From the European Molecular Biology Laboratory
(EMBL), EMBL Hamburg Outstation, Notkestrasse 85, D-22603 Hamburg,
Germany, ¶ EMBL, EMBL Heidelberg, Protein Expression and
Purification Unit, Meyerhofsrasse 1, D-69012 Heidelberg, Germany, and
Institute of Crystallography, Russian Academy of Sciences,
Leninsky pr. 59, 117333 Moscow, Russia
Received for publication, December 1, 2000, and in revised form, January 12, 2001
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ABSTRACT |
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The The molecular structure and subunit interactions in The oligomers of bacterial sHSPs are monodisperse and more rigid. Their
gel filtration profile shows a single narrow, symmetric peak, whereas
B-crystallin, a member of the small heat-shock
protein family and a major eye lens protein, is a high molecular mass
assembly and can act as a molecular chaperone. We report a synchrotron radiation x-ray solution scattering study of a truncation mutant from
the human
B-crystallin (
B57-157), a dimeric protein that comprises the
-crystallin domain of the
B-crystallin and retains a significant chaperone-like activity. According to the sequence analysis (more than 23% identity), the monomeric fold of the
-crystallin domain should be close to that of the small heat-shock
protein from Methanococcus
jannaschii (MjHSP16.5). The
theoretical scattering pattern computed from the crystallographic model
of the dimeric MjHSP16.5 deviates significantly from the experimental
scattering by the
-crystallin domain, pointing to different
quaternary structures of the two proteins. A rigid body modeling
against the solution scattering data yields a model of the
-crystallin domain revealing a new dimerization interface. The
latter consists of a strand-turn-strand motif contributed by each of
the monomers, which form a four-stranded, antiparallel, intersubunit
composite
-sheet. This model agrees with the recent spin labeling
results and suggests that the
B-crystallin is composed by flexible
building units with an extended surface area. This flexibility may be
important for biological activity and for the formation of
B-crystallin complexes of variable sizes and compositions.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
A- and
B-crystallin, which share 54% amino acid sequence
identity, build the subunits of
-crystallin, a major eye lens protein, comprising up to 40% of the total lens proteins. The structural function of the
-crystallin is to assist in maintaining transparency in the lens (1). The chaperone-like function of
B-crystallin helps to avoid formation of large light-scattering aggregates and possibly helps to prevent cataract in the lens. Moreover, neurodegenerative diseases, ischemia, or multiple sclerosis lead to an overexpression of this protein, which makes it an object of
special medical interest (2).
-crystallin as well as other mammalian small
heat-shock proteins (sHSPs)1
form large globular complexes with a diameter of about 10-25 nm.
Cryoelectron microscopy and image analysis revealed that
B-crystallin is a hollow spherical shell with variable quaternary
structure (3), and a frequent exchange of subunits between the
particles was observed. The chaperone activity of
B-crystallin is
associated with partial perturbation of the substrate protein tertiary
structure, leading to a multimeric molten globule-like state with
increased hydrophobicity (4, 5). The exposed hydrophobic regions of
-crystallin interact with substrate proteins possessing an increased surface hydrophobicity but a low degree of unfolding (6).
-crystallin have
long been under investigation. The stretches of residues promoting
formation of lower or higher molecular weight
-crystallin oligomers
have been identified. Upon addition of 1% deoxycholic acid, the
-crystallin oligomer dissociates into tetramers (7). The latter can
also be formed by deletion of amino acids 1-63 in
A-crystallin (8).
Site-directed spin-labeling demonstrated that the
A-crystallin
dimers are formed by subunit interactions along a highly conserved
-strand in the region of residues 109-121 (9). This stretch of
residues forms a sheet of two antiparallel
-strands that extends
across the dimer interface.
-crystallin,
B-crystallin, or HSP27 yield broader peaks
(10). A 2.9 Å resolution crystal structure of MjHSP16.5, a sHSP
isolated from a hyperthermophilic archaeon, has been determined (11).
The topology of the MjHSP16.5 monomer resembles an immunoglobulin fold
with a central
-sandwich of two sheets, consisting of four
-strands each. Upon dimerization, one of the sheets is extended by
an additional strand contributed by the neighboring subunit (Fig.
