From the Department of Biological Chemistry, The
Weizmann Institute of Science, Rehovot 76100, Israel, the
§ Bioénergétique et Ingéniérie des
Protéines, Centre National de la Recherche Scientifique,
IBSM-IFR1, Marseille 13402, France, the ¶ Université de
Provence, Marseille 13331, France, the
Department of
Molecular Microbiology and Biotechnology, Tel Aviv University, Ramat
Aviv 69978, Israel, and the ** Department of Food Engineering and
Biotechnology, and Institute of Catalysis Science and Technology,
Technion-Israel Institute of Technology, Haifa 32000, Israel
Received for publication, October 10, 2000, and in revised form, January 4, 2000
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ABSTRACT |
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The assembly of enzyme components into the
cellulosome complex is dictated by the cohesin-dockerin interaction. In
a recent article (Mechaly, A., Yaron, S., Lamed, R., Fierobe, H.-P.,
Belaich, A., Belaich, J.-P., Shoham, Y., and Bayer, E. A. (2000)
Proteins 39, 170-177), we provided experimental evidence
that four previously predicted dockerin residues play a decisive role
in the specificity of this high affinity interaction, although
additional residues were also implicated. In the present communication,
we examine further the contributing factors for the recognition of a
dockerin by a cohesin domain between the respective cellulosomal
systems of Clostridium thermocellum and Clostridium
cellulolyticum. In this context, the four confirmed residues were
analyzed for their individual effect on selectivity. In addition, other
dockerin residues were discerned that could conceivably contribute to
the interaction, and the suspected residues were similarly modified by
site-directed mutagenesis. The results indicate that mutation of a
single residue from threonine to leucine at a given position of the
C. thermocellum dockerin differentiates between its
nonrecognition and high affinity recognition (Ka ~ 109 M The cellulosome concept is one of the major paradigms of microbial
degradation of plant cell-wall polysaccharides (1-4). Initially, such
a multienzyme complex was described in the anaerobic thermophilic
bacterium, Clostridium thermocellum (5, 6). Additional
cellulosomes have been described subsequently for a variety of
anaerobic bacteria and fungi (7-13). The various hydrolytic enzymes
are incorporated into the cellulosome complex by virtue of a pivotal
nonhydrolytic polypeptide, called scaffoldin, which bears a collection
of cohesin modules for this purpose. Each cohesin binds a single
dockerin domain located on the enzymes, thereby generating the fully
assembled cellulosome.
In recent years, much effort has been devoted toward a better
understanding of the cohesin-dockerin interaction. In early works, it
was reported that the cellulosomal subunits are cohesively contained in
the complex of C. thermocellum (14, 15). It was also noted
that a duplicated segment within the dockerin sequence shows remarkable
similarity with the EF-hand motif of calcium binding proteins, such as
calmodulin and troponin C (16). Indeed, it was shown subsequently that
the cohesin-dockerin interaction in C. thermocellum is
calcium-dependent, but surprisingly nonspecific among the
different dockerin-bearing enzymes of this species (17, 18). It was
further demonstrated that both duplicated segments of the dockerin
domain are required for binding to the cohesin module (19, 20). The
three-dimensional structure of a dockerin is still unknown, although
related secondary structure predictions indicated that the dockerin
domain would assume an "F-helix variation" of the EF-hand motif
(21, 22). This hypothesis has recently been corroborated experimentally
by NMR spectroscopy (23). Despite the reported crystal structures for
two different C. thermocellum cohesins (24, 25), structural
information regarding the cohesin-dockerin complex and the precise
residues involved in the interaction have yet to be discovered.
We have shown that the interaction among cohesins and dockerins between
two cellulosome-producing bacteria, C. thermocellum and
C. cellulolyticum, is species-specific (21). Sequence
analysis of the various dockerin domains from the two species enabled
us to predict four residues that could unambiguously account for the
observed species specificity. Subsequent studies, in which the four
suspected dockerin residues of one species were converted to emulate
those of the other, provided experimental verification of this
prediction (26). Thus, interconversion of the selected residues created
new specificities in which the cohesins now recognized the dockerin
from the rival species. However, in each case, despite the relevant
mutations, the intra-species interaction was not destroyed, and the
modified dockerins still recognized the cohesin from the same species.
This implied that other residues might contribute more subtle secondary
interactions that would also be involved in cohesin-dockerin recognition.
The purpose of the present work was 2-fold: first, to try to determine
the minimal number of selectivity determinants in the dockerin domain
that distinguish between the two species, and second, to try to
identify additional residues that contribute secondarily to the
cohesin-dockerin interaction. The first goal was achieved by mutation
of a single dockerin residue from threonine to leucine in a C. thermocellum cellulosomal enzyme that sufficed to create the
ability to bind a cohesin from C. cellulolyticum. The second
goal was only partially achieved, in that up to eight separate dockerin
mutations served only to reduce but not to abolish self-recognition of
the C. thermocellum cohesin.
