Download Molecular Characterization by Mass Spectrometry of the Human

Molecular Characterization
by
Mass Spectrometry
of the Human
Estrogen Receptor Ligand-Binding
Domain Expressed in Escherichia
co/i
Donald A. Seielstad, Kathryn E. Carlson, John A. Katzenellenbogen, Peter J. Kushner, and Geoffrey L. Greene
Department of Chemistry
University of Illinois
Urbana, Illinois 61801
(D.A.%
K.E.C., J.A.K.)
Metabolic Research Unit (P.J.K.)
University of California
San Francisco, California 94143
Ben May Institute (G.L.G.)
University of Chicago
Chicago, Illinois 60637
The ligand binding domain of the human estrogen
receptor
(hER-LBD),
encompassing
the sequence
MDP!$,&G
. . . V,,, has been expressed
at high
levels in Escherichia co/i from a PET-23d vector,
and a purified preparation
has been characterized
both by mass spectrometry
and biochemical
methods. Inclusion bodies from the bacterial expression
were solubilized
by sonication
and the hER-LBD
was purified to near homogeneity
by affinity chromatography
over an estradiol-Sepharose
column
in urea-containing
buffer. This material
ran as a
single peak on reversed-phase
HPLC, and sodium
dodecyl
sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE)
analysis showed
a band with apparent molecular
mass of 31-32 kilodaltons
(kDa),
somewhat
smaller
than that expected
from the
construct
(36.6 kDa). Edman degradation
revealed
a single sequence
of MDPSAGDMRA,
consistent
with an intact N terminus.
Further characterization
of this material
using low resolution
matrix-assisted laser desorption
ionization
mass spectrometry indicated
an apparent
single protein species of
average mass 33,143 daltons (Da). Detailed molecular analysis by electrospray
ionization
mass spectrometry,
however, revealed that this purified hEl?LBD preparation
was actually
composed
of a
number of both modified and unmodified
LBD protein fragments
between
32$00-33,400
Da. The
bulk of this 33-kDa protein
mixture
consisted
of
three LBD protein
fragments
with C termini
at
Ala,,
(70%), Ala,
(23%), and Ser,,,
(4%), with
only a trace
amount
(3%) of the full length
expressed
LBD (MDPS,
. . . V-;
35, 602 Da).
These four protein species also appear to be partially chemically
modified
by carbamylation.
The
functional
integrity
of this hER-LBD
preparation
was investigated
by a ligand-exchange
assay,
which showed that the hER-LBD
retained
full estradiol-binding
capacity; specific, covalent labeling
was also observed
using either the electrophilic
affinity-labeling
ligand tamoxifen
aziridine
(TAZ) or
the photoaffinity-labeling
ligand
hexestrol
diazirine. Thus, this expressed
hER-LBD preparation,
while appearing
nominally
pure by conventional
biochemical
techniques
and having the expected
ligand-binding
capacity,
was shown by sensitive
high resolution
electrospray
ionization
mass spectrometty
techniques
to be largely truncated
20-26
amino acids from the expected
C terminus
and to
have a substantial
level of covalent
modification
arising from the urea. Our findings
highlight
the
power of mass spectrometric
analysis to identify
and to characterize,
at the molecular
level, the heterogeneity
that may be present
in expressed
receptor
preparations
that appear nominally
pure
by conventional
methods
of analysis. Such information on the sites and nature of cleavage
and
modification
is important
in devising
appropriate
expression
systems and purification
protocols
and
can serve as a specific guide for obtaining
preparations of the hER-LBD that are homogenous
and
potentially
better suited for nuclear magnetic
resonance
and
x-ray
crystallographic
analysis.
(Molecular
Endocrinology
9: 647-666, 1995)
0888-8809~9563.00~0
Molecular Endocrinology
Copyright 0 1995 by The Endocrine Society
647
MOL
648
END0
. 1995
INTRODUCTION
The estrogen receptor (ER) is a ligand-activated
generegulatory
protein that is a member of the nuclear
receptor superfamily (l-4). Like the other members of
this superfamily,
ER is a multidomain
protein, having
distinct amino acid sequences
associated
with the
major functions of DNA interaction
(domain C) and
ligand binding (domain E) (l-4). While the functional
characteristics
of these domains have been probed
extensively by mutagenesis
methods, direct structural
characterization
has been rather limited. The DNA
binding domains of these nuclear receptors are small,
polar, highly conserved
regions consisting
of 65-85
amino acids and have been readily expressed
in a
number of heterologous
systems. Additionally,
the ER,
glucocorticoid
receptor,
and retinoic acid receptor
DNA binding domains have been the subject of a
number of successful x-ray and nuclear magnetic resonance (NMR) structural studies and have been shown
to exist in a helix-loop-helix
topology in which a zinc
atom chelated
by four cysteines
stabilizes
the N
terminus of each helix (5-l 0).
The ligand binding domains (domain E) of the nuclear receptors, by contrast, are much longer (- 250
amino acids), and while they have been expressed
efficiently in a number of systems (1 l-13), their hydrophobic character has limited their solubility in aqueous
systems. Consequently,
the generation of stable crystals of the ligand-binding
domain suitable for x-ray
analysis has proven to be difficult, and their low
solubility has limited analysis by NMR.
It is becoming evident that conventional
means for
characterizing
protein purity and identity [sodium dodecylsulfate-polyacrylamide
gel electrophoresis
(SDSPAGE), reversed-phase
(RP)-HPLC,
N-terminal
Edman sequencing],
and functional
characterization
(ligand binding, immunological
assays, and affinity labeling), are insufficient to specify the precise molecular species that have been produced
by and purified
from heterologous
expression systems (14, 15). There
are examples where heterologously
expressed
proteins have not been fully translated,
or have been
proteolyzed,
and are thus mixtures of closely related
species (1 S), have lacked important posttranslational
modifications,
or have been subject to oxidation
or
other inadvertent
chemical derivatizations
(17-l 9). In
some cases, these changes have altered the properties of these expressed proteins sufficiently so as to
interfere with structural
studies by x-ray and NMR
analysis (16-19). The recently developed
mass spectrometry methods of matrix-assisted
laser desorption
ionization
(MALDI-MS)
and electrospray
ionization
mass spectrometry
(ESI-MS) afford new opportunities
for the characterization
of high molecular weight proteins, to ascertain both their identity and their purity
(14-20).
In this report we describe the development
of a high
yield expression
system for the production
of the ligand-binding
domain of the human estrogen receptor
Vol 9 No. 6
(hER-LBD) in Escherichia coli and a two-step scheme
for the solubilization
and purification
of this species in
a form that binds the ligand estradiol (E.-J. We have
also characterized
this species by both conventional
methods (ligand binding, affinity labeling, SDS-PAGE
analysis,
and Edman sequencing),
as well as by
MALDI-MS and ESI-MS. Through application of HPLC/
ESI-MS methods, we have identified unexpected structural and chemical alterations in the hER-LBD that have
arisen through the expression and purification procedures employed. Since the nature of these alterations
can be ascertained
by ESI-MS, this information
can
serve as a guide for obtaining homogenous preparations
of the hER-LBD.
RESULTS
Plasmid Construction,
E. co/i, and Purification
hER-LBD
Expression
in
Initial attempts to express the full length hER in bacteria suggested that expression was relatively low, as
had been reported by others (21). Consequently,
we
constructed
a series of expression
vectors in which
the ligand binding domain (LBD) and part of the hinge
region (amino acids 282-595) could be expressed in E.
co/i. These LBD vectors, in which the ER domain is
fused to a polyhistidine
region, to a short peptide
encoding an epitope from phage T7 Gene 10, or to a
three-amino
acid lead sequence (MDP, the PET-23dERG vector) are illustrated in Fig. 1. Expression of each
of these vectors in E. co/i was equally robust. The
recombinant
protein was a significant
fraction (2030%) of all the cellular proteins, as revealed by SDSPAGE analysis of total cell lysates (not shown, but see
below), and amounted to between 60 and 90 mg/liter
of bacterial culture.
