HIV-1 gp41 and gp160 are hyperthermostable proteins
in a mesophilic environment
Characterization of gp41 mutants
Tino Krell
1
, Fre
´de
´ric Greco
1
, Olivier Engel
1
, Jean Dubayle
1
, Joseline Dubayle
1
, Audrey Kennel
1
,
Benoit Charloteaux
2
, Robert Brasseur
2
, Michel Chevalier
1
, Regis Sodoyer
1
and Raphae¨ lle El Habib
1
1
Aventis Pasteur, Marcy l’Etoile, France;
2
Centre de Biophysique Mole
´culaire Nume
´rique, Faculte
´Universitaire des Sciences
Agronomiques de Gembloux, Belgium
HIV gp41(24–157) unfolds cooperatively over the pH range
of 1.0–4.0 with T
m
values of > 100 C. At pH 2.8, protein
unfolding was 80% reversible and the DH
vH
/DH
cal
ratio of
3.7 is indicative of gp41 being trimeric. No evidence for a
monomer–trimer equilibrium in the concentration range of
0.3–36 l
M
was obtained by DSC and tryptophan fluores-
cence. Glycosylation of gp41 was found to have only a
marginal impact on the thermal stability. Reduction of the
disulfide bond or mutation of both cysteine residues had
only a marginal impact on protein stability. There was no
cooperative unfolding event in the DSC thermogram of
gp160 in NaCl/P
i
,pH7.4,overatemperaturerangeof
8–129 C. When the pH was lowered to 5.5–3.4, a single
unfolding event at around 120 C was noted, and three
unfolding events at 93.3, 106.4 and 111.8 Cwereobserved
at pH 2.8. Differences between gp41 and gp160, and
hyperthermostable proteins from thermophile organisms are
discussed. A series of gp41 mutants containing single, dou-
ble, triple or quadruple point mutations were analysed by
DSC and CD. The impact of mutations on the protein
structure, in the context of generating a gp41 based vaccine
antigen that resembles a fusion intermediate state, is dis-
cussed. A gp41 mutant, in which three hydrophobic amino
acids in the gp41 loop were replaced with charged residues,
showed an increased solubility at neutral pH.
Keywords: gp160; gp41; HIV; hyperthermostability; site-
directed mutagenesis.
HIV entry is mediated by the viral envelope proteins gp41
and gp120. Both proteins are derived from the gp160
precursor following proteolytic cleavage [1]. After cleavage,
both proteins remain associated [2] and are trimeric in their
prefusogenic state [3]. gp41 anchors the protein complex to
the viral membrane [4], whereas gp120 binds to the human
cell-surface receptor CD4 and other receptors on the target
cell. This interaction leads to a dissociation of gp120 from
gp41 [5], which induces a conformational change in gp41 [2],
resulting in the fusion of viral and cellular membranes.
Both envelope proteins are vaccine candidates against
HIV. However, the results of the world’s first phase III
efficacy trial using gp120 were relatively modest [6] and
efforts are now being made to explore the vaccine potential
of gp41. The sequence of gp41 contains four functional
regions, as follows: the N-terminal fusion peptide (which
is thought to insert into the host cell membrane) is followed
by an ectodomain, a transmembrane region and a cyto-
plasmic domain.
The ectodomain of gp41 is thought to adopt at least two
different tertiary structures [7]: a prefusogenic state, and a
lower energy fusion-active (fusogenic) state. The decrease in
free energy during the transition into the latter state is
thought to account for the energy necessary for the fusion of
viral and cellular membranes. There is now evidence that the
recombinantly produced gp41 ectodomain adopts sponta-
neously this fusogenic state, and 3D structural information
on gp41 is entirely on this lower energy state [8–11]. The
fusogenic state of the gp41 ectodomain is a trimeric coiled-
coil protein in which the three helices C pack in an
antiparallel manner against the central trimer of parallel
helices N.
It has been shown that gp41 produced in Escherichia coli
forms insoluble aggregates at neutral pH [12], and aggre-
gation is proposed to occur at the loop region connecting
helices N and C [13]. This is consistent with the fact that
recombinant constructs of gp41, which have this loop
replaced with a small flexible linker (gp41 models), are
soluble at neutral pH. Therefore, a large proportion of
biochemical and biophysical studies on gp41 have been
carried out using either a gp41 model [14–16] or a
stoichiometric mixture of peptides corresponding to the
N- and C-terminal helices, which assemble to hetero-
hexamers in a native-like manner [17,18]. However, recom-
binant gp41 was shown to be soluble at a pH of < 3.5, and
several studies have been carried out at acidic pH [19,20].
