BIOCHEMICAL
Vol. 75, No. 3, 1977
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
CIRCULAR DICHROISM OF BIOLOGICAL
MEMBRANES:
PURPLE MEMBRANE OF HALOBACTERIUM HALOBIUM M. M. Long*,
D. W. Urry*
and W. Stoeckenius**
*Laboratory
of Molecular Biophysics and the Cardiovascular Research and Training Center University of Alabama Medical Center Birmingham, Alabama 35294 **University of California School of Medicine Cardiovascular Research Institute and Department of Biochemistry and Biophysics San Francisco, California 94143
Received
February
24,1977
SUMMARY: Circular dichroism spectra of purple membrane suspensions exhibit distortions which are characteristic of particulate a-helical systems with ellipticities at 224 nm of the order of 10000. We show here that the pseudo reference state approach to the correction of CD data, when applied to the purple membrane system, results in a CD spectrum which, in its entirety, is typical of a protein with about 75% a-helix, e.g. the corrected ellipticity at 224 nm is 24000 and at 190 nm is 45000. This result is consistent with electron microscopy and diffraction data on the purple membrane which indicates 70-80% a-helix. Application to the purple membrane system validates the pseudo reference state approach. We also note that 80% trifluoroethanol solubilizes the membrane but results in a similar average secondary structure of the protein. Circular average
dichroism
membrane
if,
corrections
are
common to all
membrane
compared
cantly
and variably
membrane system
and indeed
0 1977
distortions,
by Academic Press, Inc. in any form reserved.
of reproduction
a-helical
can provide
Typically
the
but display and the
cross
proteins.
on the
insight
architecture,
if,
and flattening
structure,
depending
The anomalies are
patterns
to estimate
dampened,
suspension.
(1).
the two minima,
of soluble
any attempt
in this
scattering
systems
show a-helical
to the pattern for
light
particulate
of the maximum,
of biomembranes
and changes
are made for
importantly
Copyright All rights
architecture
suspensions
wavelengths
(CD) spectra
CD spectra shifts
point
In addition,
state
suspension documented
which of bio-
to longer
over
particulate
as has been well
and only
distortions
the CD curves
are due to the
into
when and
are signifiof the
nature
of the
(l-10).
A
725 ISSN
0006-291X
Vol. 75, No. 3, 1977
relatively
simple
of membranes, (1).
BIOCHEMICAL
It
bility
is
and practical
called the
of this
purpose
described.
published
x-ray
protein
pseudo
with Recently
halobium
therefore,
reference
state
Materials
and Methods
report
for
appro
whose
and Unwin
that
contains
distorted
has been developed
the
validity
(1 -13)
and Blaurock
(14)
membrane
a-helix.
validity
of CD data
inde-
of
the single
as much as 70-80%
the correction
has been
bacteriorhodopsin,
the
and applica-
structure
of the purple
to corroborate
CD spectra
ch,
protein
studies
indicated
(15),
these
to demonstrate
microscopy
was chosen
method
state
Henderson
which
membrane
membrane,
reference
a membrane
and electron
in this
means of correcting
of this
approach
pendently
Halobacterium
the
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The purple
of the pseudo
from
particulate
systems.
