10 mm HCl, pH 2.0.
The results were expressed as the mean residue ellipticity [h]
(degÆcm
2
Ædmol
)1
), calculated from the formula [h] = 0.1 ·
h
obs
· (MRW ⁄ lc), where h
obs
is the observed ellipticity at a
given wavelength, MRW is the mean residue molecular mass
(protein molecular mass divided by the number of amino
acid residues), l is the optical path length in centimeters and
c is the protein concentration in grams per milliliter. The per-
centage content of the a-helical structure in the proteins was
estimated from the far-UV CD spectra according to Scholtz
et al. [39]. The concentrations of proteins and peptides were
determined from their UV absorbance at 280 nm using a
Perkin-Elmer Lambda-20 spectrophotometer.
NMR spectroscopy

1
H NMR spectra were collected at 500.13 MHz with a Bru-
ker Avance 500 NMR spectrometer operated by a Bruker
TopSpin platform (Bruker Gmbh, Karlsruhe, Germany)
from samples prepared in aqueous solutions (H
2
O–D
2
O,
90 : 10) with 100 mm NaCl, 70 mm phosphate buffer and
0.13 mm dioxane at a peptide concentration of 1.2 mm.
Spectra were acquired at pH 6.3 and pH 2.1 at 298 K. A set
of experiments was also performed by adding 10% (v ⁄ v)
TFE-d4 to the aqueous solution at pH 6.3. A number of
two-dimensional total correlation spectroscopy (TOCSY)
[40], double-quantum filtered correlation spectroscopy
(DQF-COSY) [41] and NOESY [42] spectra were acquired
with solvent suppression obtained by WATERGATE excita-
tion sculpting [43,44], using a 1 s steady-state recovery time
and mixing times (t
m
) of 40–50 ms for TOCSY and 160 or
250 ms for NOESY. t
1
quadrature detection was achieved
through the proportional phase incrementation (TPPI)
scheme [45]. Isotropic mixing of the TOCSY experiment
was obtained with MLEV17 [46] pulse trains at cB
2
⁄ 2p =

7–10 kHz. Acquisitions were performed over a spectral
width of 5482.4 Hz in both dimensions, with a typical matrix
size of 2048 points in t
2
and 512 points in t
1
and 32–64
scans ⁄ t
1
free induction decay (FID). Data processing and
analysis were performed using felix software (Accelrys, San
Diego, CA, USA) by applying shifted (75–90º) square sine-
bell apodization, zero-filling up to 2048 · 2048 real points
and FLATT baseline correction [47] for NOESY data. All
spectra were referenced on the internal dioxane resonance at
3.75 p.p.m. After assignment, all chemical shift values were
deposited into the BMRB databank (b.
wisc.edu/) with accession code 15565.
Collection of experimental restraints from NMR
data
Internuclear distances were quantified from the cross-peak
volumes of the NOESY spectra collected with t
m
= 250 ms,
and processed with 90º square sinebell apodization in both
dimensions. The typical cross-peaks of the a-helical struc-
tures a
i
b
i +3

and a
i
N
i +3
, or, when not available, the
N
i
N
i +1
cross-peaks were used as calibrant for an initial
tentative assessment of the internuclear separations. Subse-
quently, the distance restraints were categorized as strong
when less than 0.25 nm, medium when less than 0.35 nm
and weak when more than 0.35 nm for dynamics calcula-
tions purposes. The criterion for calibrant peak selection
was imposed by the lack of proper intraresidual connectivity
and should not introduce bias in structural calculations
because of the adopted gross quantification scheme. When-
ever recognizable cross-peak overlap exceeded 30%, the
corresponding connectivities were considered only as
qualitative proximity indicators (£ 6A
˚
, i.e. weak).
Structure generation procedures
All the available experimental information was used as
input for restrained molecular dynamics simulations, per-
formed using the program cyana 2.1 [48]. cyana runs were
performed according to the default protocol for simulated
annealing (6000 steps torsion angle dynamics, 2000 conju-
gate gradients minimizing steps) with 380 randomly gener-