A procedure
to isolate and purify the expression
product
from the PET-23d-ERG
vector, encoding
the sequence MDPS,,,
. , . V,,, was developed.
After cell growth and protein
induction
under optimized conditions,
we found the bulk of the hER-LBD
in the cell pellet after sonic lysis, perhaps trapped in
inclusion bodies. Proteins in the pellet fraction were
solubilized
by repeated sonication
in 1 M urea-containing
buffer. SDS-PAGE
analysis of the soluble
extract (Fig. 2, lane 1) reveals that the hER-LBD
is
BamH I
BamH I
CCATGGATCCATC6
-
MetAspPrO~~2;2-
Fig. 1. A Schematic
Showing
in the PET-23d-ERG
Vector
For details, see text.
the
-
-
-
-
-
GTC
-
-
-
-
-
"&5,,
hER,,,-,,,
Insertion
Site
Human
ER Ligand
Binding
kDa
649
Domain
12
il.00
31-
0.25
BOUND
0.50
0.75
13H] ESTRADIOL
Fig. 3. Scatchard
Analysis
of the
Capacity
of the Solubilized
Bacterial
sion of the PET-23d-ERG
Vector
Assay was done after a 2300-fold
text.
Analysis of Size and Purity
SDS-PAGE
Fig. 2. SDS-PAGE
Analysis
of hER-LBD
Purification
hEf?-LBD
was purified
from bacterial
extract
by specific
adsorption
onto E,-Sepharose
and elution with reduced
specific activity
rH]E,.
Bacterial
extract
(lane 1) and eluted proteins (lane 2) were analyzed
by SDS-PAGE
on a 12% gel, and
proteins
were visualized
by staining
with silver (22). A major
protein band was observed
at 31-32
kDa and a minor band
at the expected
35 kDa. Each lane contained
approximately
10 pmol ER-LBD,
as determined
by assay
on controlled
porosity
glass.
Migration
of molecular
weight
markers
is
indicated
at the left. For details, see text.
the most abundant
protein. Scatchard
analysis of
the E,-binding
activity in this solubilized
extract,
after appropriate
dilution,
indicated
an equilibrium
dissociation
constant (Kd) of 0.29 nM (Fig. 3), which
is consistent with values obtained for full length hER
(13, 21-23)
although
somewhat
above that for an
expressed
fragment
of hER containing
the LBD,
[which was assayed at higher dilution (24)].
Purification
of the hER-LBD was effected in one
step, by increasing
the urea concentration
to 3 M,
adsorbing the hER-LBD onto an E,-Sepharose
column
(25,26), washing the column with 5 M urea, and eluting
it with excess low specific activity E, in 5 M urea. In this
manner, the hER-LBD was obtained as a nearly homogenous species (Fig. 2, lane 2). Comparison
of the
amount of protein recovered from the affinity column
with that expected on the basis of the binding assay in
the solubilized
extract indicated that the purification
process was essentially quantitative.
Furthermore,
by
proteinand ligand-binding
assays, this purified
hER-LBD binds one equivalent of E,.
1 .oo
E, Binding
Cell Pellet,
dilution.
1.25
(nM)
Affinity
and
after Expres-
For details,
of the hER-LBD
see
by
When assayed for size and purity on SDS-PAGE, the
great majority of the hER-LBD migrated as a single
band with an apparent mass of 3132 kDa (Fig. 2, lane
2). Only a very minor fraction was observed to migrate
as the 35.6-kDa species expected from the hER-LBD
construct. No other protein bands were visible, even
when the gel was purposefully
overloaded
and the
proteins detected with a sensitive silver stain (27).
Because of the construction
of the PET-23d-ERG
expression plasmid, the expressed hER-LBD begins at
residue position Ser2** of the ER sequence
and is
preceded
by a sequence
of three amino acids that
differ from wild type hER. The region (amino acids
299-303)
generally considered
to be the start of the
ligand binding domain (E) (l-4, 28-30), has been characterized as being proteolytically
sensitive (31, 32).
:-tad the hER-LBD been fully expressed to the C terminus then subsequently
clipped at this site, it would
have had a molecular mass of 32,800 daltons (Da) to
33,300 Da, more consistent
with that observed
by
SDS-PAGE
(31-32 kDa). N-terminal
analysis by the
Edman method, however, showed a clear, single sequence of MDPSAGDMRA,
indicating that the N terminus was intact. The composition
of the C terminus
was elucidated
by ESI-MS, as discussed below.
Analysis
of hER-LBD
by RP-HPLC
RP-HPLC
is an effective method for analyzing the
of picomole
quantities
of proteins.
A typical
HPLC UV chromatogram
of the hER-LBD receptor
preparation
determined
at 280 nm is shown in Fig. 4.
The broad, off-scale peak that starts with the solvent front and continues for some 20 min is attributed
purity
MOL
END0
Vol 9 No. 6
. 1995
Analysis
6
4
2
0
~
0
50
100
Retention
150
Time (min)
200
250
Fig.
4. RP-HPLC
Trace of the hER-LBD
Preparation
Purified ER preparation
(150 pmol) was injected
into a 250
mm x 2.1 mm C-4 column
and eluted with the compound
gradient
of (H,O,
0.06%
CF,CO,H)
and (CH,CN,
0.05%
CF,CO,H)
(see Materials
and Methods). The eluant was monitored at 280 nm; the offscale
peak that follows
the solvent
front is urea and buffer salts; the peak at 85 min is rH]E,,
and
the peak at 114.5 min is the hER-LBD.
and other buffer salts. The strong peak at 84.3
min is identified as free E,; this was confirmed
by
subsequent
injection of rH]E, under identical conditions. The peak observed at 114.5 min, corresponding
to approximately
72% CH,CN, is attributed
to the
hER-LBD. Protein assay showed that this fraction did
indeed contain protein, and SDS-PAGE
showed the
same 3132 kDa band present in the original preparation. Thus, by both SDS-PAGE and HPLC analysis,
the expressed hER-LBD appears to be largely a single
protein species.
to urea
Analysis
of hER-LBD
by MALDI-MS
While MALDI-MS
has higher sensitivity than other
mass spectroscopic
techniques
and has a higher
mass range, it does so at the cost of resolution (14,
33). Consequently,
mixtures of large biomolecules
differing by 1 O-200 Da are difficult to resolve. Matrices of
the hER-LBD, prepared by an alternate method (34)
because of the high urea in the sample, were subjected to MALDI-MS and provided very good signals
for [M+H]+‘,
[M+2H]+‘,
and [M-I-~H]+~ ions, indicating an average molecular mass for the major receptor
component
of 33,143 Da, as shown in Fig. 5. This
mass estimate is quite consistent with both the SDSPAGE (3132
kDa) and ESI-MS
determinations
(33,093) (see below).
of hER-LBD
by ESI-MS
Since the ESI-MS cannot tolerate appreciable
salt
concentrations,
a microbore HPLC system coupled to
a LC/ESI-MS interface (see above) was used to desalt
and remove the 5 M urea present in the hER-LBD
preparation
(see Materials and Methods). Typical injections contained
150 pmol receptor (5 ~1 of a 30
pmol/pl
solution in 5 M urea) and used somewhat
steeper acetonitrile
elution gradients than shown in
Fig. 4 above; under these conditions, hER-LBD elutes
at 57 min.
Inspection of the raw ESI-MS data for this peak (Fig.
6) indicates that there are at least three major multiply
charged ion series present in the HPLC eluate at 57
min. Deconvolution
of these ion series using maximum
entropy analysis, shown in Fig. 7, identified three major series of modified proteins at 32,923 Da, 33,093
Da, and 33,423 Da, as well as a trace amount of
protein with molecular mass of 35,595 Da; an expansion of the major protein components
between 32.833.5 kDa is shown as the inset in Fig. 7. The ESI-MS
technique provides mass accuracies up to 0.05%, so
these 33-kDa proteins can be expected to have errors
2 17 Da.
