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(Received for publication, June 29, 1995; and in revised form, September 15, 1995) From the
The potassium channel T1 domain plays an important role in the
regulated assembly of subunit proteins. We have examined the assembly
properties of the Shaker channel T1 domain to determine if the domain
can self-assemble, the number of subunits in a multimer, N
Voltage-gated K The mechanisms that govern the assembly
and function of voltage-gated ion channels are poorly understood.
Recently we and others have identified a conserved molecular domain,
the T1 domain, encoded within the cytoplasmic N terminus of the
Other recent studies have questioned the
role that the T1 domain plays in K In this paper, we have developed methods to efficiently
separate T1 domain proteins into different molecular weight species, in
order to separate assembled and unassembled proteins. By combining
molecular size separation with co-precipitation techniques we have
determined the assembled species that are responsible for the
co-precipitation of untagged T1 domains. Finally, using chemical
cross-linking we have measured the number of subunit proteins assembled
in the peak assembled fraction. These results have allowed us to
formulate more specific models of the mechanisms by which the T1 domain
functions in K
In vitro translation samples were prepared by mixing 1-2 µl of in vitro translation reaction with 200 µl of bind buffer.
The sample was then cleared by centrifugation at 60,000 rpm for 20 min
at 4 °C in a mini-ultracentrifuge (Beckmann). For HPLC samples,
individual fractions were adjusted to bind buffer conditions, by the
addition of salts, CHAPS, and imidazole, before applying to the resin.
Sample supernatant was mixed with the resin for 10 min at 4 °C to
ensure complete binding of poly-His-tagged proteins to the resin.
Unbound material was then spun through the column, and the resin was
washed in a series of steps: first 1
For HPLC analysis,
samples were cross-linked at 1.5 mM DSS, as described above,
and then injected into the HPLC following quenching.
HPLC runs were
standardized by detection of protein standards using an absorbance
detector set at 280 nm, before a series of experimental runs were
performed. Because of the small amount of synthesized protein relative
to the total protein in the in vitro translation mix, T1
domain proteins were not directly detected by UV absorbance but rather
by Soluble N-terminal peptides from the Shaker type K
Figure 1:
Schematic
description of the Shaker T1 domain containing constructs that were
used in these experiments, showing the region of the parent AKv1.1a
clone that these constructs originated from. Full N-terminal constructs
are CF2-Tag1 (AKv1.1a, amino acids 1-193), 1ABC, and
1ABC(poly-His) (AKv1.1a, amino acids 1-196). T1 domain only
constructs are 1T1-T7tag (AKv1.1a, amino acids 58-196) and 31A1BC
(AKv3.1a, amino acids 1-10, followed by AKv1.1a, amino acids
67-196). Locations of the conserved subdomains A, B, and C of the
T1 domain are indicated, as well as the locations of the epitope tags
used and the restriction enzyme sites that are introduced into the
clones to facilitate chimera construction (see (11) ). The
first transmembrane domain, S1, begins at AKv1.1a,
Leu
Figure 2:
T1 domain proteins separate into two peaks
on size exclusion chromatography columns. In vitro translated
1ABC protein was separated on a Bio-Silect 250-5 (Bio-Rad)
silica-based size exclusion HPLC column. This columns separates
proteins based on the size of the molecule, with larger proteins
eluting in earlier fractions. A, autoradiography of HPLC
fractions showing the separation of
[
Figure 3:
Co-immunoprecipitation analysis of HPLC
protein peaks identifies assembled fractions. CF2-Tag1 and 1T1-T7tag
were co-translated then run on a Bio-Silect 400-5 SEC column to
separate according to size. Fractions corresponding to the positions of
the high molecular weight peak (fractions 19, 20, and 21) and the low
molecular weight peak (fractions 23, 24, and 25) were pooled
separately. Both peaks and the original unfractionated translation were
probed to determine if the proteins were co-assembled. Anti-1B is a
common antiserum to the B subdomain of the T1 domain in both proteins,
and it is a positive control for the presence of both
proteins(11) . Anti-Tag1 only recognizes CF2-Tag1; thus
co-precipitation of 1T1-T7tag by this antiserum is indicative of
assembly. The minus antibody lane(-) is the negative
control. Both proteins are found in the two peaks separated on SEC
HPLC, as verified by precipitation with anti-1B; however, 1T1-T7tag is
not co-precipitated in the low molecular weight peak. Thus only in the
high molecular weight peak are the two proteins
co-assembled.