Bacterial Strains and Vectors--
Escherichia coli
strain XL1-Blue was obtained from Stratagene (La Jolla, CA). E. coli strain MutS was obtained from CLONTECH Laboratories (Palo Alto, CA). E. coli strain BL21
pLysS( DNA Manipulation--
DNA was manipulated by standard procedures
(27, 28). DNA was transformed using either the calcium chloride method
for strain BL21(DE3)pLysS or by electroporation using GeneZapper (IBI, New Haven, CT) for strains XL-1 Blue and MutS. DNA sequencing was
performed by the DNA Sequencing Unit at the Weizmann Institute (Rehovot, Israel).
Unique-site Elimination--
Mutant mS5 (previously termed
mCelS-ct) was constructed as reported previously (26), using a
modification of the unique-site elimination procedure (29). Briefly,
the mutations were first produced in a detached enzyme-free dockerin
construct using primers S688AT689F and S720AT721L (Table
I). The DNA encoding for the entire
mutated dockerin segment was transferred en bloc to the intact parent enzyme (CelS-ct), using selection primer I (thus converting a unique EagI site to SacII) and the
entire mutated dockerin as the mutagenic primer. Mutant mS3 was
constructed using the same approach, whereby clones containing the
desired mutated dockerin segment (S688AT689F) were selected using the
appropriate restriction enzyme (MluI), and the mutated
segment was then transferred en bloc to the intact parent
enzyme using the same selection primer. The resultant mutant,
containing both the mutated dockerin and the intact catalytic domain,
was used subsequently as a template for the construction of another
mutant, mS1. In this case, the mutagenic primer comprised L689T, and
selection primer II contained the reverse mutation, which converted the
SacII site back to EagI.
Site-directed Mutagenesis by Overlap-extension PCR--
With the
exception of mS1, mS3 and mS5, the other mutations were achieved using
OE PCR1 (30, 31) as described
earlier (26). mS5 was used as a template for the construction of mS6;
the mutations were introduced in two OE PCR cycles via mutagenic
primers R696K (forward) and R728K (reverse) and flanking primers,
N-flank-AatII (forward) and C-flank-NheI (reverse). However, ligation of the mutated dockerin fragment (477 bp)
into pET9D-CelS (5979 bp) resulted in a high background of nonmutated
colonies, apparently due to the large difference in size of the two DNA
fragments and consequent difficulty in distinguishing single- from
double-digested plasmid DNA. To circumvent this problem, a
triple-fragment ligation approach was undertaken, in which an extended
insert, delimited by NcoI and NheI sites, was
extracted from the plasmid. The insert was further digested at an
internal AatII site, and the resultant
NcoI-AatII-digested fragment (1958 bp) was
ligated, together with the mutated dockerin fragment
(AatII-NheI), into the
NcoI-NheI-digested pET9d-CelS (4021 bp). The
resulting mutant, mS6, was then used as a template for the construction
of mS8, utilizing the mutagenic primers R700G (forward) and K732G
(reverse) employing the same method. For the construction of mS7,
mutagenic primers K696R (forward) and K728R (reverse) were used with
flanking primers N-ter-ScaI (forward) and
C-flank-NheI (reverse) with mS8 as template in a simple
ligation reaction. The same flanking primers were also used to
construct mS2 and mS4. In the case of mS2, mS3 was used as a template
with the mutagenic primer A688S (forward), whereas for mS4, pET9d-CelS was used as a template with primer S720AT721L (reverse).
The mutant mCelA-cc of the C. cellulolyticum cellulase A was
described in an earlier publication (26).
Expression of Recombinant Proteins--
All mutants were
expressed in BL21(DE3)pLysS cells as described previously (26). The
crude extracts were used for affinity-blotting studies without
additional purification. The cells were centrifuged (6000 rpm, 10 min),
resuspended to a final A600 of 10, and
SDS-containing sample buffer was added, prior to boiling and
SDS-PAGE.
The respective cohesin probes, Coh-cc and Coh-ct, were prepared as
described previously (17, 32).
Purification of Recombinant Proteins--
CelA-cc and its mutant
(mCelA-cc) were purified as described earlier (20, 26). CelS-ct, mS1,
mS2, mS3, mS5, mS6, and mS8 were produced and purified as follows:
E. coli clones were grown to A600 = 1.5 at 37 °C in 2 liters of Luria-Bertani medium, supplemented with
1.2% glycerol (w/v) and 50 µg·ml Protein Biotinylation--
Cohesin-containing constructs were
biotinylated using biotinyl N-hydroxysuccinimide ester
at a 15-fold molar ratio of reagent to protein as described previously
(33). The cohesin-containing constructs from both species retained
normal binding activity to both cellulose and dockerin-containing
enzymes and were recognized by avidin probes.