The expression
system was designed to produce
the hER-LBD protein MDPS282-V595r which would be
expected
to have a molecular
mass of 35,604 Da.
N-Terminal sequencing
via Edman degradation
of the
protein mixture provided only the N-terminal residues
expected for this construct (see above). The presence
of a small amount of the full-length receptor sequence
in the purified
preparation,
as indicated
by the
35,595-Da
peak by ESI-MS, confirmed that the DNA
coding sequence in this expression system is properly
constituted. Analysis using either Masslynx BioAnalysis or MacBioSpec
(v.1 .O.l , Perkin-Elmer
SCIEX Division, Thornhill, Ontario, Canada) suggested C-terminal
truncations
compatible
with the three other protein
masses detected by ESI-MS; these are shown in Table
1. C-termini are proposed at Ala,,,, Ala,,, , and Ser,,,.
Close inspection of the major zero-charge
molecular
mass peaks observed by ESI-MS (Fig. 7, inset, especially the 33,093-Da peak) revealed the presence of an
additional
series of masses at [M+n*(43)], where n =
0,1,2,3. This suggests that each component
protein
has been covalently modified in some manner, either
before or during analysis. The most likely modification
would be carbamylation,
which should manifest itself
as a series of [Ml, [M+43], [M+86], [M+129]. In this
hER-LBD preparation,
the extent of monocarbamylation and dicarbamylation
is estimated at 22% and 7%,
respectively.
Carbamylation
could result from the reaction of the lysine E-amino group or the cysteine thiol
group with cyanate ion, which is generated
as an
equilibrium
product from the high concentrations
of
urea used for the isolation and purification of the hERLBD (35, 36). The presence of the various mass components observed
by ESI-MS indicates that a large
proportion
of the proteins in the hER-LBD preparation
Human
ER Ligand
Binding
651
Domain
100
1.5E3
60
m/z
Fig. 5. The MALDI-MS
of the hER-LBD
This is a spectrum
of a sample applied to the probe by a protocol
that allows the excess
urea to be removed
by gentle
(see Materials
and Methods).
Visible are peaks due to the singly charged
and doubly
charged
species,
as the average
of 33,143 and 16,604 Da, respectively.
10(
washing
masses
oqa.5
!.l
I 182.7
8
895.
a00
6. ESI-MS of the hER-LBD
This spectrum
shows several
left, each envelope
results from
900
10
Fig.
are either truncated (within
terminus) or are chemically
or both.
Characterization
of the hER-LBD
envelopes
of peaks that
progressively
increasing
correspond
to species
charge
on each mass
-26 amino acids of the C
modified in some manner,
of the Ligand-Binding
Capacity
The ability of hER-LBD to bind E,, even in the presence of 5 M urea, is implied because it was bound by
of different
fragment,
mass
which
(cf. Fig. 7). Going from
lowers
its m/z value.
right
to
an E, affinity column under these conditions,
and it
elutes in 5 M urea only in the presence of E,; in addition, the eluted hER-LBD is bound to one equivalent of
low specific activity (0.25 CVmmol) rH]E,, as determined
by specific
adsorption
of hER-LBD-rH]E,
complex to controlled-pore
glass (CPG) beads (see Materials and Methods).
Because the purified hER-LBD is
already occupied by ligand, standard protocols cannot
be used to determine
its binding affinity. We have,
MOL
END0
. 1995
Vol 9 No. 6
652
101
7 33093.3
33200
33600
14000
mass
3366.6
34400
34600
35200
356(
35594.6
ALI
‘200
33600
34000
34400
34600
35200
35600
mass
Fig.
7. The Deconvolution
of the ES-MS
of the hER-LBD,
Shown
in Fig. 6
Main panel shows the spectrum
of zero charge
mass from 32,800 to 35,800 Da; inset is an expansion
of the 35,800 to 33,550
Da region. The four different
length species
in the hER-LBD
preparation
give their primary
peaks at 32,923,
33,093, 33,423,
and
35,595
Da, respectively.
The monoand dicarbamylated
species,
in each case, appear
as smaller
peaks at intervals
of
approximately
43 and 86 Da higher mass. These are especially
apparent
with the most intense species
(33,093).
The deconvolution routine does not always
give clear, single peaks, especially
for the peaks with weaker
intensities.
however, been able to confirm further, by exchange
assays with high specific activity pH]E,, that most of
the purified hER-LBD is active in binding E,. For example, with 2 mg/ml BSA added as carrier protein,
2000-fold
diluted hER-LBD was allowed to exchange
at either 0 C or 25 C and assayed by hydroxylapatite
(HAP) adsorption
(37). Little exchange was observed
at 0 C: 4% after 1 h, which increased slowly to 26%
after 24 h (data not shown). Exchange
proceeded
quickly at 25 C, however, reaching 49% after 1 h and
84-94%
after 4 h. It remained
constant and stable
from 4-24 h (Fig. 8). These kinetics are consistent with
E, exchange rates measured with rat uterine ER (38).
Greater than 90% of this exchange could be blocked
by the addition of excess unlabeled
E, (Fig. 8); no
specific rH]E, binding was found with the carrier BSA
alone. While the hER-LBD will undergo exchange in 5
M urea, with or without dilution, the diluted form is less
stable under these conditions.
The addition of BSA
carrier protein is not essential, but it reduces adsorption losses of diluted preparations.
Using a dye-binding
assay (39), we determined
that
the hER-LBD preparation
(before dilution and carrier
protein addition) had a protein concentration
of 1.03
mg/ml, which makes it 3.11 X lop5 M, assuming a
molecular mass of 33,100 Da. The E,-binding
capacity
measured by this exchange assay was 2.61-2.94
x
10m5 M, which is, again, consistent with a 1:l binding
stoichiometry,
the same as that found directly after
elution from the E, affinity column. Thus, according to
standard exchange binding assays, the hER-LBD-purified preparation
has the expected capacity for binding E,. As noted earlier (Fig. 3), Scatchard analysis of
the unoccupied
hER-LBD, solubilized
in 1 M urea before purification
by affinity chromatography,
indicated
that it had a dissociation
constant (Kd) of 0.29 nM.
Affinity Labeling of hER-LBD with TAZ and
Hexestrol Diazirine
The well characterized
electrophilic
affinity label, TAZ,
can be exchanged
into the ligand-binding
site of the
hER-LBD. TAZ (40) is an efficient and selective electrophilic affinity-labeling
agent for unoccupied
ER, but
its rapid action [>90% of the reactive aziridine labeling
moiety of TAZ is consumed within the first 0.5 h (40)]
makes it less efficient under the exchange conditions
needed to label the hER-LBD.
Nevertheless,
TAZ is
able to label 38% of the hER-LBD (data not shown).
Photoaffinity
ligands behave as reversible ligands
until they are exposed to light. Therefore, they can be
incubated
with the hER-LBD under exchange conditions for several hours in the dark so as to undergo
near-maximal
exchange, whereupon
brief photolysis
can effect covalent attachment of the ligand. Hexestrol
diazirine, a new photoaffinity
labeling agent (41), binds
reversibly to the hER-LBD after exchange; photolysis
then effects covalent attachment to 38% of the total
sites (or 70% of the sites occupied
after exchange;
data not shown), which is comparable
to that achieved
Human
ER Ligand
TABLE
1. Major
Binding
Protein
Fragments
Receptor
MDPS,,,AG
MDPS,,,AG
MDPS,,,AG
MDPS,,AG
.
. .
. . .
. .
N
Il.
Il.
Il.
Domain
653
Observed
In HPLWESCMS
fragment
.
ITGEAEGFPATV,,,
. DQSHLATAGSTS,,,
. . DQSHLAT&,,
. DQSHLA,,,
with full length ER from rat uterus (41). The attachment
of both reagents is stable to the conditions
of SDSPAGE and showed radioactive bands that comigrated
with the 3132 kDa species (not shown).