1, left panel). This fifth
strand labeled
6 (11), a small stretch of five residues from 93 to
97, is part of a long loop of 19 residues (84) protruding into the
structural core of the monomer. This strand is therefore crucial for
the stabilization of the MjHSP16.5 dimer.
View larger version (43K):
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Fig. 1.
Left panel, x-ray structure of the
MjHSP16.5 dimer in two perpendicular orientations. The green
and red monomers of MjHSP16.5 and the -crystallin
domain, respectively, are in the same orientation. The strand-strand
interaction at the dimer interface is indicated by orange
spheres. Right panel, V-shaped model of the
-crystallin domain dimer obtained by rigid body refinement in two
perpendicular orientations.
A dimeric model of the -crystallin domain has recently been
generated (12) by homology modeling based on the crystal structure of
MjHSP16.5. The quaternary structure of the homology model and thus the
dimeric interface are virtually identical to those of MjHSP16.5
displayed in Fig. 1, left panel. The loop-strand-loop motif
(residues 84-102 of subunit A) interacting with strand
2 (residues
45-49 of subunit B) in MjHSP16.5 has been modeled in human
B-crystallin as a large loop lacking secondary structure (residues
104-115 of subunit A) that is adjacent to strand
2 (residues 66-70
of subunit B). The homology model (12) does not show contacts between
residues 114-118 in subunits A and B, and this contradicts the
spin-labeling results (9).
The present study is aimed at the analysis of the properties and
structure of the -crystallin domain of the human
B-crystallin. A
dimeric
-crystallin domain is expressed, purified, and characterized using different biochemical and physical methods. Its quaternary structure is studied by synchrotron radiation x-ray scattering. The
latter method allows us to determine overall structures of native
biological macromolecules under nearly physiological conditions (13).
Comparisons between experimental x-ray solution scattering curves and
those evaluated from crystallographic structures are widely used to
verify structural similarity between macromolecules in crystals and in
solution (14, 15). Moreover, the use of crystallographic models of
individual subunits permits the building of structural models of
complex particles in solution by rigid body refinement against the
scattering data (16-18). Below, solution scattering is used to
demonstrate that the quaternary structure of the
-crystallin domain
dimer differs from that of sHSP from Methanococcus
jannaschii. A model of the former, constructed by rigid
body refinement, displays a new dimerization interface, suggesting that
the
-crystallin domain may be flexible in solution.
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EXPERIMENTAL PROCEDURES |
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Cloning--
The amino acid sequence alignment between human
B-crystallin and MjHSP16.5 was used as a basis (12). The residue
Ala-57 in
B-crystallin was aligned with Gln-36 in MjHSP16.5, the
N-terminal residue of the first
-strand
1. The Arg-157 in
B-crystallin was aligned with Ile-144 in MjHSP16.5, located in the
last
-strand,
10. The sequence Ala-57 through Arg-157
covered the core
-crystallin domain plus an additional nine residues
from the N-terminal domain. The
B-crystallin deletion mutant gene
was excised from plasmid
B57+pET16b at the restriction sites
NcoI and XhoI. The gene fragment was ligated into
expression vector pETM-11 at the NcoI and XhoI sites. The resulting plasmid was named p
B57-157.
Expression and Purification of the -Crystallin
Domain--
Plasmid p
B57-157 was transformed into
BL21(DE3), and 1-liter expression cultures were grown in Luria Broth
medium supplemented with kanamycin to a final concentration of 50 µg/ml. The expression was induced at
A600 = 0.8 by the addition of
isopropyl-1-thio-
-D-galactopyranoside to a final
concentration of 1 mM. After 4-6 h of induction, cells were collected by centrifugation, and the cell pellets were resuspended in 50 ml of starting buffer (20 mM sodium phosphate, 0.5 M NaCl, pH 7.4). 400 µl of lysozyme (10 mg/ml; Sigma)
were added to the cell suspension, and the suspension was stirred in an
ice-water bath for 20 min. After the addition of 1 µl of benzonase
(250 units/µl; Merck), the suspension was incubated at 23 °C for
10 min with constant stirring. Insoluble cellular debris was removed by
sedimentation at 36,000 × g for 30 min at 4 °C. The
supernatant was filtered through a 0.22-µm filter (Millipore). At a
flow rate of 1 ml/min, 25 ml of cell extract were applied on a 1-ml
Hi-Trap metal-chelating column (Pharmacia), charged with 100 mM NiCl2 solution, and equilibrated with
starting buffer. The column was washed with 10 column volumes of
starting buffer. For 10 column volumes, a linear gradient from 0 to 500 mM imidazole was applied. The His-tagged
B57-157 fusion
protein eluted at 400 mM imidazole concentration. The
positive fractions were pooled, and a His6-tagged TEV-protease was added in a 100:1 ratio (w/w). The cleavage reaction was dialyzed against starting buffer at 20 °C for 24 h. The
reaction mixture was passed through a mini-column (Bio-Rad) filled with 1 ml of Ni-NTA resin (Qiagen), which had been equilibrated with starting buffer. The flow-through was collected and analyzed for purity
on a 10% SDS-polyacrylamide gel electrophoresis (Novex) stained with
Coomassie Blue or silver staining methods. Protein samples were
concentrated using Centricon (Millipore) filter units with a molecular
mass cutoff of 3 kDa. Protein concentrations for all
measurements were determined by the Bradford method, using the Bio-Rad
protein assay reagent with bovine serum albumin as a standard.
