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Self-assembly of protein subunits from bacteriophage fr
Vol. 30, No. 5, 1968
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SELF-ASSEBBLYOF PROTEW SUBUI?ITSFROMBACXCERIOPHAGEfr R. Herrmann,
D. Schubert
Max - Planck Institut
and U. Rudolph
fiir
'Piibingen,
Virusforschung
Germany
Received February 9, 1968
It
is well
known that under
appropriate
subunits
of tobacco mosaic virus
virus-like
rods ( Schrsmm, 1943;
Fraenkel-Conrat
and Singer,
have predicted possible
that
for
only been possible degradation reaggregation
viruses.
of protein
Until
protein
solutions
should
1955;
also be
now, however,
shells
( Finch
subunits
acid ( Sugiysma et al.,
of such viruses
and Klug,
1960
in the presence
1967; Hohn,
by
) or by
of nucleic
Bancroft
1967;
it has
and Hiebert,
1.
1967
Using the bacteriophage weight
4x10~
diameter
Daltons
of nucleic
fr,
indeed possible
shells
The coat protein
in the monomeric form.
In this
to obtain
et
virus
al.,
from protein
virus-like
yield.
576
to
it
achieve
the
material
is shown that
protein
> and
in the absence
used as starting
paper
of particle
1963
) we have tried
1967
of icosahedral acid,
en icosahedral
( Hoffmann-Berling
of 260 A ( Frank,
self-assembly
high
Schramm and Zillig,
to form
1957 >. Casper and Klug ( 1962 )
to obtain
of virus
protein
( TMV ) can reaggregate
such a self-assembly
icosahedral
conditions
shells
it
was is
in a rather
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 30, No. 5, 1968
Materials
and Methods
a) fr-virus
0 The phsge was grown as described
Berling
et al.
( 1963 ), using the trypton
and Hoffmsnn-Berling obtained modified
t
for
of protein
was carried
The mixture
about 15 min. By this
was left
in en ice bath
time most of the R?JAhad precipitated
centrifugation was diluted
solution
with
had been equilibrated for
the protein
24 hours.
dialysis
20 min, the super-
3396 acetic
were collected
acid.
The fractions
and dialyzed
was concentrated
0.1
water pressure
by this
at 30,000 rpm
was dAalyzed against
pH 7.6 containing
against
to pH 7.5 with 0.02 M NaOH. Most of
centrifugation
supernatant
con-
by negative
which had formed was dissolved
of the pH. After
The
to a column of Sephadex G 50 which
with
and was brought
rpm for
( 5 min ). After
an equal volume of cold water.
The solution
the precipitate clear
at 30,000
was then applied
taining
a
1957 ). Two
end it was removed by low speed centrifugation natant
out using
acid were added to one volume of a solution
( 4%) in water.
a further
32P-RNA was
( 1966 ).
acid method ( Fraenkel-Conrat,
volumes of acetic of virus
by Heisenberg
Isolation
acetic
medium of Knippers
( 1966 ). Phage containing
as described
b) fr-protein
by Hoffmann-
adjustment
for 20 min the
0.02 M phosphate
M NaCl. The final
protein
buffer
concentration
was between 0.5 end 2 mg/ml. 32P labelled virus
solution
protein that
phage ( 1x10~ opm/mg protein to determine
preparation. after
the amount of RNA remaining
Measurement of the radioactivity
column chromatography
decreased
to less than 0.0%
material.
Before
tained
) was added to the in the showed
the counts per mg protein
of the value
column chromatography
about s RNA. The W absorbance
577
had
of the original,
the preparation ratio
con-
A260/B2So of the
BIOCHEMICAL
Vol. 30, No. 5, 1968
purified
preparations
( without
measured in 33% acetic
correction
The preparation
for
light
scattering
and purification
carried
out in an ice bath,
results
were obtained
c) Physical
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
although
sorbance
t
measurements
tion
counter.
stained
with
Mr.G. Berger generous
gift
precipitates
Ultracentrifuge
were counted
uranyl
acetate
Results
and Discussion
monomerfc protein
that
antiserum
subunits
with
for
specific
( Anderer
et al.,
partial 1959;
( Wittmann-Liebold,
is split
weight
to
determinations
method ( Schachmsn, of
16
ooo, assuming 0.75 ml/g
the protein
578
solvent
1959 ). The molecular
with the value
).
) were
1959
acid plus
volume in this
methods for 1966
to the method of Heidel-
) molecular
Wittmann,
of 16 ooo is in good agreement by chemical
fr was the
acid and in 6696 acetic a value
and
acid in the ssme manner as the
1959
o.lM NaGl we have found the protein
against
of the virus
equilibrium
In 6696 acetic
made by Dr.H.Frank
).
acetic
of TMV ( Wittmann,
negatively
The antigen-antibody
according 1943
scintilla-
I A.
the coat protein
by the sedimentation performed.