CD spectra were obtained at ambient temperature on a Cary 60 spectropolarimeter adapted with a model 6001 attachment for circular dichroism using the 100 mdeg range. UV spectra were recorded on a Cary 14 spectrophotometer with the sample cell placed close to the phototube in the cell compartment (16). The effective pathlengths of the cells were 0.233 mn for the suspensions and 1.7 mn for the membrane solutions. The final protein concentration, as determined by the Lowry method (17), was 0.53 mg/ml for the suspension and 0.053 mg/ml for the solution. Sonication was performed on ice with a Branson Sonic power sonifier at 7.5 amp for two 30 second intervals. Solubilization for the pseudo reference state was achieved by addition of stock sodium dodecyl sulfate, SDS (Eastman Organic Chemicals), to the membrane suspension first, followed by dilution to final volume with trifluoroethanol (TFE). The 80% TFE sample without SDS was filtered through a mitrex 5 urn Millipore filter giving a spurious high absorption due to absorbing material extracted by TFE from the filter itself. Trifluoroethanol, purchased from Halocarbon Products, Hackensack, N. J., was redistilled in glass with a Duflon column of glass beads (lower 3/4) and glass helices (upper l/4). The first lo-15% of distillate and last 20-30% of pot residue were discarded. NaHC03 was added to the remainder to remove traces of acid. The purple membrane was prepared as outlined by Oesterhelt and Stoeckenius (15). Results
and Discussion
The spectrum
of the
purple
is
characterized
by red
shifting
all
particulate
systems,
are
tions
(1).
distortions the magnitude is
apparent
membrane
suspension
and dampening. due to light is
are still
value
of a soluble that
corrections
the
protein
which
are
required.
726
These
scattering
Even when the suspension apparent:
(see
sonicated of is
[O]'~~
70-80%
Figure
1, Curve
features,
cotmnon to
and flattening (Figure is
1, Curve still
a-helical.
a)
only
distorb),
the
one-half
Therefore
it
Vol. 75, No. 3, 1977
BIOCHEMICAL
:. t3
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a.
Purple membrane suspension (frozen)
b.
Purple membrane sonicated I
c.
Purple membrane TFE.li20.4:I
d.
Corrected
e.
Metmyoglobin
Ellipticities
220
200
mm
240
X (nm)
FIGURE 1:
With
CD spectra of the purple membrane suspension which had been previously frozen (a) and after sonication (b) Curve d is the same corrected ellipticity as for Figure 2, Curve a. Note the marked correspondence between d and c, the membrane solubilized with 80% TFE and filtered to remove non-protein particles. Curve e is the spectrum of metmyoglobin which is 70% a-helix as determined by x-ray, reprinted with permission from Quadrifoglio, F., and Urry, D.W., J. Am. Chem. Sot. 90, 27552760, 1968. Copyright by the American Chemical Society.
the
was calculated, and Figure reference
pseudo
wavelength
3, Curve state
reference
(or
d.
state
approach
by wavelength, In this
solution)
calculation absorbances
727
the corrected
and is
shown
in Figure
the membrane (see
Figure
membrane
2),
1, Curve
suspension the
spectrum
former's
d
and pseudo CD
Vol. 75, No. 3, 1977
BIOCHEMICAL
0. Purple membrane sonicated I men.
I.0 h .5 $)0.6 0 6.u
x 0
0.6
-
0.4
-
0.2
-
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
b. Pseudo reference TFE:+O-SDS.
\
stats I:4 (0.4%)
_._ 200
220
260
240 A (nm)
FIGURE 2:
curve,
Absorbance data of the purple membrane suspension, protein/ml, 0.233 mm cell) and the purple membrane (0.053 mg protein/ml, 1.72 mm effective pathlength 4.
the
latter's
ORD curve
["torrected is evaluated
the
suspension
pseudo
a better
fit
reference than
Duysens'
correction
factor
[ml1
correction for
3, Curve
[m15”
[o]~,,~~
-k
-
Q;
ellipticity
at one wavelength
of the
Q, is
=
(Figure
(19). factor absorption
10
[ml
is
for
the
flattened
absorption
obscuring
728
used.
(1) X, k (=0.52)
the mean residue
has been shown
AF is
1 were
-AF
at wavelength
It
and Equation
Q,
.
(1,16,18),
state.
c),
(a): (0.53 m solution, (b 4 : relative to
molar
empirically absorbance
flattening
due to light
is
that
a constant rotation
[ml 5/k
at wavelength (20)
scattering.
and Q, is
is A, the
BIOCHEMICAL
Vol. 75, No. 3, 1977
AND BiOPHYSlCAL
RESEARCH COMMUNICATIONS
7
6 6
o Purple membrane sonicated I min
d I
5
-
: I ’ !