To
determine which peak(s) contain assembled proteins, we co-translated
1T1-T7tag, and CF2-Tag1 and separated the proteins on a Bio-Silect
400-5 SEC HPLC column. High molecular weight and low molecular
weight fractions were separately pooled, and assembly-tested in the two
protein populations by anti-Tag1 antiserum immunoprecipitation. Both
1T1-T7tag and CF2-Tag1 proteins are present in both peaks, as verified
by immunoprecipitation with the common anti-1B antiserum. However,
1T1-T7tag proteins are only co-immunoprecipitated from the high
molecular weight protein fractions and not from fractions corresponding
to the low molecular weight peak. Therefore, the proteins in the low
molecular weight peak are not co-assembled and thus are probably
monomeric. Identical results were obtained from proteins separated on
Bio-Silect 250-5 and 125-5 SEC columns.
Figure 4:
Ni
Ni
Figure 5:
Ni
Figure 6:
Chemical cross-linking of T1 domain
proteins. 1ABC was in vitro translated and then cross-linked
with increasing concentrations of DSS, as described under
``Experimental Procedures.'' Cross-linked proteins were run
on a 5-20% linear gradient SDS-PAGE gel, and the positions of the
proteins were determined by autoradiography. The first lane shows the migration of molecular mass standards. The monomer band
is the only band present in the non-cross-linked sample. At the lowest
level of cross-linking, a band at the expected position for a dimer is
seen. At higher cross-linker concentrations, bands are seen at the
expected positions for the trimer and tetramer, with additional
material present as a diffuse smear at high molecular weights. The
protein band at the tetramer position is broader than the other bands
and appears to be a doublet.
Figure 7:
SEC HPLC analysis of cross-linked T1
domain proteins characterizes the assembled protein peak. 1ABC was in vitro translated, then cross-linked with 1.5 mM DSS as described under ``Experimental Procedures.'' This
cross-linked material was then separated by size on a Bio-Silect
250-5 SEC HPLC column. Fractions were acetone-precipitated and
then run on a 5-20% linear gradient SDS-PAGE gel and subjected to
autoradiography to determine the migration pattern of the different
protein species. A, autoradiograph of the HPLC fractions
following separation of cross-linked 1ABC. B, densitometry
analysis of bands from the gel in panel A. Band volume for
proteins at the different molecular weight sizes was measured and
plotted without normalization versus the fraction number. The
monomer band shows two peaks at the expected high and low
molecular weight positions seen in uncross-linked samples. Dimer,
trimer, and tetramer bands all show a single peak in the fraction
corresponding to the high molecular weight peak fraction for
uncross-linked samples. The higher molecular weight smear of
cross-linked protein peaks in earlier fractions that do not correspond
to the position of protein peaks in uncross-linked samples. Therefore,
the high molecular weight T1 domain peak is identified as being
composed of T1 domain protein
homotetramers.
The SEC HPLC
separation of the proteins shows that T1 domain proteins can be
cross-linked up to the tetramer size and still migrate in the high
molecular weight peak, thus identifying the higher molecular weight
peak as the tetrameric form of the protein. Furthermore, the fact that
the dimer and trimer proteins also show a single peak at the tetramer
position indicates that they are partially cross-linked tetramer
proteins that are separated during the SDS-PAGE analysis. The T1 domain was originally identified through its role in
K HPLC
analysis shows that the T1 domain exists predominantly in two forms, a
high molecular weight form and a low molecular weight form. Both
co-immunoprecipitation and cross-linking analysis clearly identify the
high molecular weight form as an assembled state of the protein and the
low molecular weight form as the unassembled, monomeric form of the T1
domain. Chemical cross-linking shows that all the assembled T1 proteins
cross-link in an evenly spaced ladder; therefore, the T1 domain can
assemble as a homomultimer. Four different sizes of T1 domain protein
are produced by cross-linking, monomer, dimer, trimer, and tetramer.