The NMR structure of gp41, at pH 3.0 [9], and its X-ray
structure, using crystals grown at pH 4.25 [21], are identical
to structures of gp41 fragments determined at pH values of
4.6–8.0 [8,11,16,22]. This demonstrates that the structure of
gp41 is not altered by acidic pH.
Correspondence to T. Krell, Aventis Pasteur, 1541 avenue Marcel
Me
´rieux, 69280 Marcy l’Etoile, France. Fax: + 33 4 37 37 31 80,
Tel.: + 33 4 37 37 90 12, E-mail: tino.krell@aventis.com
Abbreviations: SIV, simian immunodeficiency virus; TCEP,
Tris(2-carboxyethyl)phosphine.
(Received 8 January 2004, revised 1 March 2004,
accepted 3 March 2004)
Eur. J. Biochem. 271, 1566–1579 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04068.x
Based on the evidence that the structure of gp41 is not
altered by acidic pH, and the functional importance of the
loop region [23] with its conserved cysteine residues [24],
we have carried out our analyses at acidic pH using
recombinant gp41(24–157) containing the cysteine resi-
dues.
The potent HIV entry inhibitor 5-helix [25], a recombin-
ant gp41 trimer lacking a helix C, has been shown to bind
tightly to individual C peptides. It has been proposed that
the mechanism of action of 5-helix is based on the binding to
a fusion intermediate of gp41, which is characterized by an
accessible and exposed helix C [26]. These data can be
regarded as evidence that a protein is capable of blocking
fusion intermediates of gp41, which consequently leads to
the inhibition of virus entry. Our vaccine approach is based
on the generation of gp41 mutants, which resemble the
fusion intermediate state. It is generally accepted that this
intermediate state is trimeric and that helices N and C do
not interact. Here we explore ways to stabilize the
interaction between helices N, to favour a trimeric state
and, on the other hand, to destabilize the interaction
between helices N and C in order to prevent helix contact.
The study of recombinant gp41, either as separate
glycosylated or nonglycosylated protein, or as part of
gp160, by biophysical techniques, forms the first part of this
article. In the second part, site-directed mutagenesis data of
gp41 are presented with the aim of assessing the influence of
amino acid replacements on protein stability and solubility.
Materials and methods
Materials
Gp41(24–157). Recombinant ectodomain of gp41 corres-
ponding to amino acids 537–669 of the envelope protein of
the LAI isolate (p03375) with a C-terminal extension of
amino acids GGGGSHHHHHH. For details of protein
expression and purification see below.
Gp41(34–170). Glycosylated recombinant ectodomain of
gp41 purchased from Tebu-bio (Le Perray en Yvelines
Cedex, France) corresponding to amino acids 546–682 of
the envelope protein of the HxB2 isolate (p04578). The
protein has been expressed in Pichia pastoris. The gp41
domains of isolates BH10 and HxB2 share 99% sequence
identity.
Gp160. Fusion of amino acids 30–500 of the envelope
protein of the isolate MN (comprising gp120) with amino
acids 501–744 (comprising a large part of gp41) of the LAI
isolate. The amino acid sequence of the gp120–gp41
cleavage site KAKRRVVQREKR (502–513 in the LAI
sequence) has been altered to KAQNHVVQNEHQ. This
change prevents cleavage of gp160 into gp120 and gp41.
This protein has been expressed in vaccinia virus grown on
BHK21 cells and was purified by affinity chromatography
on immobilized antibodies. Further details on the construc-
tion and biochemical characterization of this protein are
found in Kieny et al. [27]. This protein has been used for
several HIV vaccine trials [28,29].
TCEP/HCl [Tris(2-carboxyethyl)phosphine hydrochlo-
ride] was purchased from Pierce.
Cloning procedures
A 0.6 kb DNA fragment, containing the sequence encoding
the ectodomain of gp41 of HIV isolate LAI (amino acids
537–669), was obtained by PCR amplification using, as
template, a plasmid containing the gp120 sequence of MN
and the gp41 sequence of the LAI isolate. Restriction sites
for BspHI and XhoI (shown in italics) were, respectively,
included in the forward and reverse primers, as follows:
forward primer: 5¢-CTCTTTCATGACGCTGACGGTA
CAGGCC-3¢; reverse primer: 5¢-CCGCTCGAGCTAATG
GTGATGGTGATGGTGTGACCCTCCCCCTCCACT
TGCCCATTTATCTAA-3¢. The start codon in the for-
ward primer (in bold) is a naturally occurring methionine
residue. A stop codon (in bold) and the DNA sequence
encoding the extension GGGGSHHHHHH (underlined)
were added to the reverse primer. Platinum HF polymerase
(Gibco Invitrogen Corp.) was used, according to the
manufacturer’s instructions, for PCR amplification.
