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Crystallographic studies on concanavalin B
Vol. 57, No. 2, 1974
BIOCHEMICAL
CRYSTALLOGRAPHIC
Alexander
McPherson,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
STUDIES ON CONCANAVALIN
B
Joshua Geller and Alexander
Rich
Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02 139 Received
February
6.1974
ABSTRACT Concanavalin B has been grown as hexagonal needles and analyzed by x-ray
diffraction
and electron microscopy.
group P61 with -a = 8li
that the crystals interstitial
There is
and 2 = IOli.
weight as the asymmetric
The crystals
unit of the crystal.
maintain considerable
one
are of space
molecule of 30,000 molecular
Electron
micrographs
order after dehydration
demonstrate
and exhibit large
solvent regions. INTRODUCTION Concanavalin
Ensiformis)
B is one of four proteins from the Jack Bean (Canavalis
which were crystallized
by J. B. Sumner between 1918 and 1935 (1,2, 3).
The other three are urease, the only one of the four to be assigned a distinct enzymatic
function,
canavalin,
composition and arrangement diffraction
and concanavalin
A.
The hexameric
of canavalin have recently
analysis (4), and the entire three-dimensional
has been determined physiological
(5, 6). This latter
properties
due to its ability
subunit
been established by x-ray structure
of concanavalin
protein exhibits a number of interesting to bind to cell surfaces (7, 8).
The
function of concanavalin
B in the seed, like that of concanavalin A, is unknown,
and very little
research
has been conducted on its biochemical
The molecular
weight was estimated by Sumner in 1938 to be 45,000 by ultra-
centrifugal
analysis (9). 494
Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
properties
to date.
A
Vol. 57, No. 2,1974
BIOCHEMICAL
We have crystallized procedure
AND BIOPHYSICAL
concanavalin B by a modification
(1) and studied it by x-ray
and SDS-polyacrylamide
RESEARCH COMMUNICATIONS
diffraction
analysis,
of Sumner’s
electron microscopy
gel electrophoresis. METHODS
Crystallization
- One hundred grams of Jack Bean meal purchased
from Sigma Co. was extracted insoluble residue. the precipitate
with 30% acetone and centrifuged
to remove
The supernatant was brought to 50% acetone concentration,
collected and dissolved in minimal Tris-HCl
buffer pH 8. 0.
The protein solution was dialyzed
for 48 hours vs. distilled
a heavy yield of micro-crystalline
needles collected by centrifugation.
crystals
concanavalin
filtered
4-pentanediol
whereupon large hexagonal needles of
B were formed.
X-Ray
diffraction
The
were dissolved in aqueous ammonia and dialyzed for several days
against 15% Z-methyl-Z,
crystals
water at 4°C and
Analysis
and Electron
Microscopy
were sealed in quartz capillaries photographs recorded CuKo, radiation
at 40 kV and 40 mA.
- For x-ray
by the conventional
examination, means, and
on a Supper precession camera using nickel
generated by an Elliot rotating For examination
anode source operated
with the electron microscope,
microcrysta
.ls
suspended in a drop of water were placed on a carbon covered grid followed by a drop of 2% uranyl
acetate.
Micrographs
at 80kV and a magnification
of 60, 000.
Acrylamide was performed system. parallel
Gel Electrophoresis
according to Laemmli
were taken on a JOEL 100-B operating
- SDS polyacrylamide
(10) using a discontinuous tris-glycine
Eight proteins of known molecular gels and the molecular
gel electrophoresis buffer
weight were run as standards on
weight of concanavalin B calculated according to
Weber and Osborn (11). 495
Vol. 57, No. 2, 1974
Figure
BIOCHEMICAL
1 - An 8” precession
hO1 zone of the reciprocal
of concanavalin
the dark areas to stain.
photograph of (a) the hk0 zone and (b) the
lattice
Figure 2 - An electron micrograph microcrystal
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
B.
of hexagonal concanavalin
B crystals.
of a small section of a negatively
stained
The light areas correspond to protein and
The magnification
is x 750,000.
RESULTS
Electrophoresis
of concanavalin
B on SDS-polyacrylamide
gels
and comparison with known standards showed the protein to have a minimum molecular
weight of 30, OOOf500, which is about 30% lower than Sumner’s
1938 result. 496
BIOCHEMICAL
Vol. 57, No. 2, 1974
When concanavalin parallel
B crystals
enantiomorph
photographs like that shown in
The Laue symmetry
for which $ = 6n are present.
is 6, and only those
The space group is P61 ( or its
~63) and the unit cell dimensions are a = 81;; and c = 1Oli.