Larger molecular weight cross-linked complexes run in anomalous
positions on HPLC and therefore are probably artifactually created
during the cross-linking procedure. Dimer, trimer, and tetramer bands
all show a single peak on HPLC that lines up with the assembled peak
seen on uncross-linked samples. Therefore, these structures represent
variable cross-linking of a single, tetrameric assembled form of the
protein. The lack of a detectable binary assembled form in the T1
domain HPLC profile suggests that there is no particular stability
associated with this structure. This observation argues against
subunits assembling as dimers and the dimers assembling as tetramers in
an isologous association. Rather, the results suggest that T1 domain
proteins assemble by the monomeric addition of single subunit proteins
in a repeated A-B interface interaction pattern until the fourth
subunit protein completes the closed ring. Such heterologous
association has been observed in the multimerization of other membrane
proteins, such as neuraminidase(15) . Closer analysis of the
cross-linked tetrameric band, following autoradiography of SDS-PAGE
gels reveals that it often appears to be a doublet or a broader band
than any of the other T1 domain bands. This suggests that the tetramer
can be cross-linked into two different structures. One structure is
presumably the linear form, common to the monomer, dimer, and trimer
cross-linked proteins. The second structure probably represents the
fully cross-linked, native form of the assembled T1 domain. Based on
the presumed form of the functional K A closed circular structure of the assembled T1 domain would provide
additional stability to the tetramer by providing stabilizing
subunit-subunit interactions on both sides of the T1 domain, thus
requiring two separate interactions to be broken in order to disrupt
the structure. Indeed, co-purification analysis on HPLC fractions,
using a poly-His-tagged subunit protein co-translated with an untagged
subunit protein, shows that co-purification is limited to the tetramer
peak. Thus interactions between T1 domain proteins that are not
completed to the tetrameric state must dissociate within the time frame
for these experiments. Cross-subfamily interactions between T1 domains
or point mutations that disrupt the tetramer may be filtered out by
this stringent assembly requirement. Selectivity against assembly of
K
Volume 270,
Number 48,
Issue of December 1, 1995 pp. 28595-28600
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Channel T1 Domain Self-tetramerizes to a Stable
Structure (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
and the mechanism of assembly. High pressure
liquid chromatography (HPLC) size exclusion chromotography (SEC)
separates T1 domain proteins into two peaks. By co-assembly assays,
these peaks are identified to be a high molecular weight assembled form
and a low molecular weight monomeric form. To determine the N of the assembled protein peak on HPLC SEC, we
first cross-linked the T1 domain proteins and then separated them on
HPLC. Four evenly spaced bands co-migrate with the assembled protein
peak; thus, the T1 domain assembles to form a tetramer. The absence of
separate dimeric and trimeric peaks of assembled T1 domain protein
suggests that the tetramer is the stable assembled state, most probably
a closed ring structure.
channel proteins are
multisubunit ion channel proteins. The core channel consists of an
apparent tetramer of
-subunit proteins that assembles to form the
K ion-selective aqueous pore across the cell's
plasma membrane(1) . In addition,
-subunit proteins, which
are apparently not transmembrane proteins, can be attached to each
-subunit protein(2) . Molecular cloning has revealed a
large diversity of K channel subunit proteins.
Sequence comparisons among
-subunits has revealed that the
similarities among encoded proteins cluster into a variety of
K channel subfamilies(3) . Biophysical studies
have indicated that these subfamilies are in fact functional subsets of
channel proteins in that functional heteromultimeric channels have only
been formed by co-expression of two
-subunit proteins from the
same subfamily(4) .-subunit protein that plays an important role in the assembly of
K channel subunit
proteins(5, 6, 7, 8, 9, 10) .