(
UV-ab-
o.l)'C.
of preparations
of Dr. H.D.Schlumberger.
and Mac Pherson
schlieren
spectrophotometer.
were kindly
t Rabbit
berger
using
on a Packard liquid
on a Siemens-Elmiskop test
was performed
were made at (20.02
were obtained
To prove
the seme
Ultracentrifugation
Electronmicrographs
d) Serological
obtained
essentially
was measured on a Zeiss MQ III
32p-disintegrations
was usually
at room temperature.
measurements
All
).
of the protein
on a Spinco Model E Analytical optics.
acid was 0.550.59
of
16
subunit
weight
600
of fr
Vol. 30, No. 5, 1968
BIOCHEMICAL
It was apparent
from the electron
tion
to 3396 acetic
virus.
Significant
had been dialyzed therefore weight within
against
supposed that aggregates.
appeared
the limits
Essentially microscope
( Fig.
of about lmg/ml
homogeneously sediments all
after
the protein
high molecular these aggregates
1 ), having
equal to that
of the particles
virus
with
diameters,
of the intact
virus
2 ).
showing a hypershsrp intact
microscope,
particles
undegraded
pH 7.6 . It was
contained
raphs of resggregated 7 Fig. 2 ).
At concentrations sediments
buffer
the solutions
of error,
( compare with Fig.
aggregation
phosphate
dilu-
E260/E280.
were obtained
In the electron
to be spherical
Electronmicro intact virus
of experiments
solutions
after
or RNA in the preparations
and by the low ratio
opalescent
that
do not contain
of virus
by the results
virus
Highly
micrographs
acid the solutions impurities
are also excluded 32P-labelled
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and higher,
can be observed.
in the ultracentrifuge
boundary. with
( Fig.
1
) and
side to side The protein with
so - 685
Under the same contidions
the
so = 80s.
of the material
was present
protein
visible
as virus-like 579
in the electron particles.
!l!here was very
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 30, No. 5, 1968
non aggregated
little
material
ways, The sedimentation sediments
with
or material
diagrams
measurements,
more than 50% of the protein concentration
had no difficulties
and extensive
at
in reproducing
very
In comparison
similar
with
to that
techniques,
denatured
during
its
A self-assembly protein
of fr
shells.
All
icosahedron
that
in the process
surface
structure
virus.
of the protein
in neutral
the protein
demonstrated subunits
is less
be undertsken
to icosahedral of the protein.
( Kellenberger,
1966;
self-assembly.
to show whether
between the structure
minor
of the empty protein
coat of the intact
of the "A-protein"
the coat
of the virus
factor"
for
for
the formation
in the structure
"morphopoetic
and the protein
the function
its
was
the use of normal ultracen-
needed for
Hohn, 1967 > is not necessary
shell
specific
material
that
solubilitg
leads from protein
RNA as an additional
exist
apparently
of the intact
is thereby
is contained
differences
this
preparation.
information
will
end
particles.
bus also shows that
process
Experiments
that
the method of Hahn ( 1967 ) the procedure
This not only enables
trifugation
and
can be concluded
by purification
indicating
used here leads to a higher buffers.
micrographs
The reaggregated
by fr-antiserum
least
it
to virus-like
reaggregation.
precipitated is
obtained
is reaggregated
in other
show that most of the protein
the 685 peek. From the electron
the ultracentrifugation
We
aggregated
virus
and to elucidate
( Argetsinger-Steitz,
of assembly of the complete
1967
)
virus.
We are grateful to Prof. Dr. G. Schramm for his encouragement and his interest in our work. The helpful advice of and discussion with Dr. A. Anderer and Dr. H.D. Schlumberger ere gratefully acknowledged. The work was supported by the US Public Health Service grant 5 ROI Ca 04601-08. 580
Vol. 30, No. 5, 1968
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
References
24,
.540.
Frenk,H.(lq67).~ersonal communication. Heidelberger, M,s.nd Mac Pherson,C.E.C.(1943),Science 2,405. Heisenberg,M.('?966) J.Mol,Biol. '& 136. Hoffmann-Berling,H.,Marvin,D.A. and D&rwald,H. (1963),Z.Natu.r-
Academic Press, New York. Schramm,G. (1943). Naturwissenschaften 1,94. Schrsmm,G. and Zillig,W.(l955) Z.Natur &orschung lob,493. Sugiysma,!?L?.,Herbert,R.R.,and Hsrtmann,K.A.(1967)~ol. 1249.