ated starting conformations. Standard pseudo-atom
corrections were applied [49]. The 20 best structures of the
cyana ensemble were submitted to restrained minimization
using discover (msi) with the amber force field [50]. The
quality of the final structures was assayed by means of the
software aqua and procheck-nmr. All structures were
visualized within molmol [51], which has also been used
for rmsd calculations.
Secondary structure predictions
Helical propensities of the polypeptide chains of Mb and
Ngb at pH 7.0 or pH 2.0 were calculated using the agadir
algorithm [17] available at the website l-
heidelberg.de/services/serrano/agadir/agadir-start.html. The
parameters used were as follows: pH 7.0, ionic strength
L. Codutti et al. Globin helix F conformational stability
FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS 5187
0.05 m,25°C, or pH 2.0, ionic strength 0.01 m,25°C.
Predicted helical propensities were plotted along the poly-
peptide chain of the proteins.
Molecular dynamics simulations
Three molecular dynamics simulations were performed
starting from: (a) the crystal structure of Mb [Protein Data
bank (PDB) code 1YMB]; (b) the crystal structure of Mb
(PDB code 1YMB) with the heme group removed; and (c)
the conformation of chain A in the crystal structure of Ngb
(PDB code 1OJ6) with the heme group removed. Protons
were added using the pdb2gmx utility of the gromacs sim-
ulation package [52]. The protonated structure was then
used to generate a topology and coordinate file using the
psfgen utility of namd simulation software [53]. The struc-

ture was used to compute the electrostatic potential around
the molecule using the program uhbd [54]. In order to neu-
tralize the protein charge ()2 for holoMb and )4 for
apoNgb), a sodium ion was placed in the most negative
potential point on a surface enclosing the molecule at a dis-
tance of 0.7 nm from any heavy atom of the protein. The
calculation and placement of ions were repeated until the
global charge was zero. The system was then relaxed by
200 conjugate gradient minimization steps. The dielectric
was set to 10.0 in order to minimize the effect of missing
solvent and the cut-off was 1.2 nm. The minimized system
was solvated using the module solvate in the vmd software
package [55] in a box with margins at 0.5 nm distance from
any solute atom. The systems contained approximately
25 000 atoms. The solute molecules, including ions, were
fixed and the system was energy minimized by 300 conju-
gate gradient steps using periodic boundary conditions and
the particle mesh Ewald (PME) method for electrostatic
interactions [56]. PME employed a grid of 128 · 128 · 128
points. The PME tolerance was set to 10
)5
nm
)1
which,
together with the cut-off of 1.2 nm, resulted in a Ewald
coefficient of 2.57952 nm
)1
. The minimized system was
further relaxed, keeping the solute (including the ion) fixed,
by molecular dynamics simulation. The system was heated

to 300 K in 2 ps, and a further 18 ps simulation was run in
order to let the water molecules reorient, consistent with
the average lifetime of a hydrogen bond in water [57]. The
system without restraints was energy minimized by 300 con-
jugate gradient minimization steps. The system was then
heated to 300 K in 2 ps, and a further 118 ps simulation
was run in order to let the system equilibrate, and finally
the production run could start. The systems were then sim-
ulated for 3 ns. In all molecular dynamics simulations, the
temperature was kept constant through a simple velocity
rescaling procedure, and the pressure was controlled
through a Berendsen bath [58] using a relaxation time of
100 fs. The volume of the box fluctuated by about
250 nm
3
, with a standard deviation of less than 0.0025 of
its value. All structural analyses, in particular rmsds, sec-
ondary structure and angular order parameter analyses,
were performed using the program molmol [51].
Acknowledgements
This work was financially supported by MIUR
(RBNE03PX83) and EU (LSHM-CT-2005-037525).
The suggestions of Dr A. Makek are acknowledged.
References
1 Wittenberg BA & Wittenberg JB (1989) Transport of
oxygen in muscle. Annu Rev Physiol 51, 857–878.
2 Perutz MF (1990) Mechanisms regulating the reactions
of human hemoglobin with oxygen and carbon mon-
oxide. Annu Rev Physiol 52, 1–25.
3 Minning DM, Gow AJ, Bonaventura J, Braun R,