Chaperone Activity Assay--
The effect of the -crystallin
domain on protein aggregation was measured as described previously
(19). Aggregation of alcohol dehydrogenase (ADH) at 37 °C was
measured as an apparent optical density at A360
using a Kontron Uvikon 922 Spectrophotometer equipped with a
thermostated cuvette holder. In a total reaction volume of 400 µl, 5 µM yeast ADH (Sigma) was incubated with varying amounts of purified
-crystallin domain or 1 µM human
B-crystallin. The reaction buffer was a 50 mM sodium
phosphate buffer (pH 7.0), 0.1 M NaCl, and 2 mM
EDTA. The optical density in the cell was recorded every 2 min. For all
molar ratios, three independent experiments were conducted.
Sedimentation Velocity Analysis and Equilibrium Centrifugation-- A Beckman XLA analytical ultracentrifuge (Beckman Instruments, Inc., Palo Alto, CA) equipped with ultraviolet absorption optics was used for the sedimentation velocity and the sedimentation equilibrium studies at 4 °C and at 10 °C, respectively. Protein in 50 mM phosphate buffer, pH 7.0, was loaded into 12-mm double-sector aluminum cells placed in an An60 Ti rotor. For the sedimentation velocity analysis, sample volumes of 350 µl were centrifuged at 60,000 rpm. Radial scans of absorbance at 290 nm were taken at 2-min intervals. Data were analyzed to provide the apparent distribution of sedimentation coefficients by means of the programs DCDT (20) and SVEDBERG (21). The hydrodynamic modeling was done with ULTRASCAN (22).
For the sedimentation equilibrium analysis, a sample volume of 180 µl at 3 mg/ml protein in 50 mM phosphate buffer, pH 7.0, was centrifuged at 26,000 rpm. Each data point measured at 297 nm was an average of 50 measurements. The solution density (1.0029 g/ml) and the partial volume of the protein (0.7217 ml/g) were calculated with ULTRASCAN, using the known buffer composition and amino acid composition of the protein. The degree of hydration was estimated based on the amino acid composition by the method of Kuntz and Brassfield (23).
Scattering Experiments and Data Analysis--
The synchrotron
radiation x-ray scattering data were collected using standard
procedures on the X33 camera (24-26) of the European Molecular Biology
Laboratory (EMBL) on storage ring DORIS III of the Deutsches Elektronen
Synchrotron (DESY) and the multiwire proportional chambers with delay
line readout (27). The solutions were measured at protein
concentrations of 5, 10, and 25 mg/ml. The scattering curves were
recorded at a wavelength = 0.15 nm for sample detector
distance 1.4 m covering the momentum transfer range
0.40 < s < 5.1 nm
1
(s = 4
sin
/
, where 2
is the scattering
angle). The data were normalized to the intensity of the incident beam
and corrected for the detector response, the scattering of the buffer
was subtracted, and the difference curves were scaled for concentration
using the program SAPOKO.2
The curves were extrapolated to zero concentration and merged with the
data obtained at high protein concentration using standard procedures
(13).