DISCUSSION
The development
of methods for the high-level
expression of proteins in heterologous
systems and their
purification
in homogenous,
functional form are prerequisites for detailed studies of their structure
by
spectroscopic
methods. In this report, we have described the development
of an expression system for
the LBD of the hER (MDPS,,,-V,,5)
in f. co/i and a
simple and efficient purification
of this receptor domain by chromatography
on an E, affinity column in
3-5 M urea after solubilization
in 1 M urea. The purified
hER-LBD was characterized
with respect to size, purity, and ligand-binding
capacity using a variety of
classical biochemical
techniques,
as well as newly
developed
mass spectrometry
methods.
The latter
methods revealed an unexpected
heterogeneity
in this
preparation,
the nature of which has led to a redesign
of the system for expression
and purification
of the
hER-LBD.
1001
I
___-_-_-----_---_-_.
------0’ .. .- ___
*’___________
10
15
20
25
0
5
HOURSOFEXCHANGE
Fig.
6. Time Course
of Exchange
of High Specific
Activity
PH]E, into the binding
site of the hER-LBD
at 25 C. The
hER-LBD
was diluted to 10 nM ER with BSA added as carrier
protein.
This was incubated
with 43 nM rH]E,
(total) z-1 p,~
unlabeled
E, (blocked).
Exchange
was measured
by the HAP
assay and is expressed
as a percent
of the total hER-LBD.
Specific
exchange
(bold line) is the difference
between
total
(solid line) and blocked
(dashed
line).
Analysis
of Original
Receptor
Mass (Da)
(predicted)
Mass (Da)
(observed)
35603.6
33522.2
33089.9
32917.7
35594.6
33422.7
33093.3
32923.3
Preparation
(dacns)
9.0
0.5
3.4
5.6
Relative
abundance
3%
4%
70%
23%
By conventional
techniques
of SDS-PAGE,
HPLC,
and Edman N-terminal sequencing,
the purified hERLBD appeared to be an essentially homogenous
protein component
of 32 kDa, with a single, intact N
terminus. Although the hER-LBD migrated on SDSPAGE as a somewhat smaller mass than that expected
for the expressed
protein (35.6 kDa), this could be
attributed to variations in SDS association due to the
highly hydrophobic
nature of the LBD. The intact N
terminus indicated that cleavage had not occurred at
the known proteolysis-sensitive
sites in the region of
amino acids 299-303.
Both before and after its purification by E,-Sepharose
affinity chromatography,
the
hER-LBD was shown to bind E,. The eluted, purified
protein displayed the expected 1 :l ligand-binding
stoichiometry both by direct binding and by exchange,
and the photoaffinity
label, hexestrol diazirine, underwent covalent attachment
to the hER-LBD with an
efficiency nearly comparable
to that obtained with fulllength ER. These studies indicate that the hER-LBD,
by itself, has the tertiary structure
needed for high
affinity ligand binding.
When the LBD was examined by the recently developed mass spectrometry
method of MALDI-MS, the
preparation
appeared to be a homogenous
protein of
molecular
mass 33,143 Da, consistent with the estimate by SDS-PAGE.
RP-HPLC,
which is frequently
used to characterize the homogeneity
of protein preparations, showed the purified hER-LBD as a single
protein peak, eluting at high CH,CN concentration
(72%). However, when this technique was coupled to
ESI-MS, the microcomposition
of the hER-LBD eluate
was revealed. This high resolution mass spectrometry
method showed that the hER-LBD preparation
consisted of four components
sharing a common N terminus, but having various C termini: full-length
hERLBD ending at Valsss, and three fragments truncated
at Ser,,,, Ala,,, , and Ala,,,. Their observed molecular
masses (and relative abundances)
were 35,595 Da
(3%), 33,423 Da (4%), 33,093 Da (70%), and 32,923
Da (23%), respectively.
These truncations
could arise either from incomplete translation or from physical or enzymatic cleavage of the intact hER-LBD. The occasional
presence
of codons in eukaryotic genes that are rarely used in
prokaryotes
does not usually result in premature truncation, although clusters of rarely used codons may
account for reduced expression yields (42, 43). Of the
nucleotide
sequences
that code for the amino acid
pairs spanning the three observed premature termina-
MOL
END0
1995
Vol 9 No. 6
654
tion sites: Ala,,,-Thr,,,
(GCC-ACU),
Ala57,-Gly572
(GCG-GGC),
and Ser,,,-Ser,,G
(UCA-UCG), only the
Ser UCG codon is infrequently
used in f. co/i genes
(44, 45); however, it is also infrequently
used in human
genes, and this Ser-Ser sequence
does not correspond to the predominant
site of cleavage, which is at
Ab,.
Although physical cleavage of the hER-LBD during
its solubilization
by sonication is possible, this is not a
well documented
phenomenon.
C-Terminal truncation
of expressed
proteins by proteolysis
is well known;
however, the sequences at the three C-terminal truncation sites do not correspond
to the substrate specificity of bacterial proteases
most commonly
implicated in the degradation
of heterologously
expressed
proteins (46-48). The sequence
in the region of the
hER-LBD that suffers cleavage at these three sites is
unusual, being rich in serine and threonine residues,
and it bears considerable
similarity to signal peptide
leader sequences
found at the N termini of proteins
that are directed to the periplasmic space (e.g. ompT).
In E. cob, these leader sequences
are cleaved by a
recently characterized
leader peptidase (SPase I, 49),
which has a preference for small residues at the P, and
P, sites, and a helix-breaking
residue (Gly, Pro, or Ser)
at the P,-, sites. The sequence in this region fulfills
this motif at all of the three cleavage sites (indicated by
a co/on) and would account for the predominant
cleavage at As7,: Dse4 Q S H L A:T A:G S T S:S,,,
The signal leader peptidase
is an integral inner
membrane protein that normally cleaves the N-terminal signal sequence of proteins during their secretion
into the periplasmic space. Thus, this peptidase would
cofractionate
with the hER-LBD in the pellet fraction
after sonic lysis of the cells, and it may be cleaving the
LBD at the three C-terminal
cryptic cleavage sites
during the solubilization
process that involves repeated sonication
in 1 M urea. This supposition
is
supported
by the observation
that hER-LBD that is
solubilized in the initial rounds of sonication contains a
greater amount of the 35kDa species (the size corresponding to the intact LBD), while the final phases of
solubilization
release material that is almost exclusively 3132 kDa (G.L. Greene, unpublished).
As the
signal leader peptidase is very difficult to inhibit (49), it
is unlikely that this cleavage can be entirely blocked.
Analysis of the hER-HBD by ESI-MS also revealed
that all four length components
were chemically modified by mono- and dicarbamylation
to the extent of
22% and 7%, respectively.
It is known that cyanate
ion, generated
via a slow equilibrium
reaction from
aqueous urea solutions and therefore often present as
a contaminant,
can readily carbamylate
cysteine
y-thiol and lysine E-amino side chains (35, 36).
Therefore, although appearing
to be homogenous
and functionally active, in terms of E,-binding
capacity
and affinity-labeling
specificity, this hER-LBD preparation is, in fact, a mixture of peptides truncated adventitiously 20-26 residues from the C terminus, with
significant modification
by carbamylation.
It is known
that microheterogeneity
of heterologously
expressed
proteins can affect solubility
and can interfere with
crystallization,
thereby complicating
structural studies
by x-ray and NMR (16-l 9). Therefore, it is essential to
fully characterize
the purity and the identity of expressed proteins at the molecular level. Conventional
methods for ascertaining
purity and identity, such as
SDS-PAGE,
Edman
N-terminal
sequencing,
and
HPLC, can reveal major structural anomalies, but are
unable to detect variations in microstructure;
by contrast, molecular composition
is readily disclosed
by
the more sensitive and higher resolution mass spectrometry technique
ESI-MS. A detailed knowledge
of
chemical constitution
at this level may be critical in
designing
and obtaining
expressed
preparations
of
steroid receptors that are suitable for characterization
by NMR and x-ray crystallographic
analysis. In fact, on
the basis of the specific information
on the C-terminal
cleavage and carbamylation
of the hER-LBD preparation that we analyzed in this report, we have redesigned the expression construct so as not to include
the C-terminal cleaved region (which is actually in the
F-domain), and we have modified the purification protocol to minimize exposure of the LBD to cyanate. As
will be described in full elsewhere, this revised expression and purification
protocol results in an hER-LBD
preparation that now appears to be homogeneous
and
functional by all of the criteria used in this report.