Dewhirst M, Goldberg DE & Stamler JS (1999) Ascaris
hemoglobin is a nitric oxide-activated ‘deoxygenase’.
Nature 401, 497–502.
4 Flo
¨
gel U, Merx MW, Go
¨
decke A, Decking UK &
Schrader J (2001) Myoglobin: a scavenger of bioactive
NO. Proc Natl Acad Sci USA 98, 735–740.
5 Burrmester T, Weich B, Reinhardt S & Hankein T
(2000) A vertebrate globin expressed in the brain.
Nature 407, 520–523.
6 Kawada N, Kristensen DB, Asahina K, Nakatani K,
Minamiyama Y, Seki S & Yoshizato K (2001) Charac-
terization of a stellate cell activation-associated protein
(STAP) with peroxidase activity found in rat hepatic
stellate cells. J Biol Chem 276, 25318–25323.
7 Pesce A, Dewilde S, Nardini M, Moens L, Ascenzi P,
Hankein T, Burmester T & Bolognesi M (2003) Human
brain neuroglobin structure reveals a distinct mode of
controlling oxygen affinity. Structure 11, 1087–1095.
8 Hughson FM, Wright PE & Baldwin RL (1990) Struc-
tural characterization of a partly folded apomyoglobin
intermediate. Science 249, 1544–1548.
9 Eliezer D & Wright PE (1996) Is apomyoglobin a
molten globule? Structural characterization by NMR.
J Mol Biol 263, 531–538.
10 Fontana A, Zambonin M, Polverino de Laureto P, De
Filippis V, Clementi A & Scaramella E (1997) Probing

the conformational state of apomyoglobin by limited
proteolysis. J Mol Biol 266, 223–230.
11 Picotti P, Marabotti A, Negro A, Musi V, Spolaore B,
Zmbonin M & Fontana A (2004) Modulation of the
structural integrity of helix F in apomyoglobin by
single amino acid replacements. Protein Sci 13, 1572–
1585.
12 Nishimura C, Dyson HJ & Wright PE (2002) The
apomyoglobin folding pathway revisited: structural
heterogeneity in the kinetic burst phase intermediate.
J Mol Biol 322, 483–489.
Globin helix F conformational stability L. Codutti et al.
5188 FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS
13 Picotti P, De Franceschi G, Frare E, Spolaore B, Zam-
bonin M, Chiti F, Polverino de Laureto P & Fontana
A (2007) Amyloid fibril formation and disaggregation
of fragment 1–29 of apomyoglobin: insight into the
effect of pH on protein fibrillogenesis. J Mol Biol 367,
1237–1245.
14 Gilmanshin M, Gulotta M, Dyer RB & Callender RH
(2001) Structures of apomyoglobin’s various acid-desta-
bilized forms. Biochemistry 40, 5127–5136.
15 Reymond MT, Merutka G, Dyson HJ & Wright PE
(1997) Folding propensities of peptide fragments of
myoglobin. Protein Sci 6, 706–716.
16 Nishimura C, Prytulla S, Dyson HJ & Wright PE
(2000) Conservation of folding in evolutionary distant
globin sequences. Nat Struct Biol 7, 679–686.
17 Munoz V & Serrano L (1997) Development of the mul-
tiple sequence approximation within the AGADIR

model of alpha-helix formation: comparison with
Zimm–Bragg and Lifson–Roig formalisms. Biopolymers
41, 495–509.
18 Wu
¨
thrich K (1986) NMR Spectroscopy of Proteins and
Nucleic Acids. Wiley & Sons, New York.
19 Laskowski RA, Rullmann JAC, MacArthur MW,
Kaptein R & Thornton JM (1996) AQUA and PRO-
CHECK-NMR: programs for checking the quality of
protein structures solved by NMR. J Biomol NMR 8,
477–486.
20 Wishart DS, Bigam CG, Holm A, Hodges RS & Sykes
BD (1995)
1
H,
13
C and
15
N random coil NMR chemical
shifts of the common amino acids. I. Investigations of
nearest-neighbor effects. J Biomol NMR 5, 67–81.
21 Wishart DS, Sykes BD & Richards FM (1992) The
chemical shift index: a fast and simple method for the
assignment of protein secondary structure through
NMR spectroscopy. Biochemistry 31, 1647–1651.
22 Sirangelo I, Tavassi S, Martelli PL, Casadio R & Irace
G (2000) The effect of tryptophanyl substitution on
folding and structure of myoglobin. Eur J Biochem 267,
3937–3945.