Our studies have suggested that the T1 domain, translated by itself,
can self-assemble(6) . Sucrose density gradients reveal the
formation of a high molecular weight complex; co-immunoprecipitation
studies show that a tagged T1 domain protein can co-precipitate another
un-tagged T1 domain protein. In addition, the T1 domain contains the
molecular recognition sequences required for the subfamily-specific
assembly of voltage-gated K
channel
proteins(11) . Chimeras made with swapped N-terminal sequences
show the assembly specificity of the N-terminal donor; the soluble T1
domain translated by itself only co-assembles with T1 domain proteins
made from the same subfamily. These results have prompted our
hypothesis that the T1 domain is the primary site for organized
tetramerization of K
channel subunit proteins along
subfamily-specific lines.
channel subunit
assembly. Subunit proteins synthesized with deleted N termini have been
shown to function in Xenopus oocyte expression
systems(9, 10, 12, 13) . In
addition, another group has suggested that S1, the first transmembrane
domain, is necessary for assembly, based on their hydrodynamic and
co-precipitation studies(7) . In fact, no studies have directly
examined the assembled T1 domain protein to show that it is forming a
tetramer on its own. Tetramerization has only been inferred based on
the migration rate of the protein in sucrose gradients or gel
filtration columns. In addition, the molecular species responsible for
the co-immunoprecipitation signal has not been identified. It is not
clear to what extent dimers, trimers, tetramers, larger octomers, or
complexes with other proteins contribute to the co-immunoprecipitation
signal. Finally, what role the T1 domain plays in the normal assembly
of other, non-Shaker type potassium channels has not been directly
examined.
channel assembly.
T1 Domain Constructs
The constructs CF2-Tag1,
1ABC(no tag), 31A1BC, and 1T1-T7tag were described
previously(6, 11) . In experiments not taking
advantage of epitope tags, 1ABC(no tag) is abbreviated 1ABC. All clones
contain stop codons immediately following the expressed sequence. For
1ABC, the stop codon was inserted into the EcoRI site of pCITE
2A (Novagen) by oligonucleotide-directed mutagenesis. 1ABC(poly-His)
construct is identical to 1ABC, except it lacks a stop codon at the EcoRI site. This puts the potassium channel sequence in frame
with the poly-His (6-His) tag in pCITE 2A (Novagen), with the stop
codon provided by the vector immediately following the 6-His stretch.In Vitro Translation
In vitro translation
procedures were described previously(11) . All proteins were
synthesized with [S]Met. Microsomal membranes
were not needed in these experiments, since all constructs lacked
transmembrane domains.
SDS-PAGE and Autoradiography
Electrophoresis and
autoradiography procedures were performed similarly to those described
previously(11) . Gradient gels were poured in Bio-Rad minigel
multicasters, from the bottom, as recommended by the manufacturer.
Acetone precipitations were performed to concentrate large volume
samples using bovine serum albumin (BSA) as a carrier, as described
previously(11) . In cross-linking experiments, lactalbumin was
substituted for BSA to avoid interference with the higher molecular
weight bands.Immunoprecipitations
Immunoprecipitation
experiments were performed using procedures described
previously(11) .Ni
Affinity columns were prepared by placing
Ni Affinity
Chromatography
affinity resin (Novagen), sufficient to form a
50-µl bed volume, into a 0.65-µm Ultrafree-MC microcentrifuge
spin filter (Millipore Corp.). The resin was washed in distilled
H
0 to remove storage buffer. All Ni column buffers were removed by brief centrifugations (<2 min)
at 4,000 rpm in a microcentrifuge at 4 °C. Washed resin was then
charged with Ni
by equilibration in 250 µl of
charge buffer (50 mM NiSO
) for 10 min at 4 °C.
Charge buffer was removed and exchanged with 250 µl of bind buffer
(500 mM NaCl, 20 mM Tris, pH 7.9, 1% CHAPS, (
)5 mM imidazole) to prepare the column for use.
Following removal of the bind buffer, the column was ready for use.