The PCR amplified fragment was cloned directly into the
vector pM1800 using the restriction sites NcoIandXhoI.
The expression vector pM1800 is a derivate of pET28c
(Novagen), in which the F1 origin of replication has been
deleted and replaced with the Cer fragment, allowing for
multimer resolution.
Site-directed mutagenesis: single and multiple point
mutations
The various point mutations were created using the Quick-
change site-directed mutagenesis kit (Stratagene), using the
instructions provided by the manufacturer. Mutations were
carried out directly on the expression vector containing the
sequence of native gp41, and multiple mutations were
introduced in a sequential manner.
NC and CN fusion mutants
The NC and CN gp41 fusion constructs were generated
using three consecutive PCRs. In the first PCR (for the NC
constructs) the following two partially overlapping DNA
fragments were generated: a fragment corresponding to
helix N containing a C-terminal extension encoding the first
six amino acids of the helix C and a fragment corresponding
to helix C containing an N-terminal extension coding for the
last six amino acids of helix N. In the second reaction, a
stoichiometric mix of both partially overlapping fragments
(no primers added) was submitted to 10 PCR cycles. The
third reaction was carried out with the product of the second
reaction in the presence of primers containing NcoIorXhoI
restriction sites and which are complementary to the 5¢end
of helix N and to the 3¢end of helix C. An equivalent
approach was used to generate the CN fusion constructs.
Expression
For protein expression, E. coli BL21(DE3) was transformed
with the corresponding plasmids. Typically, 1 L cultures
were grown at 37 C on LB (Luria–Bertani) broth supple-
mented with kanamycin (25 lgÆmL
)1
). Protein expression
was induced at an attenuance (D)of0.6at600nm,by
the addition of isopropyl thio-b-
D
-galactoside (Q-BIOgene,
FEBS 2004 Studies on HIV envelope proteins (Eur. J. Biochem. 271) 1567
Illkirch, France) to a final concentration of 1 m
M
.Bacteria
were harvested, 4 h after protein induction, by centrifuga-
tion. For certain mutants, protein expression was optimized
by the replacement of LB medium with Terrific Broth
medium, induction with 0.02–0.1 m
M
isopropyl thio-b-
D
-galactoside and a growth temperature of 30 C.
Protein purification
The bacterial pellet resulting from a 1 L culture was
resuspended at room temperature in 95 mL of 50 m
M
Tris/HCl containing 100 l
M
CompleteTM EDTA-free pro-
tease inhibitor cocktail (Roche Molecular Biochemicals) and
100 lgÆmL
)1
lysozyme (Sigma-Aldrich), pH 8.0, and gently
agitated for 30 min. The bacterial suspension was then
placed on ice and cell lysis was achieved by ultrasound
treatment (4 ·2 min) using a Branson-Sonifer 450. After-
wards, MgCl
2
and Benzonase (Merck) were added to final
concentrations of 1 m
M
and 1 UÆmL
)1
, respectively. The
resulting solution was then centrifuged (20 000 g,30min,
4C). Aliquots of the resulting supernatant and pellet were
analysed by SDS/PAGE and recombinant proteins were
detected by Western blot analysis using a monoclonal
antibody raised against poly-histidine (Novagen). Recom-
binant protein was found to be almost exclusively present in
the pellet. The pellet was resuspended in 100 mL of buffer A
(50 m
M
Tris/HCl, 8
M
urea, 500 m
M
NaCl, 10 m
M
imida-
zole, pH 8.0) and agitated at 4 C for 30 min. After filtration
using a 0.45 lm cut-off filter, protein was loaded onto a 5 ml
Hi-Trap Chelating column (Amersham Pharmacia Biotech),
previously equilibrated in buffer A. After washing with
50 mL of buffer A, protein elution was achieved using a
buffer comprising 50 m
M
Tris/HCl, 8
M
urea, 500 m
M
NaCl
and 500 m
M
imidazole, pH 8.0. Protein refolding was
achieved by dialysis with 50 m
M
formate, pH 2.8. Protein
was then sterile filtered and stored at )45 C.