The maximum dimension Dmax of the protein was estimated from the experimental data using the orthogonal expansion program ORTOGNOM (28). The radius of gyration Rg was evaluated using the Guinier approximation, Iexp(s) = I(0)exp(-s2Rg2/3), which is valid for (sRg) < 1.3 (13), and also from the entire scattering curve using the indirect transform package GNOM (29, 30). The Porod volume (31) Vp was calculated from the experimental data as described previously (13).
The coordinates of the crystallographic model of the HSP16.5 from
M. jannaschii were taken from the Protein Data Bank, entry 1shs (11). The scattering curves from the x-ray structure and
the homology models were calculated using the program
CRYDAM,3 a modified version
of the program CRYSOL (32). The program takes into account the
scattering from the solvation shell at the surface of the protein
model. The latter is covered by a 0.3-nm-thick hydration layer with an
adjustable density b, which may differ from that of the bulk
solvent. The scattering intensity is
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(Eq. 1) |
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(Eq. 2) |
Molecular Model Building--
The x-ray structure of MjHSP16.5,
Protein Data Bank entry 1shs (11), was used as a template for molecular
modeling. The monomer of the -crystallin domain was modeled in close
structural homology to the crystal structure of MjHSP16.5, using the
residues 36-144 with the exception of the dimerization motif. All
models were built and energy minimized using the program O (33). The sequence alignment used in the molecular modeling is identical to that
of Muchowski et al. (12), with one exception. The largest gap in the entire alignment was reduced from eight to seven residues to
optimize the interaction at the putative dimer interface (Fig. 2).
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RESULTS |
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Characterization and Chaperone Activity--
The -crystallin
domain corresponds to the peptide 57-157 from
B-crystallin, which
was isolated after a tryptic digest of
B-crystallin (34). It was the
smallest peptide identified after trypsinization, which comprises the
-crystallin domain. With the expression of p
B57-157 and after
proteolytic cleavage of the His-tagged fusion protein, the
-crystallin domain had an additional three N-terminal,
vector-derived amino acids (Gly, Ala, and Met). The untagged form of
the
-crystallin domain eluted with a >98% purity in the
flow-through of the Ni-NTA column (Fig. 3). Proper folding of the protein was
confirmed by far-ultraviolet circular dichroism spectroscopy (Fig.
4). The spectrum showed negative
ellipticity with a minimum at 215 nm, typical for a
-sheet structure
and in agreement with secondary structure predictions made for the
-crystallin domain of
B-crystallin (35-37) and with far-ultraviolet CD spectra observed for wild type
B-crystallin (12)
and other sHSPs (38).
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The functionality of the -crystallin domain was also confirmed by
the chaperone activity assay (Fig. 5).
The ADH aggregation was effectively suppressed with a 5-fold molar
excess of
-crystallin domain over ADH and was comparable to the
protection by wild type
B-crystallin in a ratio of ADH (5 µM):
B-crystallin (1 µM). When comparing
the aggregation values of the ADH control and ADH (5 µM):
-crystallin domain (5 µM) at the 32 min time point, the control showed 100% ADH aggregation, whereas the
ADH in the presence of
-crystallin domain aggregated only to 45%.
At a 10-fold molar excess of ADH over
-crystallin domain, no
protection of ADH aggregation was observed. The decrease of absorption
for ADH (5 µM) and ADH (50 µM):
-crystallin domain (5 µM) beyond the
34 min time point was caused by precipitation of large protein
aggregates in the cuvette. With an average of 32 subunits/wild type
B-crystallin molecule, the protection from aggregation per dimer was
100% at a 3.2-fold molar excess of
B-crystallin over ADH. With the
-crystallin domain dimer, a 5-fold molar excess was effective in
protecting the same amount of ADH. Although the chaperone activity of
the wild type
B-crystallin dimer exceeded that of the
-crystallin domain dimer by about 1.5-fold, the effectiveness of the
-crystallin domain in preventing ADH aggregation remains high.
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Solution Structure of the B-Crystallin Domain--
The
molecular mass of the
-crystallin domain estimated from the
analytical ultracentrifugation (22 ± 2 kDa) indicated that the
protein is dimeric in solution. The velocity sedimentation data
confirmed that the solution was monodisperse. The sedimentation coefficient 1.82 ± 0.06 (s20,W) and the hydrodynamic modeling suggested that the dimer is rather elongated with an estimated axial
ratio of 1:10.