Assumption asymmetric
beam
When rotated by 90” about the spindle axis, the
pattern in figure lb was observed. 001 reflections
RESEARCH COMMUNICATIONS
were alligned with the x-ray
with the long axis of the crystal,
figure la were recorded.
of v ,=3.
AND BIOPHYSICAL
of one molecule of 30,000 molecular
unit of the crystal
weight as the
implies a unit cell volume to protein mass
18. This value is within the range of crystalline
proteins compiled
by Matthews (12) although it is rather high, and indicates a large solvent volume in the crystals.
According
solvent volume for concanavalin To verify
to the formula derived by Matthews
B crystals
would be 61%.
that this was the case, microcrystals
were examined in the electron microscope a small portion of the microcrystal.
crystal
of concanavalin
by negative staining.
Figure 2 is
between molecules,
evident from the extensive distribution
The long dimension of the crystal
is the crystallographic
of the electron beam is approximately
along the 110 direction,
onto the 100 plane.
join to form striking
longitudinal
lines running the length of the crystal
as cross striations,
The crystallographic
Regions of heavy staining
lines,
period is the
62i. These dimensions represent
of 32% and 23% along 2 and 2 respectively.
497
as well
period along -c is the distance between
cross lines which is 68A0. Along a - the crystallographic
distance between longitudinal
state.
-c axis,
and the protein is seen projected
alternate
It is
of stained areas in the
that there must be a large solvent regions in the fully hydrated
the direction
B
The light areas correspond to protein
and dark areas to the stain which fills intersticies immediately
the
shrinkages
The volume of the dehydrated
unit
BIOCHEMICAL
Vol. 57, No. 2, 1974
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
This represents
cell is 2, 26 x 105i3.
of solvent of 61%, exactly
a shrinkage in unit cell volume after loss
that predicted
that the precise equivalence
by the formula
of the two values is partly
of Matthews. coincidental,
We suspect since some
small volume in the dry state must be occupied by stain, but the agreement is sufficient
evidence that the asymmetric X-ray
diffraction
intensities
extended to less than 3. Oi resolution, crystals
unit is one molecule. were observed in still photographs that and no appreciable
with exposure time was evident for 60 hours.
concanavalin
B, therefore,
degradation of the
The crystals
of
seem suitable for a three dimensional structure
analysis.
ACKNOWLEDGEMENT Wethank Mrs.
Elaine Lenk for her technical assistance and Dr. Jon King
for the use of his facilities. National Institutes
This research
was supported by grants from the
of Health and the National Science Foundation.
BIBLIOGRAPHY
1.
Sumner,
J. B.
J. Biol.
Chem. (1919) -37, 137 - 142.
2.
Sumner,
J. B.
J. Biol.
Chem. (1926) -69, 435 - 440.
3.
Sumner,
J. B. and S. F. Howell.
4.
McPherson,
5.
Edelman, G. M., B. A. Cunningham, G. N. Reeke, J. W. Becker, M. J. Waxdal and J. L. Wang. Proc. Nat. Acad. Sci. U. S. (1972) -6% 2580-2584.
6.
Hardman, K. D., M. K. Wood, M. Schiffer, A. B. Edmundson, and C. F. Ainsworth. Cold Spring Harbor Symposium on Quantitative Biology XXXVI ( 1971). 271-276.
7.
Goldstein, 1, J. , C. E. Hollerman (1965) 4, 876883.
A. and A. Rich.
J. Biol.
J. Biochem.
498
Chem (1936) 113, 607 - 610. (1973) f 74 155- 160.
and E. E. Smith.
Biochemistry
Vol. 57, No. 2, 1974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
8.
Inbar,
M. and L. Sachs, Nature 223, 710 - 712 (1970).
9.
Sumner, J. B., N. Gralen and I. Eriksson-Quensal. (1938) 125, 45 - 48.
10.
Laemmli,
11.
Weber,
12.
Matthews,
J. Biol.
Chem.
V. K. Nature (1970) 227, 680 - 682. K. and M. Osborn. B. W.
J. Mol.
J. Biol. Biol.
499
Chem. (1969) 244, 4406 - 4412.
(1968) -33, 1491 - 1497.