MATERIALS
AND METHODS
Materials
Radioligands
were
obtained
from
the following
sources:
vH]E, ([6, 7-3H]estra-l,
3, 5,(10)-triene-3,176-diol)
51-53 Ci/
mmol;
PH]TAZ
([ring-3H]-(Z)-[4-[2-(N-aziridinyl)
ethoxylphenyl]]-1,2-diphenyl-1
-butene),
20 Ci/mmol
from Amersham
Coro (Arlinaton
Heiahts.
IL): f Hlhexestrol
diazirine
(PHl-3azidutyl
2, s-bis(4-h~droxyphenyljpentyl
sulfide), 32 CGmmol
prepared
in our laboratory
(41); unlabeled
ligands:
E, from
Sigma; TAZ (40) and hexestrol
diazirine
(41) were prepared
in
our laboratory
as previously
reported.
The following
compounds
were obtained
from the sources
indicated:
HAP and
Kaleidoscope
prestained
mol wt standards
(Bio-Rad,
Hercules CA); CPG beads (500 A pore size; Electra-Nucleonics,
Inc., Fairfield,
NJ); pET‘vecto&,
E. co/i strains,
and growth
broth
(Novagen
Inc., Madison
WI). The E,-Sepharose
(ESeph) affinity
column
was prepared
as previously
reported
(25).
Construction
of Expression
Vectors
for hEF?-LBD
To construct
a fusion protein of the ER-LBD
to a leader with
an epitope
from phage T7 Gene
10 or to a polyhistidine
leader, a BarnHI-EcoRI
fragment
spanning
the domain (S,,,V,,,) from HE 14 (50) was inserted
into PET-1 7b and PET-1 9b
vectors
(Novagen
Inc.), respectively.
These constructions
incorporated
the Val,,,
mutation
present
in the original
ER
cDNA
(51) but were converted
to the wild type Gly,,,
by
substitution
of an Ncol-EcoRI
fragment
or a HindIll-EcoRI
fragment,
respectively,
from pCMV-ER
[a gift from Dr. Benita
S. Katzenellenbogen
(52,53)].
To construct
a fusion protein of
the ER sequences
to the three-amino
acid leader (MDP), an
Ncol-EcoRI
fragment
spanning
the C-terminal
portion
of the
Human
ER Ligand
Binding
655
Domain
domain from pCMV-ER
was inserted
into a PET-23d
vector
(Novagen
Inc.). An Ncol fragment
incorporating
the leader
sequences
and amino-terminal
portion from HE 14 were subsequently
inserted
to complete
the construction
of PET-23dERG expression
vector. This plasmid
contains
the sequence
from the carboxy-terminal
region of wild type
Sebdhg5
hER, preceded
by an MDP sequence,
and has Gly at position
400 (MDPS,,,-V,,,).
Expression
and Purification
of hER-LBD
Conditions
suggested
in the manual provided
by the makers
of the PET vectors
(Novagen
Inc.) for cell growth
and protein
induction
were followed
precisely,
except
that medium
was
2x LB. hER-LBD
was overexpressed
in BL21 (DE3 A lysogen)
cells transformed
with the PET-23d-ERG
vector.
Cells were
grown in selective
medium
(2x LB + ampicillin)
to an absorbance
of 2.0 (@ 600 nm) and then induced
with 0.6 mM
isopropyl+-thiogalactopyranoside
for 3 h at room temperature (23 C). Cells were pelleted by centrifugation,
snap frozen,
and stored
at -75
C. Each liter of culture
yielded
a 5-ml
pellet. Frozen cells were thawed
and lysed by sonication
(4 x
20 set) in four volumes
(20 ml) of lysis buffer [50 mM Tris; 50
mM NaCI. 1 mM EDTA. 1 mM dithiothreitol
(D7-n. 1 M urea. oH
7.41. This procedure
‘was repeated
several
tknes for each
pellet. Clarified
fractions
were obtained
by centrifugation
at
30,000
x g for 30 min, and 61 ml pooled
extracts
were
adjusted
to contain
3 M urea and 0.4 M NaCl (loading
buffer)
and then applied to a 20-ml column
of E,-Sepharose
(2526).
The column
was washed
successively
with loading
buffer
(240 ml) and loading buffer that contained
5 M urea (50 ml).
Bound hER-LBD
was eluted with 2 x 1 O-5 M rH]E,
(specific
activity
0.25 Ci/mmol)
in 30 ml of a buffer that contained
25
mM Tris, 100 mM NaCI, 1 mM EDTA, 1 mM DlT, and 5 M urea,
pH 7.4. The yield of hER-LBD,
as determined
by specific
adsorption
of bound PH]E, to CPG beads (54) was 156 nmol
(100% recovery).
The protein
content,
as determined
by the
method
of Bearden
(39) with BSA as the protein
standard,
was 4.9 mg, which agreed closely
with the predicted
yield of
5.0 mg for a determined
hER-LBD
mass of 33 kDa. hER-LBD
purity was also assessed
on silver-stained
SDS-polyacrylamide gels and by Western
blot analysis
with the H222
monoclonal
rat antibody
(55, 56). The major eluate fraction
from the E,-Sepharose
column
was supplemented
with 5%
glycerol
and concentrated
by centrifugation
through
an Amicon Centriprep-10
filter to a final volume
of 3.5 ml and an
hER-LBD
concentration
of 31 nmol/ml
[31.3 PM, containing
62 PM E, (0.25 Ci/mmol)].
Aliquots
were snap frozen
and
stored at -75 C.
hER-LBD
Binding
For exchange
in the sample
was routinely
Assay
and Affinity
SDS-PAGE
Cell lysate fractions
and purified
hER-LBD
were analyzed
by
SDS-PAGE
as described
previously
(25) except
that a 12%
reducing
gel was used. Samples
for N-terminal
sequencing
were electrophoresed
using the Tricine
system
(59). Gels
were either stained with Coomassie
Blue G (59) or silver (27)
for visualization
or electroblotted
to polyvinylidene
fluoride
membrane
for N-terminal
sequencing.
Molecular
weight
prestained
standards
were from Bio-Rad.
N-Terminal
Microsequencing
Gel patterns
were electroblotted
to Pro-Blott
polyvinylidene
fluoride
membranes
and stained
with Coomassie
Blue R,
following
the recommendations
of the manufacturer
for the
Pro-Blott
membrane
(Applied
Biosystems,
Inc). Bands of interest
were excised
with a razor blade and N-terminal
sequenced
by Edman
chemistry
on an Applied
Biosystems
model 477A protein
sequencer
coupled
to a model
120A
on-line
PTH analyzer
by Dr. K.-L. Ngai and the personnel
in
the Genetic
Engineering
Facility
of the University
of Illinois.
Protein
Determination
Total protein was assayed
using
Standard
curves
were generated
Analysis
The equilibrium-binding
affinity
of hER-LBD
for [3H]E2 was
determined
by saturation
analysis
of an unpurified
bacterial extract
that was diluted
(2300-fold)
to contain
approximately
1 nM unliganded
hER-LBD
in binding
buffer (50 mM
Tris, 50 mM NaCI,
1 mM EDTA,
1 mM DlT,
and 0.1%
ovalbumin,
pH 7.4).
Increasing
amounts
of [6,7-3H]E,
(0.2-20
nM; 53 Ci/mmol)
were added,
and solutions
were
incubated
overnight
at 4 C. Bound
[3H]E2 was determined
by specific
adsorption
of [3H]E,-hER-LBD
to CPG beads
(54):Free
[3H]E2 was determined
by subtraction
of bound
f3H1E, from total added
f3H1E,. Nonspecific
bindina
was
mea&red
in the presence
of a 200-f&d
excess
of-unlabeled
E,. An equilibrium
dissociation
constant
(Kd) was
calculated
by the method
of Scatchard
(57).