MALDI-TOF MS
Mass spectrometric analyses were carried out on a Biflex III
MALDI-TOF mass spectrometer (Bruker Daltonics, Wiss-
embourg, France). Samples of native and mutant gp41
(1–3 mgÆmL
)1
)in50m
M
formate, pH 2.8, were diluted
with 30% acetonitrile (v/v) containing 0.07% trifluoroacetic
acid to a final concentration of 0.4 mgÆmL
)1
(protein
solution). A saturated solution of sinapic acid (Sigma-
Aldrich) in 70% acetonitrile (v/v) containing 0.1% trifluoro-
acetic acid was prepared and subsequently diluted
four-fold with the same solvent (matrix solution). Droplets
(1 lL) of a 1 : 1 (v/v) mixture of protein and matrix solution
were deposited on the sample slide and allowed to dry at
room temperature. Positive ion mass spectra were acquired
in the linear mode with pulsed ion extraction. Mass
assignments were based on an external calibration of the
instrument.
DSC
DSC experiments were performed on a MicroCal VP-DSC
apparatus (MicroCal, Northampton, MA, USA). Prior to
analysis, proteins were exhaustively dialyzed against the
buffer stated in the legend of each figure, and degassed. The
dialysis buffer was used for baseline scans and was present
as a reference buffer for the protein scans. The system was
allowed to equilibrate at 5 C for 15 min, and temperatures
from 5 to 129 C were scanned at a rate of 85 C/h.
Thermograms obtained were analysed using the MicroCal
version of
ORIGIN
. The standard deviation indicated for
each parameter corresponds to the error of curve fitting.
Details concerning the calculation of thermodynamic
parameters and instrumentation have been published pre-
viously [30,31].
CD
Far-UV CD measurements were made at 25 CinaJasco
J-810 spectropolarimeter (Tokyo, Japan) using cuvettes
with a pathlength of 0.1 mm. Proteins were exhaustively
dialyzed against 50 m
M
formate, pH 2.8. All proteins were
analysed at a concentration of 60 l
M
and spectra were
corrected using the spectra of the dialysis buffer.
Fluorescence spectroscopy
Measurements of the intrinsic tryptophan fluorescence
were carried out at 25 C using a Kontron SFM 25
spectrofluorimeter (Kontron, Zurich, Switzerland). Unless
stated otherwise, proteins were present at a concentration
of 1 l
M
in 50 m
M
formate, pH 2.8. Emission spectra
between 300 and 400 nm were collected after excitation at
295 nm. Spectra were corrected using the spectra of the
buffer.
Results
Protein expression of native and mutant gp41(24–157)
Histidine tagged gp41(24–157), and a substantial number of
mutants, have been expressed in E. coli. After centrifugation
of the cell lysate, all proteins were present in the pellet, which
was solubilized in buffer containing 8
M
urea. Purification
was also carried out in the presence of chaotropic agents
and refolding was achieved by a simple dialysis into 50 m
M
formate, pH 2.8. The DSC analysis of gp41(24–157) did not
provide evidence of partially or wrongly folded protein (see
below).
Typically, a yield of 60 mg of pure protein per litre of cell
culture was observed for native gp41(24–157). This yield
appeared to be lower for certain gp41 mutants. Multiple
point mutations to the loop region, e.g. as in the triple
mutant L91K/I92K/W103D (see below), did not change
the protein yield.
Analysis of gp41(24–157) by CD, DSC and MALDI-TOF MS
The far-UV CD spectrum of gp41(24–157) in 50 m
M
formate, pH 2.8, is shown in Fig. 1. The spectrum is
literally superimposable to the CD spectrum of glycosylated
gp41(21–166) at pH 7.5, as reported by Weissenhorn et al.
[32]. Both spectra show minima at around 208 and 222 nm,
a crossover in sign at 202 nm, and a maximum at 193 nm,
which are typical characteristics of a protein largely
dominated by a-helix. From the molecular ellipticity at
222 nm, an a-helix content of 75% has been calculated [33],
1568 T. Krell et al.(Eur. J. Biochem. 271)FEBS 2004
which is consistent with the a-helix content of 80%
determined by Weissenhorn et al. for glycosylated gp41(21–
166) at pH 7.5 [32].
Figure 2A (upper trace) shows the DSC thermogram of
gp41(24–157) after the renaturation process by dialysis into
50 m
M
formate, pH 2.8 (see the Materials and methods).