BIOCHEMICAL
CRYSTALLOGRAPHIC
Alexander
McPherson,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
STUDIES ON CONCANAVALIN
B
Joshua Geller and Alexander
Rich
Department of Biology Massachusetts Institute of Technology Cambridge, Massachusetts 02 139 Received
February
6.1974
ABSTRACT Concanavalin B has been grown as hexagonal needles and analyzed by x-ray
diffraction
and electron microscopy.
group P61 with -a = 8li
that the crystals interstitial
There is
and 2 = IOli.
weight as the asymmetric
The crystals
unit of the crystal.
maintain considerable
one
are of space
molecule of 30,000 molecular
Electron
micrographs
order after dehydration
demonstrate
and exhibit large
solvent regions. INTRODUCTION Concanavalin
Ensiformis)
B is one of four proteins from the Jack Bean (Canavalis
which were crystallized
by J. B. Sumner between 1918 and 1935 (1,2, 3).
The other three are urease, the only one of the four to be assigned a distinct enzymatic
function,
canavalin,
composition and arrangement diffraction
and concanavalin
A.
The hexameric
of canavalin have recently
analysis (4), and the entire three-dimensional
has been determined physiological
(5, 6). This latter
properties
due to its ability
subunit
been established by x-ray structure
of concanavalin
protein exhibits a number of interesting to bind to cell surfaces (7, 8).
The
function of concanavalin
B in the seed, like that of concanavalin A, is unknown,
and very little
research
has been conducted on its biochemical
The molecular
weight was estimated by Sumner in 1938 to be 45,000 by ultra-
centrifugal
analysis (9). 494
Copyright 0 1974 by Academic Press, Inc. All rights of reproduction in any form reserved.
properties
to date.
A
Vol. 57, No. 2,1974
BIOCHEMICAL
We have crystallized procedure
AND BIOPHYSICAL
concanavalin B by a modification
(1) and studied it by x-ray
and SDS-polyacrylamide
RESEARCH COMMUNICATIONS
diffraction
analysis,
of Sumner’s
electron microscopy
gel electrophoresis. METHODS
Crystallization
- One hundred grams of Jack Bean meal purchased
from Sigma Co. was extracted insoluble residue. the precipitate
with 30% acetone and centrifuged
to remove
The supernatant was brought to 50% acetone concentration,
collected and dissolved in minimal Tris-HCl
buffer pH 8. 0.
The protein solution was dialyzed
for 48 hours vs. distilled
a heavy yield of micro-crystalline
needles collected by centrifugation.
crystals
concanavalin
filtered
4-pentanediol
whereupon large hexagonal needles of
B were formed.
X-Ray
diffraction
The
were dissolved in aqueous ammonia and dialyzed for several days
against 15% Z-methyl-Z,
crystals
water at 4°C and
Analysis
and Electron
Microscopy
were sealed in quartz capillaries photographs recorded CuKo, radiation
at 40 kV and 40 mA.
- For x-ray
by the conventional
examination, means, and
on a Supper precession camera using nickel
generated by an Elliot rotating For examination
anode source operated
with the electron microscope,
microcrysta
.ls
suspended in a drop of water were placed on a carbon covered grid followed by a drop of 2% uranyl
acetate.
Micrographs
at 80kV and a magnification
of 60, 000.
Acrylamide was performed system. parallel
Gel Electrophoresis
according to Laemmli
were taken on a JOEL 100-B operating
- SDS polyacrylamide
(10) using a discontinuous tris-glycine
Eight proteins of known molecular gels and the molecular
gel electrophoresis buffer
weight were run as standards on
weight of concanavalin B calculated according to
Weber and Osborn (11). 495
Vol. 57, No. 2, 1974
Figure
BIOCHEMICAL
1 - An 8” precession
hO1 zone of the reciprocal
of concanavalin
the dark areas to stain.
photograph of (a) the hk0 zone and (b) the
lattice
Figure 2 - An electron micrograph microcrystal
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
B.
of hexagonal concanavalin
B crystals.
of a small section of a negatively
stained
The light areas correspond to protein and
The magnification
is x 750,000.
RESULTS
Electrophoresis
of concanavalin
B on SDS-polyacrylamide
gels
and comparison with known standards showed the protein to have a minimum molecular
weight of 30, OOOf500, which is about 30% lower than Sumner’s
1938 result. 496
BIOCHEMICAL
Vol. 57, No. 2, 1974
When concanavalin parallel
B crystals
enantiomorph
photographs like that shown in
The Laue symmetry
for which $ = 6n are present.
is 6, and only those
The space group is P61 ( or its
~63) and the unit cell dimensions are a = 81;; and c = 1Oli.