Affinity Blot Analysis--
The affinity blotting procedure was
carried out essentially as described previously (26). Briefly, samples
of the mutated dockerin constructs were separated by 10% SDS-PAGE and
transferred electrophoretically onto nitrocellulose membranes. The
blots were reacted with biotinylated cohesin-containing constructs from
either C. thermocellum or C. cellulolyticum
(Coh-ct and Coh-cc, respectively) and developed using
ExtrAvidin-horseradish peroxidase complexes (1:2000 dilution, Sigma
Chemical Co., St. Louis, MO).
Surface Plasmon Resonance--
Experiments were performed as
described earlier (20), using 10 mM Tris-maleate (pH 6.8),
0.15 M NaCl, 2.5 mM CaCl2, 0.005% P20 (BIAcore, Uppsala, Sweden) as running buffer (flow rate 25 µl
min The cellulosome is a cohesive enzyme complex of exceptional
stability. To dissociate the cellulosome from C. thermocellum into its component parts, the presence of detergent
at elevated temperatures is required (14, 34). The observed stability of the cellulosome is a function of the very high affinity interaction between two different types of module; i.e. the single
dockerin domain of the individual enzyme subunits and the multiple
copies of the cohesin domain on the scaffoldin subunit (35, 36). Recent attempts in our laboratory to control the specificity of the
cohesin-dockerin interaction have met with partial success. By
interconverting four predicted recognition residues of the dockerins
from C. thermocellum and C. cellulolyticum (21),
a new specificity was created whereby the modified dockerin of one species now recognized the cohesin of the other (26).
Following these studies, it was of interest to try to refine this
interaction further, by pinpointing whether fewer than four residues
may account for the specificity of binding between the two species. On
the other hand, the inability to neutralize completely the
cohesin-dockerin interaction within the same species
implicated additional residues as auxiliary specificity determinants,
and we thus revisited the potential contribution of other dockerin sites to the observed specificity.
Refinement of Critical Recognition Residues--
To determine the
minimal changes necessary to create the new interspecies specificity,
progressive mutations of the four putative recognition residues were
designed. These particular mutations were carried out at positions 10 and 11 of the two duplicated segments of the dockerin domain in both
species (Table II). Thus, mutants of the
C. thermocellum CelS dockerin exhibited single mutations at
these positions in the first duplicated segment (mutants mS1 and mS2),
mutations of both of the latter positions in each duplicated segment
alone (mS3 and mS4), and the four-residue mutant (mS5 in the present
study, equivalent to mCelS-ct as described previously (26)). The mutant
dockerin construct of the C. cellulolyticum CelA enzyme
(mCelA-cc) was used as a cross-species reference and corresponded to
the previously reported (26) four-residue mutation.
The various C. thermocellum mutant dockerins were
overexpressed and subjected to SDS-PAGE and Western blotting, and the
resultant blots were challenged with a cohesin probe derived from
either species. The results are presented in Fig.
1. Strikingly, the conversion of a single
amino acid residue in the CelS dockerin was enough to create a new
specific interaction with the C. cellulolyticum cohesin
(Fig. 1A). In this context, it was clear that the common denominator for interaction of a dockerin mutant with the cross-species cohesin was the presence of a leucine instead of a threonine at position 11 in the first duplicated segment. Thus, mutants mS2, mS3,
and mS5 interacted strongly with Coh-cc, whereas mS1 and mS4 failed to
do so. Indeed, this result reflects the conservation of
hydroxy-amino acids in this particular position of the known C. thermocellum dockerins and hydrophobic residues of the
C. cellulolyticum dockerins (Table
III). It is not clear, however, why
mutation of the other conserved residues in positions 10 and 11 (notably the interconversion of serine to alanine in position 10)
appears not to be strictly required for interspecies recognition
between these two species of dockerin.
Attempts to Reduce Self-recognition between Mutant Dockerins and
Coh-ct--
In our previous study (26), we showed that the
four-residue mutant, mS5, clearly recognized the cross-species cohesin
probe. Nevertheless, the strong binding of its own cohesin was
retained. In the present work, we sought additional sites that might
reduce or abolish such intra-species self-recognition while maintaining the newly created binding specificity between the two species. For this
purpose, positions 18 and 22 of the two duplicated segments of the CelS
dockerin were chosen as targets for site-directed mutagenesis. The
basis for selection of these four positions was 2-fold. First, the
character of these residues is quite well conserved, especially in the
C. thermocellum dockerins (Table III). Second, according to
PHD prediction (Profile network prediction HeiDelberg) of solvent
accessibility (37-39), these four residues are predicted to be in
exposed positions in the dockerin structure (Table
IV). According to the prediction,
positions 17 may also be exposed (Table IV), but the dockerins of
both C. thermocellum and C. cellulolyticum almost
invariably show a lysine at this position (position 21), and
interconversion in this case would thus be irrelevant.