Two unfolding transitions at 110.4 and 119.5 C are seen
(Table 1), demonstrating that this protein is hyperther-
mostable at low pH. The thermal unfolding was highly
cooperative with an DH
vH
/DH
cal
ratio of 3.7 for the major
unfolding event, which is consistent with a cooperative
unfolding of a gp41 trimer. Evidence that gp41 is trimeric
at pH 2.5–3 has previously been demonstrated using gel
filtration [13], analytical ultracentrifugation [19] and NMR
[9].
A major peak corresponding to the monomer, and two
smaller peaks corresponding to covalently linked dimeric
and trimeric forms of gp41(24–157), are seen in the
MALDI-TOF spectrum (Fig. 2B) of the same sample. It
is generally accepted that MALDI-TOF analysis results in
the disruption of all noncovalent interactions and that the
observed multimers correspond to covalently linked multi-
mers. gp41(24–157) contains two cysteine residues which are
involved in an intrasubunit disulfide bond in the native
protein [34]. The reaction of this protein sample with
Ellman’s reagent [35] showed that the two cysteine residues
are engaged in disulfide bonds (data not shown). This is
consistent with the monomer, observed by MS, having an
intrasubunit disulfide bond, whereas the dimer and trimer
peaks indicate the presence of intersubunit disulfide bonds
Fig. 1. Far UV CD spectra of native gp41(24–157) (––) and of the
quadruple mutant W6OA/I124D/I131D/Q142N (- - - -). Proteins were
analyzed at a concentration of 60 l
M
in 50 m
M
formate, pH 2.8.
Fig. 2. Analysis of native gp41(24–157) by DSC and MALDI-TOF
MS. (A) DSC thermograms of gp41(24–157) before and after reduc-
tion with tris(2-carboxyethyl)phosphine (TCEP). Derived thermo-
dynamic parameters are shown in Table 1. (B) MALDI-TOF mass
spectra of gp41(24–157) before and after reduction with TCEP. In
panels (A) and (B), the same samples were used for analysis. The
sequence-derived mass of gp41(24–157) is 16801.5 l. Spectra indicate
that the N-terminal methionine residue has been processed. (C) Study
of the reversibility of the thermal unfolding of gp41(24–157). Shown
are segments of two consecutive DSC up-scans from 5 to 129 C.
Reversibility was defined as: % reversibility ¼(DH
cal2
/DH
cal1
)·
100%, with DH
cal2
being the change of enthalpy from the second
up-scan and DH
cal1
the change of enthalpy from the first up-scan of the
same protein sample. Reversibility data are given in Table 1. For
clarity reasons, DSC thermograms and mass spectra are moved
arbitrarily on the y-axis. Proteins were analyzed in 50 m
M
formate,
pH 2.8.
FEBS 2004 Studies on HIV envelope proteins (Eur. J. Biochem. 271) 1569
which have been described previously for glycosylated
gp41(21–166) [32]. To verify the hypothesis of the presence
of intersubunit disulfide bonds, a gp41(24–157) sample was
analysed after reduction with TCEP, a reagent known to
reduce disulfides selectively over a pH range of 1.5–8.5 [36].
After reduction, only a single unfolding transition was
observed by DSC, which was characterized by a downshift in
T
m
of 1.3 C, with respect to the major peak before reduction
(Fig. 2A, Table 1). This event was equally very cooperative
(DH
vH
/DH
cal
¼3.3), demonstrating that reduction does not
alter the trimeric state of gp41. The peaks corresponding
to covalent dimers and trimers in the mass spectrum of this
sample were significantly decreased as compared to the
sample analysed before reduction (Fig. 2B). These minor
peaks can probably be attributed to the formation of
covalent multimers during ionization in the instrument, a
well-known phenomena of this technique, because multimer
peaks of similar size are observed in the spectrum of the
cysteine-free double mutant, C87S/C93S (data not shown).
This implies that the single transition seen in DSC after
reduction corresponds to the unfolding of noncovalently
associated trimers with reduced disulfide bonds, whereas
the major unfolding event before reduction represents the
unfolding of noncovalently associated trimers with intra-
subunit disulfide bonds. The effect of disulfide reduction on
the thermal stability of gp41 trimers is thus relatively modest
(downshift in T
m
of 1.3 C). The absence of the second
unfolding transition after reduction demonstrates that this
event represents the unfolding of gp41 trimers characterized
by one or several intersubunit disulfide bonds.