581
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
SELF-ASSEBBLYOF PROTEW SUBUI?ITSFROMBACXCERIOPHAGEfr R. Herrmann,
D. Schubert
Max - Planck Institut
and U. Rudolph
fiir
'Piibingen,
Virusforschung
Germany
Received February 9, 1968
It
is well
known that under
appropriate
subunits
of tobacco mosaic virus
virus-like
rods ( Schrsmm, 1943;
Fraenkel-Conrat
and Singer,
have predicted possible
that
for
only been possible degradation reaggregation
viruses.
of protein
Until
protein
solutions
should
1955;
also be
now, however,
shells
( Finch
subunits
acid ( Sugiysma et al.,
of such viruses
and Klug,
1960
in the presence
1967; Hohn,
by
) or by
of nucleic
Bancroft
1967;
it has
and Hiebert,
1.
1967
Using the bacteriophage weight
4x10~
diameter
Daltons
of nucleic
fr,
indeed possible
shells
The coat protein
in the monomeric form.
In this
to obtain
et
virus
al.,
from protein
virus-like
yield.
576
to
it
achieve
the
material
is shown that
protein
> and
in the absence
used as starting
paper
of particle
1963
) we have tried
1967
of icosahedral acid,
en icosahedral
( Hoffmann-Berling
of 260 A ( Frank,
self-assembly
high
Schramm and Zillig,
to form
1957 >. Casper and Klug ( 1962 )
to obtain
of virus
protein
( TMV ) can reaggregate
such a self-assembly
icosahedral
conditions
shells
it
was is
in a rather
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 30, No. 5, 1968
Materials
and Methods
a) fr-virus
0 The phsge was grown as described
Berling
et al.
( 1963 ), using the trypton
and Hoffmsnn-Berling obtained modified
t
for
of protein
was carried
The mixture
about 15 min. By this
was left
in en ice bath
time most of the R?JAhad precipitated
centrifugation was diluted
solution
with
had been equilibrated for
the protein
24 hours.
dialysis
20 min, the super-
3396 acetic
were collected
acid.
The fractions
and dialyzed
was concentrated
0.1
water pressure
by this
at 30,000 rpm
was dAalyzed against
pH 7.6 containing
against
to pH 7.5 with 0.02 M NaOH. Most of
centrifugation
supernatant
con-
by negative
which had formed was dissolved
of the pH. After
The
to a column of Sephadex G 50 which
with
and was brought
rpm for
( 5 min ). After
an equal volume of cold water.
The solution
the precipitate clear
at 30,000
was then applied
taining
a
1957 ). Two
end it was removed by low speed centrifugation natant
out using
acid were added to one volume of a solution
( 4%) in water.
a further
32P-RNA was
( 1966 ).
acid method ( Fraenkel-Conrat,
volumes of acetic of virus
by Heisenberg
Isolation
acetic
medium of Knippers
( 1966 ). Phage containing
as described
b) fr-protein
by Hoffmann-
adjustment
for 20 min the
0.02 M phosphate
M NaCl. The final
protein
buffer
concentration
was between 0.5 end 2 mg/ml. 32P labelled virus
solution
protein that
phage ( 1x10~ opm/mg protein to determine
preparation. after
the amount of RNA remaining
Measurement of the radioactivity
column chromatography
decreased
to less than 0.0%
material.
Before
tained
) was added to the in the showed
the counts per mg protein
of the value
column chromatography
about s RNA. The W absorbance
577
had
of the original,
the preparation ratio
con-
A260/B2So of the
BIOCHEMICAL
Vol. 30, No. 5, 1968
purified
preparations
( without
measured in 33% acetic
correction
The preparation
for
light
scattering
and purification
carried
out in an ice bath,
results
were obtained
c) Physical
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
although
sorbance
t
measurements
tion
counter.
stained
with
Mr.G. Berger generous
gift
precipitates
Ultracentrifuge
were counted
uranyl
acetate
Results
and Discussion
monomerfc protein
that
antiserum
subunits
with
for
specific
( Anderer
et al.,
partial 1959;
( Wittmann-Liebold,
is split
weight
to
determinations
method ( Schachmsn, of
16
ooo, assuming 0.75 ml/g
the protein
578
solvent
1959 ). The molecular
with the value
).
) were
1959
acid plus
volume in this
methods for 1966
to the method of Heidel-
) molecular
Wittmann,
of 16 ooo is in good agreement by chemical
fr was the
acid and in 6696 acetic a value
and
acid in the ssme manner as the
1959
o.lM NaGl we have found the protein
against
of the virus
equilibrium
In 6696 acetic
made by Dr.H.Frank
).
acetic
of TMV ( Wittmann,
negatively
The antigen-antibody
according 1943
scintilla-
I A.
the coat protein
by the sedimentation performed.