The synchrotron x-ray scattering curve from the -crystallin domain
is presented in Fig. 6a. The
maximum dimension of the particle was 7.0 ± 0.2 nm, and the
radius of gyration = 2.27 ± 0.03 nm. The Porod volume
(i.e. the excluded volume estimate) of 42 ± 3 nm3 was compatible with the expected excluded volume of a
hydrated dimer and incompatible with a monomeric protein. It can thus
be concluded that the
-crystallin domain is dimeric in solution, in
agreement with the above-mentioned results of the analytical ultracentrifugation. The dimeric structure has been found at all protein concentrations.
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The comparison between the scattering curve computed from the dimeric
x-ray model of the sHSP (11) (Fig. 1, left panel) and the
experimental scattering curve is presented in Fig. 6a, fit
(1). The two curves display noticeable systematic deviations (the discrepancy between the experimental and calculated data is
= 1.89). To check whether the data can be fitted by minor modifications of the crystallographic structure, several tentative models were generated by rigid body movements (up to ± 0.2 nm) and rotations (up to ± 10 degrees) of the second monomer. All these models yielded poor fits with
between 1.8 and 2.5. This suggests that the quaternary structure of the
-crystallin domain of
the human
B-crystallin differs from that of HSP16.5 from M. jannaschii.
The focus of the molecular modeling of the B-crystallin mutant was
the generation of an interface between two subunits, which is in
agreement with (i) the sequence alignment, (ii) the spin-labeling study
(9), and (iii) the general molecular interactions involved in protein
stabilization and fits the experimental scattering data. In keeping
with the low resolution of solution scattering, the modeling of the
dimer of
B-crystallin was performed in terms of rigid body movements
and rotations of the monomeric subunit derived by homology modeling
from the crystal structure of MjHSP16.5. The amino acid sequence of the
-crystallin domain of human
B-crystallin and MjHSP16.5 is more
than 23% identical, and more than 50% of the residues are chemically
similar. Furthermore, secondary structure prediction indicated a
similar arrangement of secondary structure elements in both proteins
(results not shown). In the crystal structure of MjHSP16.5, the strands
5 and
7 are connected by a loop-strand-loop motif ranging from
residues 84-102. Within this motif, the short strand
6 (residues
93-97) from one subunit interacts with strand
2 (residues 45-49)
from the neighboring subunit. The corresponding region in
B-crystallin was modeled as a strand-turn-strand ranging from
residues 105 to 116 and designated as
6.1 and
6.2. The
dimerization between the monomers takes place along strand
6.2,
contributed by the two subunits A and B, which built a four-stranded,
antiparallel, intersubunit composite
-sheet with the following
strand order: A
6.1-A
6.2-B
6.2-B
6.1. The modeled strands
6.1 and
6.2 are separated from
5 and
7 by one residue only,
Glu-105 and Glu-117, respectively. Both residues are in a nonextended
main chain dihedral conformation, resulting in a kink between the sheet
made up by
5 and
7 and the
6.1-
6.2 sheet. The angle between
the core
-sandwich and the intersubunit composite sheet varies
depending on the actual main chain dihedrals of Glu-105, Glu-117, and
their flanking residues. Accordingly, the dimer may have a more closed,
compact or a more open, elongated shape. Five different dimer models
have been tested (Fig. 6b), ranging from a dimer with an
open L-shape (top; (2)) to more compact structures
and, finally, a V-shaped dimer (bottom; (6)). The fits to
the experimental data computed by CRYDAM are presented in Fig.
6a, curves (2-6). All these models yield
better fits than that provided by the crystallographic model of the
MjHSP16.5 dimer (curve (1)). The most compact,
V-shaped model providing the best agreement with the experimental data
is also displayed in Fig. 1 (right panel) for comparison
with the crystallographic model of MjHSP16.5.
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DISCUSSION |
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Probably the most intriguing functional finding in our study of
the dimeric -crystallin domain from human
B-crystallin is that
the chaperone activity does not require a multimeric
B-crystallin complex. The existence of two sites of interactions between sHSP monomers has been proposed previously (39). Our results confirm that
after deletion of the N-terminal domain of
B-crystallin no large
oligomers are formed and that the interaction site in the C-terminal
domain promotes the assembly of a stable dimer. Unlike HSP16.2 from
Caenorhabditis elegans,
B-crystallin maintains chaperone-like activity without its N-terminal domain and, moreover, does not require multimeric assembly to be active (40). The chaperone
activity at different levels of oligomerization has also been observed
for murine HSP25, a member of the mammalian sHSP family (41).