In summary, gp41(24–157) is, after renaturation, mainly
present in its native conformation, defined by noncovalently
associated trimers with intrasubunit disulfide bonds. Based
on this result, all subsequent analyses were carried out using
protein taken after renaturation (nonreduced). The param-
eters of the major unfolding transition were used for data
analysis. Furthermore, thermal unfolding of gp41(24–157)
is highly reversible, as calculated from two consecutive scans
of the protein (Fig. 2C).
pH dependence of the thermal unfolding
of gp41(24–157)
gp41(24–157) has been shown to be hyperthermostable in
50 m
M
formate, pH 2.8. To study the pH dependence of
protein unfolding, samples were analysed by DSC, CD and
fluorescence spectroscopy over a pH range of 0.5–5.0.
Protein was dialyzed into 50 m
M
formate and adjusted to
the pH indicated by the addition of concentrated HCl or
NaOH.
The DSC scans of gp41(24–157) at different pH values
areshowninFig.3A.AplotofT
m
and DH
vH
/DH
cal
,asa
function of pH, is shown in Fig. 3B. Cooperative unfolding
events at temperatures of > 100 C are seen in the pH range
of 1.0–4.0. The variation of T
m
as a function of pH is of a
slight valley shape, with the minimum at pH 2.25 (102 C).
The DH
vH
/DH
cal
ratio at pH 1.0–2.5 was 1and
increased to 3–4 at pH 2.8–3.5. This sudden increase was
accompanied by an increase in T
m
(Fig. 3B). To evaluate
whether the increase in DH
vH
/DH
cal
from 1to3
represents an association of monomers to trimers, or
whether this increase corresponds to an increase in coop-
erativity of the unfolding of gp41 trimers, 14 samples of
gp41 at different pH values (1.0–4.0) were analysed by
fluorescence spectroscopy. A gp41 monomer contains eight
tryptophan residues. Three are involved in a buried
tryptophan cluster [11], characterized by the tight packing
of W60 of helix N between W117 and W120 located at
helix C of a neighbouring monomer. Trimer dissociation
into monomers disrupts this cluster, leading to an exposure
of the three buried tryptophan residues to the solvent, giving
rise to a shift in the maximum of the emission spectrum, as
shown for the gp41 model [14]. The maximum of the
fluorescence emission spectrum of gp41 at pH 2.8 was
349 nm (data not shown). This maximum of fluorescence
emission for the analysis of gp41 at pH between 1.0 and 4.0
(data not shown) was unchanged (349 ± 1 nm), suggesting
no disruption of the tryptophan cluster and no trimer
dissociation at this pH range.
Samples of gp41 (all at 60 l
M
) at this pH range have also
been analysed by CD. Over the range of pH 1.0–4.0, spectra
are closely superimposable and no shift in the minima or
maxima is observed (data not shown). Differences in
molecular ellipticity are in the range of the error associated
with determination of the protein concentration.
Monomer–trimer equilibrium
A monomer–trimer equilibrium has been described for the
cysteine double mutant of simian immunodeficiency virus
Table 1. Thermodynamic parameters derived from the DSC analysis of gp41(24–157), gp41(34–170) and gp160 (Figs 2 and 5). ND, not determined.
Sample
T
m
(C)
DH
cal
(kcalÆmol
)1
)
DH
vH
(kcalÆmol
)1
)DH
vH
/DH
cal
Reversibility
(%)
gp41(24–157) nonreduced
a
110.4 61 ± 0.4 228 ± 2 3.7 80
119.5 21 ± 0.5 165 ± 5 7.8 85
gp41(24–157) reduced (36 l
M
)
b
109.1 94 ± 0.4 308 ± 2 3.3 85
gp41(24–157) reduced (1 l
M
)
b
109.1 91 ± 1.0 303 ± 4 3.3 ND
gp41(34–170), glycosylated
c
112.6 38 ± 0.5 201 ± 4 5.3 25
gp160
c
93.3 71 ± 0.6 134 ± 1 1.9 95
106.4 32 ± 1.5 218 ± 6 6.8 100
111.8 41 ± 1.5 191 ± 7 4.6 0
a
Non-reduced protein corresponds to gp41(24–157) after the dialysis step into 50 m
M
formate at pH 2.8 (Materials and methods).
b
Reduction was carried out by overnight dialysis of the protein into 50 m
M
sodium formate, 150 l
M
Tris(2-carboxyethyl)phosphine (TCEP)
at 4 C.
c
See the Materials and methods for a description of the protein.
1570 T. Krell et al.(Eur. J. Biochem. 271)FEBS 2004