Each column was used for only one purification.
wash with 350 µl of
bind buffer, followed by 3 washes with 350 µl of wash
buffer (bind buffer with 20 mM imidazole). Samples were eluted
in 350 µl of elution buffer (bind buffer with 1 M imidazole). All fractions were collected and analyzed by SDS-PAGE
gel and autoradiography. Samples were concentrated for gel analysis
either by acetone precipitation or immunoprecipitation with the common
anti-1B antiserum(11) .Chemical Cross-linking
Samples were prepared for
cross-linking by adding 8 µl of in vitro translation
reaction to 800 µl of cross-link buffer (150 mM NaCl, 20
mM HEPES, pH 7.9, 1% CHAPS, 5 mM imidazole).
Hemoglobin, the dominant protein in the in vitro translation
mix, was removed by passing the material through charged Ni affinity resin equilibrated in cross-link buffer. Hemoglobin
binds to the resin under these conditions, and non-poly-His tagged T1
domain proteins pass through the resin. Disuccinimidyl suberate (DSS,
Pierce) was added to alloquots of cleared sample (total volume 100
µl), from a 25 mM stock in dimethyl sulfoxide, to vary DSS
concentration from 0 to 2.5 mM. The cross-linking reaction was
incubated on ice for 30 min. The reaction was quenched by adding 5
µl of 1 M Tris and incubating for 60 min at room
temperature. Samples were analyzed by SDS-PAGE gradient gels
(5-20%) and autoradiography. Samples were concentrated for gel
analysis either by acetone precipitation or immunoprecipitation with
the common anti-1B antiserum(11) .
Size Exclusion HPLC
Silica-based size exclusion
HPLC chromatography columns and guard columns were obtained from
Bio-Rad. HPLC was performed on a Waters HPLC at 0.5 ml/min with one
fraction collected per min. Columns were equilibrated in standard
buffer (150 mM NaCl, 20 mM Tris, pH 7.6, 1 mM EDTA) at 4 °C. Samples were prepared for injection by mixing 5
µl of in vitro translation reaction with 100 µl of
standard buffer and cleared by centrifugation at 60,000 rpm at 4 °C
for 30 min. The supernatant was filtered through a 0.45-µm
microcentrifuge spin filter (Millipore) and injected into the HPLC. The
column was checked before each series of runs by injection of molecular
weight size standards. Three sets of standards were used: TGOM standard
(thyroglobulin, globulin, ovalbumin, myoglobin, and vitamin B-12
to mark the total volume; FCAC standard (ferratin, catalase, albumin,
and chymotrypsinogen); and TAOR standard (thyroglobulin, aldolase,
ovalbumin, and ribonuclease A). The position of the hemoglobin peak was
detected at 280 nm to verify the consistent separation of our samples. In vitro translated proteins could only be detected by gel
analysis of fractions collected from the column. All fractions from the
excluded volumn to the void volumn were collected and analyzed by
SDS-PAGE gel and autoradiography. Samples were concentrated for gel
analysis either by acetone precipitation or immunoprecipitation with
the common anti-1B antiserum (11) .
Data Quantitation and
Analysis
S-Labeled samples were analyzed by either
film densitometry of autoradiographs or direct PhosphorImager analysis
of gels. Phosphor image capture was performed on a Molecular Dynamics
PhosphorImager. Film images were captured using an Envisions ENV25Pro
scanner. All images were analyzed using the Molecular Dynamics
ImageQuant version 3.3 software package. Bands were quantified using
volume integration with background subtraction from a neighboring empty
location. Both methods were extensively compared and found to give
comparable results. The data were plotted with Deltagraph and fitted
with cubic spline curves for presentation.
S autoradiography of SDS-PAGE gels run on the
fractionated column eluate. To calculate the distribution coefficient, K
, which describes the average access of a protein
to the total column volume, we needed to measure the void and total
volumes for the columns. Void volume was detected as the first
appearance of protein aggregates; the total volume was detected by the
appearance of vitamin B-12. K is (peak volume
- void volume)/(total volume - void volume)(14) .