4-
b. Pseudo stole
reference - TFE:,+O-SD! 1'4 10.4%)
5
c. Pseudo State
reference - ORD c*w*
4
d. Corrected
* b-3
Ellipticities 3
e b x
x 52
2
-
I
-
/
2-
T
I
0
I
2
200
220
240
A (nm)
FIGURE 3:
CD spectra of the purple membrane sonicated suspension (this is a different sample from Figure 1, Curve b), the purple membrane solution (a), and pseudo reference state (b), the pseudo reference state ORD (c), and the corrected ellipticity (d). The calculated percent of o-helix using Curve d is 73% for the purple membrane - in good agreement with the x-ray data indicating between 70 and 80% a-helix.
The TFE - SDS molecularly meets
the
Equations
criteria
for
an appropriate
state pseudo
(mds)
of the
reference
state
purple
membrane
by obeying
2 and 3.
224
[@Imds
In the
dispersed
initial
>
224
[OICl
approximation
224 [@Isusp
= (Qi2')'
indicated
by the
729
.
(2)
Q;,,
subscript,
CI,
to [CII’~~,
Q, is
Vol. 75, No. 3, 1977
BIOCHEMICAL
taken
as 1, making
about
10 to 20% less
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
a conservative than
[o];::
I
.
I
value,
In this
which
case it
generally
should
be
was 9% less.
(3)
In this
approximation
conservative
value;
be within
(Figure
is the
d).
its
With pseudo
the
corrected
o-helix
a calculated indicated
Becher
(22),
and Cassim
CD data
of membranes.
extrema
match
spectrum
is
spectrum
(Figure
well.
the
uncorrected
which
The correspondence
is
similar
quantity
calculated and the
effective
soluble
is
striking
of a-helix protein
with
of the
purple
importance
the
by comparing curve
the
estimate
membrane
shape
at the
of the
corrected
the metmyoglobin for
bacteriorhodopsin. result
protein
in
a
bacterior-
metmyoglobin.
membranes
with
detergents
and other
730
has proven particulate
especially
systems
by
raw
magnitudes
corrections
the membrane
membrane's
of correcting
absolute
but also
for
gives
(11-13).
to be compared
the
This
70 and 80% a-helix
corrected
that
of an
x 10'.
purple
d of
studies
from
the calculated
showing
ellipticity
the
the
evident
e) with
the
Curve
microscopy
CD spectrum
do the
and in combination
in solubilizing
is
73% o-helix,
This
1, Curve
This
underscores
with
appropriate.
between
1 and 3,
spectrum;
-3.3
(224
3c) were
ellipticity
and electron
was calculated
agrees
Not only
a protein
is
should
absorption
(Figures
with
is
it
(Figure
suspension
CD spectrum
X-ray
which
curve
a
wavelengths
The characteristic
of the
Usually
ORD curve
membrane
b).
.
also
suspension
in good agreement
--in situ
of 73% a-helix
TFE alone
is
state
ellipticity
a is the
(Curve
bacteriorhodopsin
from
and membrane
corrected
which
[O],$l
[@Ii:"
met at critical
reference
of 73% a-helix.
of 45% calculated
hodopsin
pseudo
state
CD spectrum
in 25% of
state
224 nm extremum
value
The CD value corrected
reference
3, Curve
reference
as 1, making
the CD requirements
spectrum
at the
was within
complete
In Figure
pseudo
it
2) and the
used to obtain Curve
here
lo-30%.
and 190 nm) the curves
at 190 nm, Q, is taken
(1,23-27).