(
UV-ab-
o.l)'C.
of preparations
of Dr. H.D.Schlumberger.
and Mac Pherson
schlieren
spectrophotometer.
were kindly
t Rabbit
berger
using
on a Packard liquid
on a Siemens-Elmiskop test
was performed
were made at (20.02
were obtained
To prove
the seme
Ultracentrifugation
Electronmicrographs
d) Serological
obtained
essentially
was measured on a Zeiss MQ III
32p-disintegrations
was usually
at room temperature.
measurements
All
).
of the protein
on a Spinco Model E Analytical optics.
acid was 0.550.59
of
16
subunit
weight
600
of fr
Vol. 30, No. 5, 1968
BIOCHEMICAL
It was apparent
from the electron
tion
to 3396 acetic
virus.
Significant
had been dialyzed therefore weight within
against
supposed that aggregates.
appeared
the limits
Essentially microscope
( Fig.
of about lmg/ml
homogeneously sediments all
after
the protein
high molecular these aggregates
1 ), having
equal to that
of the particles
virus
with
diameters,
of the intact
virus
2 ).
showing a hypershsrp intact
microscope,
particles
undegraded
pH 7.6 . It was
contained
raphs of resggregated 7 Fig. 2 ).
At concentrations sediments
buffer
the solutions
of error,
( compare with Fig.
aggregation
phosphate
dilu-
E260/E280.
were obtained
In the electron
to be spherical
Electronmicro intact virus
of experiments
solutions
after
or RNA in the preparations
and by the low ratio
opalescent
that
do not contain
of virus
by the results
virus
Highly
micrographs
acid the solutions impurities
are also excluded 32P-labelled
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
and higher,
can be observed.
in the ultracentrifuge
boundary. with
( Fig.
1
) and
side to side The protein with
so - 685
Under the same contidions
the
so = 80s.
of the material
was present
protein
visible
as virus-like 579
in the electron particles.
!l!here was very
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Vol. 30, No. 5, 1968
non aggregated
little
material
ways, The sedimentation sediments
with
or material
diagrams
measurements,
more than 50% of the protein concentration
had no difficulties
and extensive
at
in reproducing
very
In comparison
similar
with
to that
techniques,
denatured
during
its
A self-assembly protein
of fr
shells.
All
icosahedron
that
in the process
surface
structure
virus.
of the protein
in neutral
the protein
demonstrated subunits
is less
be undertsken
to icosahedral of the protein.
( Kellenberger,
1966;
self-assembly.
to show whether
between the structure
minor
of the empty protein
coat of the intact
of the "A-protein"
the coat
of the virus
factor"
for
for
the formation
in the structure
"morphopoetic
and the protein
the function
its
was
the use of normal ultracen-
needed for
Hohn, 1967 > is not necessary
shell
specific
material
that
solubilitg
leads from protein
RNA as an additional
exist
apparently
of the intact
is thereby
is contained
differences
this
preparation.
information
will
end
particles.
bus also shows that
process
Experiments
that
the method of Hahn ( 1967 ) the procedure
This not only enables
trifugation
and
can be concluded
by purification
indicating
used here leads to a higher buffers.
micrographs
The reaggregated
by fr-antiserum
least
it
to virus-like
reaggregation.
precipitated is
obtained
is reaggregated
in other
show that most of the protein
the 685 peek. From the electron
the ultracentrifugation
We
aggregated
virus
and to elucidate
( Argetsinger-Steitz,
of assembly of the complete
1967
)
virus.
We are grateful to Prof. Dr. G. Schramm for his encouragement and his interest in our work. The helpful advice of and discussion with Dr. A. Anderer and Dr. H.D. Schlumberger ere gratefully acknowledged. The work was supported by the US Public Health Service grant 5 ROI Ca 04601-08. 580
Vol. 30, No. 5, 1968
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
References
24,
.540.
Frenk,H.(lq67).~ersonal communication. Heidelberger, M,s.nd Mac Pherson,C.E.C.(1943),Science 2,405. Heisenberg,M.('?966) J.Mol,Biol. '& 136. Hoffmann-Berling,H.,Marvin,D.A. and D&rwald,H. (1963),Z.Natu.r-
Academic Press, New York. Schramm,G. (1943). Naturwissenschaften 1,94. Schrsmm,G. and Zillig,W.(l955) Z.Natur &orschung lob,493. Sugiysma,!?L?.,Herbert,R.R.,and Hsrtmann,K.A.(1967)~ol. 1249.
581