For in vitro translated proteins, peak protein fraction
volumes were set at the midpoint volume for the fraction and normalized
back to the absorbance detector by subtracting the volume in the line
after the detector up to the fraction collector. In internal control
for this correction was the peak hemoglobin fraction. Protein peaks
that were evenly divided between two fractions were considered to peak
at the point where the fraction changed. Because these K measures for T1 domain proteins were only accurate to half a
fraction volume, these numbers are considered approximations.
channel subunit protein AKv1.1, containing the T1 domain (see Fig. 1) were synthesized in vitro using a rabbit
reticulocyte lysate system, as described
previously(6, 11) . All proteins translated
efficiently without the addition of microsomal membranes and were
soluble without added detergent. Full N-terminal peptides that were
studied include CF2-Tag1, an epitope tagged N-terminal
peptide(6) ; 1ABC(no tag), the full N-terminal protein with
restriction sites added between subdomains of the T1
domain(11) ; and 1ABC(poly-His), the 1ABC protein cloned in
frame with the C-terminal 6-His sequence of the pCITE-2A vector
(Novagen). In addition, two constructs encoding the Shaker T1 domain
with minimal additional sequences were examined: the epitope-tagged T1
domain protein, 1T1-T7tag(11) , which uses the pCITE-2A
initiator Met, and 31A1BC(11) , which uses the AKv3.1a
initiator Met and 10 amino acids, before the Shaker T1 domain. There
were no detectable differences in the behavior of any of these
constructs except for epitope tag sensitivity and longer retention of
T1 only proteins on SEC HPLC due to their smaller size. The results
presented are based on over 110 individual HPLC runs, with a minimum of
three experiments for any individual result.
.
HPLC Size Exclusion Chromatography of N-terminal
Proteins
Synthesized proteins were size-fractionated by size
exclusion chromatography on silica-based HPLC columns (Bio-Rad).
Columns with three different pore sizes were tested, Bio-Silect
125-5 (fractionation range 5-100 kDa), Bio-Silect
250-5 (fractionation range 10-300 kDa), and Bio-Silect
400-5 (fractionation range 20-1000 kDa). Fig. 2shows an autoradiogram and a densitometry plot of a
typical separation of 1ABC protein on a Bio-Silect 250-5 column.
Two peaks of 1ABC protein are readily apparent, suggesting that the
protein is primarily found as two different sized species. Based on the
fraction that the protein peaks were located in, approximate values
were obtained for the fraction of the total volume that the protein has
access to, K(14) . For full-length
N-terminal proteins, such as 1ABC or CF2-Tag1, on a Bio-Silect
250-5 column, the low molecular weight protein peak has an
approximate K of 0.53, whereas the high molecular
weight protein peak has an approximate K of 0.24.
For full length N-terminal proteins on Bio-Silect 400-5 columns,
the K for the low molecular weight peak is
approximately 0.66, and the high molecular weight peak is approximately
0.46. Bio-Silect 125-5 columns do not adequately fractionate the
higher molecular mass peak for full length N-terminal proteins (K < 0.1), suggesting that it is >100 kDa,
as expected for a tetramer. For the T1 domain only proteins, such as
31A1BC or 1T1-T7tag, similar results are obtained except that the two
peaks are retained for slightly longer times, as expected for the
smaller size of these proteins. Fractionations of soluble T1 domain
proteins were unchanged by the addition of 1% CHAPS detergent to the
sample or running buffer. We next sought to determine directly whether
the two protein peaks correspond to differently assembled states of the
T1 domain proteins.
S]Met-labeled 1ABC protein through this column.
The first fraction run on the gel is the void volume; the last fraction
is the total volume. B, quantitation of the autoradiograph shown in panel A. Band volumes were measured for 1ABC
protein in each fraction and plotted versus the fraction
number. As is clear from the autoradiograph and the quantitation, the
1ABC protein separates into two distinct populations of differing
apparent size during the chromatography.