23 Onufriev A, Case DA & Bashford D (2003) Structural
details, pathways, and energetics of unfolding apomyo-
globin. J Mol Biol 325, 555–567.
24 Blagdon DE & Goodman M (1975) Letter: mechanisms
of protein and polypeptide helix initiation. Biopolymers
14, 241–245.
25 Shoemaker KR, Kim PS, Brems DN, Marqusee S,
York EJ, Chaiken IM, Stewart JM & Baldwin RL
(1985) Nature of the charged-group effect on the stabil-
ity of the C-peptide helix. Proc Natl Acad Sci USA 82,
2349–2353.
26 Aurora R & Rose GD (1998) Helix capping. Protein
Sci 7, 21–38.
27 Ota M, Isogai Y & Nishikawa K (1997) Structural
requirement of highly-conserved residues in globins.
FEBS Lett 415, 129–133.
28 Wright PE, Dyson HJ & Lerner RA (1988) Conforma-
tion of peptide fragments of proteins in aqueous solu-
tion: implications for initiation of protein folding.
Biochemistry 27, 7167–7175.
29 Eliezer D, Yao J, Dyson HJ & Wright PE (1998) Struc-
tural and dynamic characterization of partially folded
states of apomyoglobin and implications for protein
folding. Nat Struct Biol 5, 148–155.
30 Jamin M & Baldwin RL (1996) Refolding and unfold-
ing kinetics of the equilibrium folding intermediate of
apomyoglobin. Nat Struct Biol 3, 613–618.
31 Jamin M (2005) The folding process of apomyoglobin.
Protein Pept Lett 12, 229–234.
32 Bashford D, Chothia C & Lesk AM (1987) Determi-

nants of a protein fold: unique features of the globin
amino acid sequences. J Mol Biol 196, 199–216.
33 Krebs H, Schmid FX & Jaenicke R (1983) Folding of
homologous proteins: the refolding of different
ribonucleases is independent of sequence variations,
proline content and glycosylation. J Mol Biol 169,
619–635.
34 Stackhouse TM, Onuffer JJ, Matthews CR, Ahmed SA
& Miles EW (1988) Folding of homologous proteins:
conservation of the folding mechanism of the alpha
subunit of tryptophan synthase from Escherichia coli,
Salmonella typhimurium and five interspecies hybrids.
Biochemistry 27, 824–832.
35 Nishimura C, Dyson HJ & Wright PE (2008) The
kinetic and equilibrium molten globule intermediates of
apoleghemoglobin differ in structure. J Mol Biol 378,
715–725.
36 Nishimura C, Lietzow MA, Dyson HJ & Wright PE
(2005) Sequence determinants of a protein folding path-
way. J Mol Biol 351, 383–392.
37 Dewilde S, Kiger L, Burmester T, Hankeln T,
Baudin-Creuza V, Aerts T, Marden MC, Caubergs R &
Moens L (2001) Biochemical characterization and
ligand binding properties of neuroglobin, a novel mem-
ber of the globin family. J Biol Chem 276, 38949–38955.
38 Fields GB & Noble RL (1990) Solid phase peptide
synthesis utilizing 9–fluorenylmethoxycarbonyl amino
acids. Int J Pept Protein Res 35
, 161–214.
39 Scholtz JM, Hong Q, York EJ, Stewart JM & Baldwin

RL (1991) Parameters of helix-coil transition theory for
alanine-based peptides of varying chain lengths in
water. Biopolymers 31, 1463–1470.
40 Braunschweiler L & Ernst RR (1983) Coherence trans-
fer by isotropic mixing: application to proton correla-
tion spectroscopy. J Magn Reson 53, 521–528.
41 Piantini U, Sorensen OW & Ernst RR (1982) Multiple
quantum filters for elucidating NMR coupling net-
works. J Am Chem Soc 104, 6800–6801.
42 Jeener J, Meier BH, Bachmann P & Ernst RR (1979)
Investigation of exchange processes by two-dimensional
NMR spectroscopy. J Chem Phys 71, 286–292.
L. Codutti et al. Globin helix F conformational stability
FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS 5189
43 Piotto M, Saudek V & Sklenar V (1992) Gradient-tai-
lored excitation for single-quantum NMR spectroscopy
of aqueous solutions. J Biomol NMR 2, 661–665.
44 Hwang TL & Shaka AJ (1995) Water suppression that
works. Excitation sculpting using arbitrary waveforms
and pulsed field gradients. J Magn Reson 112, 275–279.
45 Marion D & Wu
¨
thrich K (1983) Application of phase
sensitive two-dimensional correlated spectroscopy
(COSY) for measurements of proton–proton spin–spin
coupling constants in proteins. Biochem Biophys Res
Commun 113, 967–974.
46 Bax A & Davis DG (1985) MLEV-17 based two dimen-
sional homonuclear magnetization transfer spectros-
copy. J Magn Reson 65, 355–360.