The final model of the -crystallin domain dimer in Fig. 1
(right panel) provides an excellent fit to the solution
scattering data in a wide angular range. A nominal resolution of the
data is 1.2 nm, and details of the tertiary structure can obviously not
be validated experimentally at this level of resolution, which is why
we restricted ourselves to rigid body modeling. Solution scattering is
rather sensitive to rigid body movements of structural domains and has
successfully been used to model the quaternary structure of proteins
(15-17). The use of rigid body modeling is justified by a high
sequence homology between monomeric
B-crystallin and MjHSP16.5.
Because no reasonable fit to the scattering data could be obtained in
the vicinity of the crystallographic model of MjHSP16.5 (Fig. 1,
left panel), the arrangement of the monomers as shown in
Fig. 1 (right panel) was the only possibility to construct a
sound model compatible with the spin-labeling results (9) and with the
solution scattering data. The modeling in the form of a grid search
permitted us to systematically explore plausible dimeric
configurations. An automated rigid body refinement would have been a
better option for larger proteins (42), in which the influence of the
bound solvent scattering is less pronounced.
The present study is not the first example of proteins with
similar tertiary structures but totally different quaternary
structures. In particular, tertiary structures of monomeric and
chemokines (e.g. interleukin-8 and human macrophage
inflammatory protein-1
) are very similar, but the dimers are formed
by different sets of residues (43). One should also mention a number of
thiamine diphosphate-dependent enzymes (transketolase,
pyruvate oxidase, and pyruvate decarboxylase) consisting of identical
or nearly identical monomers with molecular masses of about 60 kDa that form very different biologically active dimers and tetramers (42).
The proposed model of the -crystallin domain dimer displays a
potentially flexible dimer interface. This flexibility may be the
reason why our protein crystallization attempts were not successful,
despite extensive trials.4
The flexibility might also be an essential property enabling variable
sizes and compositions of the multimeric complex of
B-crystallin and
yielding mixed sHSP species (i.e.
A-crystallin,
B-crystallin, and HSP27) in a single hetero-oligomeric complex (44).
It should be stressed that the potential flexibility of the protein is
problematic for crystallographic analysis but not for solution
scattering modeling (the experimental scattering pattern corresponds to
an average position of the monomers).
The dimer interface in the proposed model contains the residue Arg-116
that causes autosomal dominant congenital cataract in humans when
mutated to Cys. Circular dichroism and
1,1'-bi(4-anilino)naphtalene-5,5'-disulfonic acid fluorescence spectra
indicate that R116C A-crystallin is structurally different from the
wild type protein (45). Arg-116 is an identically conserved residue
between
A- and
B-crystallin. According to the present model, this
residue plays an essential role in dimerization, and detrimental
effects on the quaternary structure and biological activity can be
expected after mutation.
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ACKNOWLEDGEMENTS |
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We thank John Clark and Paul Muchowski for
supplying plasmid B57+pET16b and Santi Canela for technical assistance.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ To whom correspondence may be addressed: European Molecular Biology Laboratory (EMBL), EMBL Hamburg Outstation, c/o Deutsches Elektronen Synchrotron, Notkestrasse 85, D-22603 Hamburg, Germany. Tel.: 49-40-89902-118; Fax: 49-40-89902-149; E-mail: Feil@EMBL-Hamburg.DE.
** To whom correspondence may be addressed: European Molecular Biology Laboratory (EMBL), EMBL Hamburg Outstation, c/o Deutsches Elektronen Synchrotron, Notkestrasse 85, D-22603 Hamburg, Germany. Tel.: 49-40-89902-125; Fax: 49-40-89902-149; E-mail: Svergun@EMBL-Hamburg.DE.
Published, JBC Papers in Press, January 12, 2001, DOI 10.1074/jbc.M010856200
2 D. I. Svergun and M. H. J. Koch, unpublished data.
3 M. Malfois and D. I. Svergun, manuscript in preparation.
4 I. K. Feil, J. Hendle, and H. van der Zandt, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: sHSP, small small heat-shock protein; ADH, alcohol dehydrogenase.
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