Exchange
C), giving a final concentration
of 3 nmol/ml
ER and 0.5 M
urea. The preparation
could also be exchanged
without
dilution (i,e. at 31 nmol/ml
ER, 5 M urea). The 3H ligand at a
specific
activity
of 10-15 Ci/mmol
was added to be 2- to 3
times the ER concentration.
A control
was always
run in
parallel
where
the specific
exchange
was blocked
by a
lo-fold,
or greater,
excess
of unlabeled
E,,
For the time course of [3H]E2 exchange,
the hER-LBD
was
diluted more extensively
so as to ensure that the E, solution
would be saturating.
It was diluted with 0.05 M Tris, 0.15 M
NaCI, 20% glycerol,
1 mM DTT, and 0.1 mM EDTA, pH 7.4, at
25 C to give 10 nM ER. This resulted
in 20 nM low specific
activity
E, present
in solution
as carry-over
from the chromatographic
purification.
BSA was added as a carrier protein
(2 mg/ml),
and reversible
binding
was measured
using the
HAP assay (37).
To label with TAZ, samples
were incubated
0.5 h at 25 C.
For exchange
with E, or the photoaffinity
ligand, hexestrol
diazirine,
incubations
were conducted
for 2 h at 25 C. After
exchange
with hexestrol
diazirine,
covalent
attachment
was
effected
by 15 min of photolysis
at greater than 315 nm (41).
Covalent
attachment
for either affinity ligand was assayed
by
the EtOH-disc
assay (56).
Labeling
of the low specific
activity
PH]E, that is present
with high specific
activity
ligands,
the hER-LBD
diluted 1 :lO with 50 mM Tris buffer (pH 7.4 at 25
Liquid
Chromatography
the method
of Bearden
using BSA.
(39).
(HPLC)
HPLC analyses
were performed
on four reversed-phase
columns using two systems.
HPLC separations
of the original
protein
preparation
were performed
with either a Vydac
2.1
mm x 250 mm C-4 reversed-phase
column
(5 pm particles,
300 A pore size) or a Vydac 2.1 mm x 250 mm C-6 reversedphase
column
(5 pm particles,
300 A pore size) using a
SpectraSYSTEM
(ThermoSeparations
Inc., Carlsbad,
California) HPLC system
consisting
of a model AS3000 autosample
injector,
a model P4000 gradient
(piston)
pump, and a model
UV2000
dual wavelength
UV detector
outfitted
with a standard analytical
(9 ~1, 6 mm path) flow cell. UV detection
was
followed
at 216 nm and 280 nm to observe
amide and aromatic absorbances,
respectively.
This HPLC system
was additionally
outfitted
with an P-RAM
model 2 (IN/US
Systems
Inc., Tampa,
FL) continuous-flow
scintillation
counter
for the
on-line detection
of 3H-labeled
receptor
ligands.
The HPLC interfaced
with the ESI-MS
probe used microbore RP-HPLC
columns
from Brownlee-Applied
BioSystems
MOL
656
END0
1995
Vol 9 No. 6
(1 .O mm id C-l 8, 7 pm particles,
300 A pore size), either 100
mm or 250 mm long; the longer column
permitted
loading of
more injected
material,
while maintaining
the low 40 yllmin
eluent flow rate. This microbore
system
consisted
of an Applied BioSvstems
(Foster Citv. CA) model 140b dual svrinae
pump equipped
with two 101ml syringes
and very low dead
volume
mixing tee, and an Applied
BioSystems
model 759a
single wavelength
UV detector
with 2.5~~1 flow-through
detection
cell. Detection
was set for the UV amide absorbance
between
21 O-21 6 nm.
Binary gradient
elution conditions
were used for all analyses. Compound
linear gradients
were generated
for elution
from 2% CH,CN
to 100% CH,CN
over 60-180
min; binary
components
for these gradients
were comprised
of solvent A
(H,O, 0.06”/,
CF,COOH)
and solvent
B (CH,CN,
0.05”/,
CF,COOH).
Solvents
and reagents
used for the HPLC and
ESI-MS analyses
were typically
HPLC grade and were used
without
further
purification.
Water
used was Milli-Q grade
distilled
before use. Acetonitrile
was Optima (HPLC) grade as
supplied
by Fisher
(Pittsburgh,
PA). Trifluoroacetic
acid
(CH,COOH,
TFA), protein sequencing
grade (99% pure), was
obtained
from Aldrich Chemical
Co (Milwaukee,
WI) and used
as supplied.
ESI-MS
A VG Quattro
(quadrupole-hexapole-quadrupole,
QHQ) mass
spectrometer
system
(Fisons
Instruments,
VG Analytical;
Manchester,
UK) was used for the analysis
of the receptor
protein.
Data aquisition
and processing
were controlled
by
the VG MassLynx
(version
2.0) data system;
MAXEnt
(Maximum Entropy)
software
was used for the processing
and
analysis
of zero charge state electrospray
data for the receptor and large protein fragments.
Collection
of all electrospray
data was performed
on samples
coupled
through
an HPLC/
ESI-MS
interface
with an Applied
BioSystems
microbore
HPLC (see above) operating
at 40 pl/min. Postcolumn
eluent
was split using static pressure
balancing
through
a low dead
volume
mixing tee and a variable
length of 75 pm id fused
silica tubing.
Empirically
determined
split ratios of 1:6 to 1:8
were established
such that -5 pl/min HPLC eluent was directed to the ESI-MS probe for nebulization.
This postcolumn
splitting
resulted
in ESI-MS
analysis
of lo-30%
of the injected
sample
and permitted
the collection
of 70-90%
of
desalted
and purified protein fragments.
HPLC sample injections of 5 ~1 were comprised
typically
of 15-30
pmol/pI
receptor
in buffer solution
(2-5 M urea). The minimum
quantity
of receptor
required
for adequate
detection
under these conditions
appears
to be approximately
lo-20
pmol at the ESI
probe in a 5 $/min
eluent flow.
Cesium
clusters
[(Csl),Cs+],
generated
from
freshly
prepared
solutions
of cesium
iodide (3 mg/ml) in .50:50 H,O:
CH,CN,
were used for external
calibration
of the ESI-MS from
m/z 300-2300.
Typical
ESI data aquisition
was performed
for
the protein
using 4.9-set
scans
on 5.0-set
intervals;
the
resulting
total ion chromatograms
(TIC) cover m/z 300-2300
(408.2 U/set).
MALDI/Time
of Flight
(TOF)-MS
A VG TOFSPEC
matrix-assisted
laser desorption/TOF
mass
spectrometer
(Fisons
Instruments,
VG Analytical;
Manchester, UK) using a 322 nm N, laser and 22 kV acceleration
voltage
was used for the analysis
of the hER-LBD.
BSA was
used as an external
calibration
standard
for all proteins
assayed. [2-(4-Hydroxyphenylazo)benzoic
acid] (HABA, Aldrich
Chemical
Co.) was found to be an effective
matrix
for the
analysis
of these
protein
solutions
containing
2-5 M urea,
provided
a modification
was made to the matrix deposition
procedure
(34): A saturated
solution
of HABA in 50:50 EtOH:
CH,CN
was spotted
and dried on the target support;
this
matrix was overlayed
with the hER-LBD
sample,
diluted
lo-
to 30-fold
with 50:50 H,O-CH,CN,
by the spotting
of 2-3 ~1
of a l-3 pmol/$
solution
of protein.
After having been dried,
the target spot was rinsed two to three times with 5 ~1 H,O to
remove
excess
urea and other buffer salts and then allowed
to redry. Samples
thus prepared
were analyzed
by averaging
20-25
laser pulses.
Observed
masses
of proteins
prepared
using this overlay/rinse
procedure
were generally
100300
Da
higher than expected
based on the BSA calibration
and the
masses
observed
by ESI-MS analysis.