47 Gu
¨
ntert P & Wuthrich K (1992) FLATT – a new proce-
dure for high-quality baseline correction of multidimen-
sional NMR spectra. J Magn Reson 96, 403–407.
48 Gu
¨
ntert P (2004) Automated NMR protein structure
calculation with CYANA. Methods Mol Biol 278, 353–
378.
49 Wu
¨
thrich K, Billeter M & Braun W (1983) Pseudo-
structures for the 20 common amino acids for use in
studies of protein conformations by measurements of
intramolecular proton–proton distance constraints with
nuclear magnetic resonance. J Mol Biol 169, 949–961.
50 Weiner SJ, Kollman PA, Case DA, Singh UC, Ghio C,
Alagona G, Profeta S Jr & Weiner P (1984) A new
force field for molecular mechanical simulation of
nucleic acids and proteins. J Am Chem Soc 106, 765–
784.
51 Koradi R, Billeter M & Wu
¨
thrich K (1996) MOLMOL:
a program for display and analysis of macromolecular
structure. J Mol Grap 14, 51–55.
52 Berendsen HJC, van der Spoel D & van Drunen R
(1995) Gromacs: a message-passing parallel molecular
dynamics implementation. Comput Phys Commun 91,

43–56.
53 Kale L, Skeel R, Bhandarkar M, Brunner R, Gursoy A,
Krawetz N, Phillips J, Shinozaki A, Varadarajan K &
Schulten K (1999) NAMD2: greater scalability for paral-
lel molecular dynamics. J Comput Phys 151, 283–312.
54 Madura JD, Briggs JM, Wade R, Davis ME, Luty BA,
Ilin A, Antosiewicz JA, Gilson MK, Bagheri B, Ridg-
way Scott L et al. (1995) Electrostatics and diffusion of
molecules in solution: simulations with the University
of Houston Brownian Dynamics program. Comput
Commun Phys 91, 57–95.
55 Humphrey W, Dalke A & Schulten K (1996) VMD
visual molecular dynamics. J Mol Graph 14, 33–38.
56 Darden T, York D & Pedersen L (1993) Particle mesh
Ewald. An N log(N) method for Ewald sums in large
systems. J Chem Phys 98, 10089–10092.
57 Keutsch FN & Saykally RJ (2001) Water clusters:
untangling the mysteries of the liquid, one
molecule at a time. Proc Natl Acad Sci USA 98,
10533–10540.
58 Berendsen HJC, Postma JPM, van Gunsteren WF,
Di Nola A & Haak JR (1984) Molecular dynamics
with coupling to an external bath. J Chem Phys 81,
3684–3690.
Supporting information
The following supplementary material is available:
Fig. S1. Restraint distribution along the sequence of
the NgbF-wt peptide obtained at pH 6.3 and 10%
TFE.
Fig. S2. Secondary structure diagnostic restraints

obtained at pH 6.3 and 10% TFE.
Fig. S3. Limited proteolysis of peptide NgbF-wt at
neutral pH.
Table S1. Validation parameters for NMR structures.
This supplementary material can be found in the
online article.
Please note: As a service to our authors and readers,
this journal provides supporting information supplied
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may be re-organized for online delivery, but are not
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from supporting information (other than missing files)
should be addressed to the authors.
Globin helix F conformational stability L. Codutti et al.
5190 FEBS Journal 276 (2009) 5177–5190 ª 2009 The Authors Journal compilation ª 2009 FEBS