Assumption asymmetric
beam
When rotated by 90” about the spindle axis, the
pattern in figure lb was observed. 001 reflections
RESEARCH COMMUNICATIONS
were alligned with the x-ray
with the long axis of the crystal,
figure la were recorded.
of v ,=3.
AND BIOPHYSICAL
of one molecule of 30,000 molecular
unit of the crystal
weight as the
implies a unit cell volume to protein mass
18. This value is within the range of crystalline
proteins compiled
by Matthews (12) although it is rather high, and indicates a large solvent volume in the crystals.
According
solvent volume for concanavalin To verify
to the formula derived by Matthews
B crystals
would be 61%.
that this was the case, microcrystals
were examined in the electron microscope a small portion of the microcrystal.
crystal
of concanavalin
by negative staining.
Figure 2 is
between molecules,
evident from the extensive distribution
The long dimension of the crystal
is the crystallographic
of the electron beam is approximately
along the 110 direction,
onto the 100 plane.
join to form striking
longitudinal
lines running the length of the crystal
as cross striations,
The crystallographic
Regions of heavy staining
lines,
period is the
62i. These dimensions represent
of 32% and 23% along 2 and 2 respectively.
497
as well
period along -c is the distance between
cross lines which is 68A0. Along a - the crystallographic
distance between longitudinal
state.
-c axis,
and the protein is seen projected
alternate
It is
of stained areas in the
that there must be a large solvent regions in the fully hydrated
the direction
B
The light areas correspond to protein
and dark areas to the stain which fills intersticies immediately
the
shrinkages
The volume of the dehydrated
unit
BIOCHEMICAL
Vol. 57, No. 2, 1974
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
This represents
cell is 2, 26 x 105i3.
of solvent of 61%, exactly
a shrinkage in unit cell volume after loss
that predicted
that the precise equivalence
by the formula
of the two values is partly
of Matthews. coincidental,
We suspect since some
small volume in the dry state must be occupied by stain, but the agreement is sufficient
evidence that the asymmetric X-ray
diffraction
intensities
extended to less than 3. Oi resolution, crystals
unit is one molecule. were observed in still photographs that and no appreciable
with exposure time was evident for 60 hours.
concanavalin
B, therefore,
degradation of the
The crystals
of
seem suitable for a three dimensional structure
analysis.
ACKNOWLEDGEMENT Wethank Mrs.
Elaine Lenk for her technical assistance and Dr. Jon King
for the use of his facilities. National Institutes
This research
was supported by grants from the
of Health and the National Science Foundation.
BIBLIOGRAPHY
1.
Sumner,
J. B.
J. Biol.
Chem. (1919) -37, 137 - 142.
2.
Sumner,
J. B.
J. Biol.
Chem. (1926) -69, 435 - 440.
3.
Sumner,
J. B. and S. F. Howell.
4.
McPherson,
5.
Edelman, G. M., B. A. Cunningham, G. N. Reeke, J. W. Becker, M. J. Waxdal and J. L. Wang. Proc. Nat. Acad. Sci. U. S. (1972) -6% 2580-2584.
6.
Hardman, K. D., M. K. Wood, M. Schiffer, A. B. Edmundson, and C. F. Ainsworth. Cold Spring Harbor Symposium on Quantitative Biology XXXVI ( 1971). 271-276.
7.
Goldstein, 1, J. , C. E. Hollerman (1965) 4, 876883.
A. and A. Rich.
J. Biol.
J. Biochem.
498
Chem (1936) 113, 607 - 610. (1973) f 74 155- 160.
and E. E. Smith.
Biochemistry
Vol. 57, No. 2, 1974
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
8.
Inbar,
M. and L. Sachs, Nature 223, 710 - 712 (1970).
9.
Sumner, J. B., N. Gralen and I. Eriksson-Quensal. (1938) 125, 45 - 48.
10.
Laemmli,
11.
Weber,
12.
Matthews,
J. Biol.
Chem.
V. K. Nature (1970) 227, 680 - 682. K. and M. Osborn. B. W.
J. Mol.
J. Biol. Biol.
499
Chem. (1969) 244, 4406 - 4412.
(1968) -33, 1491 - 1497.