Co-immunoprecipitation of Proteins Separated on
HPLC
The first experiment was to employ a co-immunoprecipitation
assay on fractions corresponding to the high and low molecular weight
T1 domain protein peaks following separation on SEC HPLC. In this
co-immunoprecipitation assay, CF2-Tag1 was co-translated with the T1
domain only protein 1T1-T7tag. Co-assembly of these proteins was
measured by the ability of the anti-Tag1 antiserum to co-precipitate
the 1T1-T7tag protein, which it does not directly recognize, based on
the stable assembly of this protein with CF2-Tag1, which is recognized
by the antiserum. A typical result for this assay is seen in the
immunoprecipitation of unfractionated proteins in Fig. 3.
Ni
In order to determine a more complete profile
of the migration pattern for assembled T1 domain proteins on SEC HPLC,
we developed a more efficient and rapid co-purification assay based on
the ability of poly-His-tagged T1 domain proteins to selectively bind
to a NiAffinity Column Analysis of
Subunit Assembly
affinity resin. Without poly-His tagging, T1
domain proteins do not bind to the Ni
affinity resin (Fig. 4A). By co-translating untagged, and poly-His-tagged
T1 domain proteins, the co-assembly of these proteins can rapidly be
determined by the purification of untagged proteins on the
Ni
affinity column. Fig. 4B shows the co-purification of 1ABC(no tag), with 1ABC(poly-His) on
a Ni
affinity column. Therefore, the poly-His tag
does not disrupt the ability of the proteins to assemble.
affinity column
co-purification analysis demonstrates T1 domain protein assembly. T1
domain proteins were in vitro translated and then applied to
Ni
affinity columns. The columns were washed at
increasing stringency, and then bound proteins were eluted at a high
imidazole concentration (1 M imidazole). A, affinity
chromatography of 1ABC(no tag) on Ni
affinity resin. L, sample loaded; F, column flow-through, B,
bind buffer wash. W, washes with wash buffer. E,
elution with elution buffer. 1ABC(no tag) does not bind to the
Ni
affinity resin; therefore, no protein is present
in the elution step. B, affinity chromatography of 1ABC(no
tag) co-translated with 1ABC(poly-His) on Ni
affinity
resin. Although 1ABC(no tag) does not directly bind to the
Ni
affinity resin, it is present in the elution step
because of its stable assembly with 1ABC(poly-His), which does bind to
the column.
Column Analysis of Proteins Separated on
HPLC-The Ni
affinity column
co-purification analysis was next applied to fractions from the HPLC
separation of co-translated 1ABC(no tag) and 1ABC(poly-His). The raw
fractions, before application to the Ni
affinity
resin, show that both proteins are separated in similar manners,
suggesting that the proportion of assembled and unassembled proteins
was similar for each. The fractions were applied to Ni
affinity columns, washed, and eluted. Elution off the
Ni
column shows that 1ABC(poly-His) proteins are
purified identically to their original profile; however, 1ABC(no tag)
proteins are only co-purified in the higher molecular weight fractions.
Quantitation of the counts for the various proteins, following
Ni
affinity column co-purification analysis on the
different fractions, shows that 1ABC(no tag) T1 domain protein
co-purifies as a monophasic peak that is identical to the high
molecular weight peak identified above (see Fig. 5). This
suggests that the high molecular weight peak is the sole form of stably
assembled T1 domain proteins and the low molecular weight peak is
completely composed of monomeric proteins.
affinity column
co-purification analysis of HPLC fractions shows assembled protein
separation profile. 1ABC(no tag) and 1ABC(poly-His) were co-translated
and separated on a Bio-Silect 250-5 SEC HPLC column. Fractions
from the column were split in half; half the fraction was analyzed for
the protein migration, and the other half was applied to a
Ni
affinity column. A, autoradiography of
proteins separated on a Bio-Silect 250-5 SEC column. B,
band volume quantitation of experiment in panel A. The
co-translation autoradiograph shows the chromatography of the T1 domain
proteins before application to the Ni
affinity
columns. OT lane shows the unfractionated translation. The
following lanes are successive fractions from the column,
starting at the void volume. Upper band is 1ABC(poly-His), and
the lower band is 1ABC(no tag). Both proteins are present in
the translation and show essentially identical SEC separation patterns
with high and low molecular weight protein peaks. The Ni
column flow-through for the HPLC fractions shows only a single
protein at the 1ABC(no tag) molecular weight. In the Ni
column elution, both 1ABC(no tag) and 1ABC(poly-His) are present
in fractions corresponding to the high molecular weight peak; however,
only 1ABC(poly-His) is present in the fractions corresponding to the
low molecular weight peak. Therefore, T1 domain proteins in the
assembled state are completely contained within the high molecular
weight peak fractions.