Acknowledgments
We are grateful
to Dr. Richard
Milberg
MALDI-MS
and ESI-MS and to Drs. C. G.
for helpful discussions.
Dr. Greene gratefully
technical
assistance
of Chris Hospelhorn
Terminal
analysis
was performed
by the
Facility at the University
of Illinois (Urbana,
for assistance
with
Miller and K.-L. Ngai
acknowledges
the
and Lin Cheng.
NGenetic
Engineering
Illinois).
Received
December
13, 1994. Revision
received
February
21, 1995. Accepted
March
13, 1995.
Address
requests
for reprints
to: Dr. John A. Katzenellenbogen,
461 Roger Adams
Laboratory
37-5,
Department
of
Chemistry,
University
of Illinois, 600 South Mathews
Avenue,
Urbana,
Illinois 61801.
This work was supported
in part by grants
from the NIH
(PHS 5R37DK-15556
to J. A. K., and PHS 5ROl CA-02897
to
G. L. G), the U.S. Army
Medical
Research
Core (DAMD
17-94-J-44228
to G. L. G.), and the American
Cancer
Society
(BEGIE to P. J. K.). Mass spectrometric
analysis
was performed
on instruments
whose
acquisition
was supported
in
part with a grant from the Division
of Research
Resources,
NIH (RR-07141).
REFERENCES
5.
6.
7.
8.
9.
10.
11.
Parker MG 1991 Nuclear
Hormone
Receptors.
Academic
Press, London
Evans RM 1988 The steroid
and thyroid
hormone
receptor superfamily.
Science
240:889-895
Beato M 1988 Gene regulation
by steroid
hormones.
Cell
56~335-344
Wahli W, Martinez
E 1991 Superfamily
of steroid
nuclear
receptors:
positive
and negative
regulators
of gene expression.
FASEB J 5:2243-2249
Hard T, Kellenbach
E, Boelens
R, Maler BA, Dahlman
K,
Freedman
LP,
Carlstedt-Duke
J, Yamamoto
KR,
Gustafsson
J-A, Kaptein
R 1990 Solution
structure
of
glucocorticoid
receptor
DNA-binding
domain.
Science
249:157-l
60
Schwabe
JWR,
Neuhaus
D, Rhodes
D 1990 Solution
structure
of the DNA-binding
domain
of the oestrogen
receptor.
Nature 348:458-461
Lee MS, Kliewer SA, Provencal
J, Wright
PE, Evans RM
1993 Structure
of the retinoid
x receptor
(Y DNA binding
domain:
a helix required
for homodimeric
DNA binding.
Science
169:1117-l
121
Luisi
BF, Xu WX,
Otwinowski
Z, Freedman
LP,
Yamamoto
KR, Sialer PB 1991 Crvstalloaraphic
analvsis
of the interaction-of
the glucocbrticoid
receptor
with
DNA. Nature 352:497-505
Freedman
LP 1992 Anatomy
of the steroid
receptor
zinc
finger region. Endocr
Rev 13:129:145
Schwabe
JWR, Chapman
L, Finch JT, Rhodes
D 1993
The crystal structure
of the estrogen
receptor
DNA-binding domain
bound to DNA: how receptors
discriminate
between
their response
elements.
Cell 75:567-578
Srinivasan
G 1992 Overexpression
of receptors
of the
Human
ER Ligand
Binding
Domain
steroid/thyroid
family. Mol Endocrinol
6:857-860
12. Ahrens
H, Schuh
TJ, Rainish
BL, Furlow
JD, Gorski
J,
Mueller GC 1992 Overproduction
of full-length
and truncated
human
estrogen
receptors
in fscherichia
co/i.
Receptor
2:77-92
13. Wooge
CH, Nilsson
GM, Heierson
A, McDonnell
DP,
Katzenellenbogen
BS 1992 Structural
requirements
for
high affinity ligand binding by estrogen
receptors:
a comparative
analysis
of truncated
and full length estrogen
receptors
expressed
in bacteria,
yeast, and mammalian
cells. Mol Endocrinol
6861-869
14. Chait BT 1994 Mass spectrometry-a
useful tool for the
protein X-ray crystallographer
and NMR spectroscopist.
Structure
2:465-467
15. Carr SA, Hemling
ME, Bean MFRoberts
GD 1991 Integration
of mass spectrometry
in analytical
biotechnology. Anal Chem 63:2802-2824
16. Bayer EA, Ben-Hur
H, Hiller Y, Wilchek
M 1989 Postsecretory modifications
of streptavidin.
Biochem
J 258:369376
17. Chait BT, Kent SBH 1992 Weighing
naked proteins.
High
accuracy
mass measurement
of peptides
and proteins.
Science
257:1885-l
894
18. Aebersold
R 1993 Mass spectrometty
of proteins
and
peptides
in biotechnology.
Curr Opin Biotechnol5:77-84
19. Wang R, Chait BT 1990 Rapid, sensitive
analysis
of protein mixtures
by mass spectrometry.
Proc Natl Acad Sci
USA 87:6873&877
20. Hillenkamp
F, Karas M, Beavis
RC, Chait BT 1991 Matrix-assisted
laser desorption/ionization
mass spectrometry of biopolymers.
Anal Chem 63:1193A-1203A
21. Wittliff
JL, Wenz LL, Dong J, Nawaz
Z, Butt TR 1990
Expression
and characterization
of an active human estrogen receptor
as a ubiquitin
fusion protein from Escherichia co/i. J Biol Chem 26522016-22022
22. Grill H-J, Manz B, Belovsky
0, Krawielitzki
B, Pollow K
1983 Comparison
of [3H]oestradiol
and [‘251]oestradiol
as
ligands
for oestrogen
receptor
determination.
J Clin
Chem Biochem
21 :175-l 79
23. Reese JC, Wooge CH, Katzenellenbogen
BS 1992 Identification
of two cysteines
closely
positioned
in the ligand-binding
pocket
of the human
estrogen
receptor:
roles in ligand binding
and transcription
activation.
Mol
Endocrinol
6:2160-2166
24. Salomonsson
M, Carlsson
B, Haggblad
J 1994 Equilibrium hormone
binding
to human
estrogen
receptors
in
highly diluted cell extracts
is non-cooperative
and has a
K, of approximately
10 PM. J Steroid
Biochem
Mol Biol
50313318
25. Greene G, Nolan C, Engler J, Jensen
E 1980 Monoclonal
antibodies
to human estrogen
receptor.
Proc Natl Acad
Sci USA 77:5115-5119
26. Nardulli A, Greene
G, Shapiro
D 1993 Human estrogen
receptor
bound to an estrogen
response
element
bends
DNA. Mol Endocrinol
7:331-340
27. Heukeshoven
J, Dernick
R 1985 Simplified
method
for
silver staining
of proteins
in polyacrylamide
gels and the
mechanism
of silver staining.
Electrophoresis
6:103-l
12
28. Greene GL, Gilna P, Waterfield
M, Baker A, Hort Y, Shine
J 1986 Sequence
and expression
of human
estrogen
receptor
complementary
DNA. Science
231 :1150-l
154
29. Green S, Walter P, Kumar V, Krust A, Bornert
JM, Argos
P, Chambon
P 1986 Human oestrogen
receptor
cDNA:
Sequence,
expression
and homology
to v-erb-A.
Nature
320:134-l
39
30. Krust A, Green S, Argos P, Kumar V, Walter
P, Bornett
JM, Chambon
P 1986 The chicken
estrogen
receptor
sequence:
Homology
to v-erbA and the human estrogen
and glucocorticoid
receptors.
EMBO J 5:891-897
31. Thole HH, Jungblut
PW 1994 The ligand-binding
site of
the estradiol
receptor
resides
in a non-covalent
complex
of two consecutive
peptides
of 17 and 7 kDa. Biochem
Biophys
Res Commun
199:826-833
32. Thole HH 1993 Assignment
of the ligand binding site of
657
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
the porcine
estradiol
receptor
to the N-terminal
17 kDa
part of domain
E. FEBS Lett 320:92-96
Cotter RJ 1992 Time-of-flight
mass spectrometry
for the
structural
analysis
of biological
molecules.