BIOCHEMICAL
Vol. 75, No. 3, 1977
It
is
interesting
TFE without the
spectrum.
secondary
in the membrane. details
that
SDS and filtered
corrected
average
to note
of their
ACKNOWLEDGMENTS: of Health Program
This
the membrane (Figure
This
structural
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
suggests
1, Curve that,
properties
conformation
c)
solubilized
has a CD spectrum
in a membrane
of the
does not necessarily
membrane
suspension
proteins
mean that
are
with
80%
close
TFE solution, similar
polypeptides
to the
to those retain
the
in TFE.
This work was supported in part by the National Project Grant numbers HL-11310 and HL-06285.
Institutes
REFERENCES: 1. :: 4. z: 7. 8. 1;: i:: 13. Z: 16. 17. 18. 19.
2 2 24. 25. 26. 27.
Urry, D.W. (1972) Biochim. Biophys. Acta 265, 115-168. Urry, D.W. and Ji, T.H. (1968) Arch. Biochem. Biophys. 128, 802-807. Schneider, A.S., Schneider, M.T. and Rosenheck, H. (197rProc. Natl. Acad. Sci. 66-, 793-798. Kornguth, S.E., Flangas, A., Perrin, J., Geison, R. and Scott, G. (1972) Prep. Biochem. 2, 167-192. Litman, B.J. (1972) B&hem. 11, 3243-3247. Singer, J.A. and Morrison, M.T972) Biochim. Biophys. Acta 274, 64-70. Juliano, R.L. (1973) Biochim. Biophys. Acta 300, 341-378. Rottem, S. and Hayflick, L. (1973) Can. J. Biochem. 51, 632-636. Storey, B.T. and Lee, C.P. (1973) Biochim. Biophys. Eta 292, 554-565. i$ni;;; B.J. and Barenholz, Y. (1975) Biochim. Biophys. Acta 2, Henderson, R. and Unwin, P.N. (1975) Nature (London) 257, 28-32. Unwin, P.N. and Henderson, R. (1975) J. Mol. Biol. %325-440. Henderson, R. (1975) J. Mol. Biol. 93, 123-138. Blaurock, A.E. (1975) J. Mol. Biol. 93, 139-158. Oesterhelt, D. and Stoeckenius, W. (m74) Methods in Enzymology, Volume 31 (Colowick, S.P. and Kaplan, N.O., eds.) p. 667-678, Academic Press, New York. Urry, D.W. and Long, M.M. (1974) Methods in Membrane Biology, Volume 1 (Korn, E.D., ed.) p. 105-141, Plenum Press, New York. Lowry, O.H., Rosenbrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Urry, D.W. (1974) Methods in Enzymology, Volume 32 (Colowick, S.P. and Kaplan, N.O., eds) p. 220-234, Academic Press, New York. Urry, D.W. and Long, M.M. (1977) in The Physiological Basis for Disorders of Biomembranes (Andreoli, T.E., Hoffman, J.F., Fanestil, D.D., eds.) (in press) Plenum Press, New York. Duysens, L.N.M. (1956) Biochim. Bioph s. Acta 19, 1-12. Quadrifoglio, F. and Urry, D.W. (1968 Y J. Am. fiem. Sot. 90, 2755-2760. Becher, B. and Cassim, J.Y. (1976) Biophys. J. l&, 1183-lzb0. Long, M.M. and Urry, D.W. (1976) In Membrane Spectroscopy (Grell, E., ed.) (in press) Springer-Verlag, Heidelberg. Masotti, L., Urry, D.W. and Llinas, R. (1973) Acta Vitamin Enzymol. 2, 154-158. Masotti, L., Urry, D.W., Krivacic, J.R. and Long, M.M. (1972) Biochim. Biophys. Acta 266, 7-17. Urry, D.W., Masotti, L. and Krivacic, J.R. (1971) Biochim. Biophys. Acta 241, 600-612. Urry, D.W., Masotti, L. and Krivacic, J.R. (1970) Biochem. Biophys. Res. Commun. 4l-, 521-524.
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