, 1ABC(poly-His):Ni
column eluted;
, 1ABC(no tag):Ni column
eluted;
, 1ABC(no tag):Ni column
flow-through.
Chemical Cross-linking of T1 Domain Proteins
In
order to directly measure the N of the assembled
T1 domain protein complex, we chemically cross-linked in vitro translated T1 domain proteins. Fig. 6shows the
cross-linking of 1ABC with varying concentrations of DSS. SDS-PAGE
gradient gel analysis shows that cross-linked 1ABC protein runs as a
regularly spaced ladder of four clear bands, with additional
cross-linked material visible at higher molecular weights. The lowest
band is the 1ABC monomer, visible in the absence of cross-linker. The
three higher bands are even sized multiples of the 1ABC monomer and
most likely represent multimerically cross-linked 1ABC proteins.
Consistent with this interpretation, the dimer band is the first to
appear, with progressive movement of protein to trimer and
tetramer-sized molecular weights with additional cross-linking. The
tetramer band is always more diffuse than the monomer, dimer, or trimer
bands and sometimes appears to be a doublet, suggesting that at least
two different cross-linkings of the tetramer are possible. Higher
molecular weight complexes represent either further multimerizations of
the basic monomer or coupling of 1ABC to other proteins, possibly
artifacts of the coupling procedure. Identical results were obtained
with other T1 domain proteins.
HPLC Analysis of Chemically Cross-linked T1 Domain
Proteins
We next combined chemical cross-linking with separation
of proteins on SEC HPLC to directly characterize the specific molecular
composition of the two peaks of T1 domain proteins observed above and
to determine if higher molecular weight complexes, created during
cross-linking, co-migrate with the T1 domain protein peaks or migrate
at novel positions, suggestive of artifactual couplings. In Fig. 7, 1ABC domain proteins were cross-linked in 1.5 mM DSS and then run on a Bio-Silect 250-5 column. Protein in
the monomer band shows the characteristic biphasic peaks at identical
positions as uncross-linked samples. The dimer, trimer, and tetramer
peaks are monophasic and co-migrate with the higher molecular weight
monomer peak. Higher molecular weight cross-linked species do not
co-migrate with either peak; rather they migrate at novel higher
molecular weight positions and thus probably represent species that are
artifactually linked during the cross-linking.
channel subunit assembly(5, 6) .
Later, it was shown to be a critical regulator of subunit
heteromultimerization, by controlling the subfamily specific
heteromultimerization of K
channel subunit
proteins(11) . This work has sought to examine the T1 domain,
separate from the K
channel transmembrane domains, to
measure directly the self-assembly properties of the T1 domain.
channel, with an
axis of symmetry down a central aqueous pore, and the unique stability
of the fully assembled state of the T1 domain, the native form of the
assembled T1 domain is probably a closed circular, ring, structure.
channel subunit proteins across subfamilies or
disruption of assembly by mutagenesis can therefore occur either
through incompatible interface amino acids or through subtle structural
differences between proteins with compatible interfaces that prevent
the closing of the ring, either through torsion or interface angle
incompatibility.
)
We give special thanks to Melinda M. Boyer for
assistance in molecular biology, Shawn Neff for oligonucleotide
synthesis, Dr. Mark Perin for anti-Tag1 antisera and helpful
discussions, Dr. Biao Zhao for anti-1B antisera, and Dr. Larry Starke
and N. Vivienne Shen for helpful discussions on this project.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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