Anal Chem
64:1027A
Vorm 0, Roepstorff
P, Mann M 1994 Improved
resolution
and very high sensitivity
in MALDI TOF of matrix surfaces
made by fast evaporation.
Anal Chem 66:3281-3287
Stark GR, Stein WH, Moore
S 1960 Reactions
of the
cyanate
present
in aqueous
urea with amino acids and
proteins.
J Biol Chem 235:3177-3181
Stark GR 1964 On the reversible
reaction
of cyanate
with
sulfhydryl
groups
and the determination
of NH,-terminal
cysteine
and cystine
in proteins.
J Biol Chem 239:141 l1414
Williams
D, Gorski J 1974 Equilibrium
binding of estradiol
by uterine cell suspensions
and whole uteri in Vitro. Biochemistry
13:5537-5542
Katzenellenbogen
JA, Johnson
Jr HJ, Carlson
KE 1973
Studies
on the uterine,
cytoplasmic
estrogen
binding
protein. Thermal
stability
and ligand dissociation
rate. An
assay of empty and filled sites by exchange.
Biochemistry 12:4092-4099
Bearden
JCJr 1978 Quantitation
of submicrogram
quantities of protein
by an improved
protein-dye
binding assay. Biochim
Biophys
Acta 533:525-529
Katzenellenbogen
JA, Carlson
KE, Heiman
DF, Robertson DW, Wei LL, Katzenellenbogen
BS 1983 Efficient
and highly selective
covalent
labeling
of the estrogen
receptor
with [3H]tamoxifen
aziridine.
J Biol Chem 258:
3487-3495
Bergmann
KE, Carlson
KE, Katzenellenbogen
JA 1994
Hexestrol
diazirine
photoaffinity
labeling
reagent
for the
estrogen
receptor.
Bioconjug
Chem 5:141-150
Springer
BA, Sligar SG 1987 High-level
expression
of
sperm
whale myoglobin
in Escherichia
co/i. Proc Natl
Acad Sci USA 84:8961-8965
Sorensen
MA, Kurland
CG, Pedersen
S 1989 Codon
usage
determines
translation
rate in Escherichia
co/i.
J Mol Biol 207:365-377
Zhang S, Zubay G, Goldman
E 1991 Low-usage
codons
in Escherichia
co/i, yeast,
fruit fly and primates.
Gene
105:61-72
deBoer
HA, Kastelein
RA 1986 Biased codon
usage: an
exploration
of its role in optimization
of translation.
Biotechnol
Ser 9:225-285
Sodeoka
M, Larson
CJ, Chen L, Lane WS, Verdine
GL
1993 Limited proteolysis
and site-directed
mutagenesis
of the NF-KB
~50 DNA-binding
subunit.
Bioorg
Med
Chem Lett 3: 1095-l
100
Silber KR, Sauer RT 1994 Deletion
of the prc (tsp) gene
provides
evidence
for additional
tail-specific
proteolytic
activity
in Escherichia
co/i K-12.
Mol Gen Genet
242:
237-240
Gottesman
S 1990 Minimizing
proteolysis
in Escherichia
co/i: genetic
solutions.
Methods
Enzymol
185:119-129
Dalbey RE, von Heijne G 1992 Signal peptidases
in prokaryotes
and eukaryotes-a
new protease
family. Trends
Biochem
Sci 171475-478
Kumar V, Green S, Stack G, Berry M, Jin JR, Chambon
P
1987 Functional
domains
of the human estrogen
receptor. Cell 51:941-951
Tora L, Mullick A, Metzger
D, Ponglikitmongkol
M, Park
I, Chambon
P 1989 The cloned human oestrogen
receptor contains
a mutation
which alters its hormone
binding
properties.
EMBO J 8:1981-l
986
Reese JC, Katzenellenbogen
BS 1991 Mutagenesis
of
cysteines
in the hormone
binding
domain
of the human
estrogen
receptor:
alterations
in binding
and transcriptional activiation
by covalently
and reversibly
attaching
ligands.
J Biol Chem 266:10880-l
0887
Reese JC, Katzenellenbogen
BS 1991 Differential
DNAbinding abilities
of estrogen
receptor
occupied
with two
Vol 9 No. 6
MOL END0 . 1995
658
classes of antiestrogens: studies using human estrogen
receptor overexpressed in mammalian cells. Nucleic
Acids Res 19:6595-6602
54. Greene G, Harris K, Bova R, Kinders R, Moore B, Nolan
C 1988 Purification of T47D human progesterone receptor and immunochemical characterization with monoclonal antibodies. Mol Endocrinol 2:714-726
55. Landel C, Kushner P, Greene G 1994 The interaction of
the human estrogen receptor with DNA is modulated
by receptor-associated
proteins. Mol Endocrinol 8:
1407-1419
56. Kushner P, Hart E, Shine J, Baxter J, Greene G 1990
Construction of cell lines that express high levels of the
International
human estrogen receptor and are killed by estrogens.
Mol Endocrinol 4:1465-l 473
57. Scatchard G 1949 The attractions of proteins for small
molecules and ions. Ann NY Acad Sci 51:660-672
58. Katzenellenbogen JA, Ruh TS, Carlson KE, lwamoto HS,
Gorski J 1975 Ultraviolet photosensitivity of the estrogen
binding protein from rat uterus. Wavelength and ligand
dependence. Photocovalent attachment of estrogens to
protein. Biochemistry 14:231 O-231 6
59. Schagger H, von Jagow G 1987 Tricine-sodium dodecyl
sulfate-polyacrylamide
gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal
Biochem 166:368-379
Workshop
on Heterogeneity
of Calcium Microdomains
and Cell Activation
Villa Olmo-Como,
Italy, November 34, 1995
Sponsored
by The Calcium Club-European
Chapter, Institute of Pharmacological
Sciences, Milan Fondazione Giovanni Lorenzini, Milan and Houston. Program and lectures are
as follows.
November 3, 1995-Friday
Afternoon:
1400-I 800: T. Meyer (Durham, USA) “Source of
nuclear calcium signals,” R. Rizzuto (Padua, I) “Measuring
organelle calcium with recombinant aequorin,”
A. Konnerth (Homburg,
D) “Dynamics
and localization
of calcium microdomains
during synaptic activity,” and W. Mason (Cambridge,
UK) “Real time confocal
and video imaging of spatial and temporal heterogeneity
in intracellular
and intranuclear
calcium signaling.”
November 4, 1995-Saturday
Morning: 0900-1230:
A. Marty (Goettingen,
D) “Calciuminduced calcium release in cerebellar
Purkinje cells,” K.-H. Krause (Geneve, CH) “Ca*+
microdomains
and site of lysosomal exocytosis in neutrophils,”
Speaker to be announced,
and Selected Oral Communications.
The schedule for Saturday Afternoon
(1330-1830)
is as follows. Poster Session: F. Grohovaz (Milan, I) “Intracellular
calcium mapping by analytical electron microscopy”
and S.
Bolsover (London, UK) “Nuclear calcium dynamics in nerve cells.” Round Table chaired by
Prof. E. Carafoli: “Independent
regulation
of Ca*+ in the nucleus: yes or no?” Invited
discussants:
M. Mazzanti, T. Meyer, R. Rizzuto, L. Santella (plus selected brief communications).
Send requests for information
regarding Program and Registration
Form to Organizing
Secretariat:
Daniela Galli, The Calcium Club, Institute of Pharmacological
SciencesUniversity of Milan, Via G. Balzaretti g-20133 Milan (Italy), Phones: (+ 39/2) 20.488.320,
20.488.312,
20.488.306.
Fax: (+39/2) 2940.4961.
Information regarding scientific aspects
of the program may be requested from Scientific Secretary: Simonetta Nicosia.