In view of the above, mutations in positions 18 and 22 of the
duplicated segments were performed to accompany the mutations at
positions 10 and 11 (Table II). Thus, the arginines in positions 18 were mutated to lysines in mutant mS6, and the basic residues at positions 22 of the two duplicated segments (arginine and lysine, respectively) were mutated to glycines in mS7. Mutant mS8 comprised the
combined four-residue mutation in addition to the mutations at
positions 10 and 11. In the latter two mutants, mutation to glycine was
chosen for position 22 of segment 1 instead of alanine as in the CelA
dockerin from C. cellulolyticum, because glycine is more
prevalent at this position among all of the known cellulosomal enzymes
of this species (Table III).
The consequences of these mutations are shown in Fig. 1. In accord with
the above bioinformatics-based prediction, mutants mS7 and mS8 (mutated
in positions 10, 11, 22, and/or 18) indeed displayed a visible
reduction in their self-recognition of the C. thermocellum
cohesin (Coh-ct), compared with those of the native CelS-ct and mutants
mS1 through mS5 (Fig. 1B). On the other hand, mutant mS6
(mutated in positions 10, 11, 18, but not 22) retained a higher level
of interaction with Coh-ct. It thus follows that position 22 is an
important determinant in dockerin recognition. Nevertheless, unlike the
native C. cellulolyticum-derived enzyme (CelA-cc) that
failed to bind Coh-ct, both mutants mS7 and mS8 bound clearly to their
own cohesin (Coh-ct) as described above. This indicates that, in the
C. thermocellum system, other residues, in addition to the
eight designated positions, still play a role in the recognition process.
Determination of Binding Constants--
On the basis of the
affinity blotting data, several of the key mutants were overexpressed,
purified, and subjected to analysis by surface plasmon resonance, using
immobilized forms of the two species of cohesins and the BIAcore
system. The results are presented in Table
V. In general, the data are in total
accord with the blots and provide numerical values for the binding of
the individual mutant dockerins to either species of cohesin. It is
worthwhile noting that the observed affinity characteristics for the
cohesin-dockerin interaction in C. thermocellum were found
to be much higher than those of C. cellulolyticum. The
calculated affinity constant for the mesophilic C. cellulolyticum system (~1010
M
With respect to the selectivity of cohesin-dockerin recognition, the
critical mutation in mutant mS2 (in which the threonine at position 11 of the first duplicated segment was converted to leucine) generated a
novel high affinity interaction with the C. cellulolyticum
cohesin. Compared with the native dockerin-bearing enzyme, this single
mutation differentiated between a lack of detectable binding and a
tenacious cross-species cohesin-dockerin interaction, characterized by
a measurable dissociation constant of 5 × 10
With respect to intra-species cohesin-dockerin recognition, the
preference of a given dockerin mutant to either of the two species of
cohesins could be quantified by comparing the ratio of affinity
constants (see Fig. 2). Due to the very
high affinity constants for the C. thermocellum system, most
of the CelS-derived mutants showed a clear preference for
self-recognition of Coh-ct. However, as the number of mutations
increased, a trend toward preference for Coh-cc prevailed. Indeed,
mutant mS8 exhibited a detectable preference for cross-species
recognition. In the reverse direction, the CelA-derived mutant
(mCelA-cc) clearly favored interaction with the rival cohesin. It is
interesting to note that this mutant is counter-equivalent to mS5 in
its mutation pattern (i.e. in positions 10 and 11). Again,
the measurable difference in cross-species recognition between these
two mutants may be attributed to the higher affinity of the
cohesin-dockerin interaction of the thermophilic system.
Concluding Perspectives--
The exchange of a single threonine
for leucine in the C. thermocellum dockerin generated
cross-species recognition with a C. cellulolyticum cohesin.
This implies that the major difference between these two residues,
i.e. the lack of a hydroxyl group in leucine compared with
threonine, represents the molecular basis for the newly gained
specificity. The presence or absence of this single decisive hydroxyl
group at the specified position in the C. thermocellum
dockerin thus appears to be critical to the specificity of the
cohesin-dockerin interaction in this species. It is remarkable that
such a small difference would differentiate between a very high
affinity interaction and essentially no detectable interaction at all.
This phenomenon also implies a complementary distinction on the
surfaces of the two cohesin species. Specifically, it may be expected
that the C. cellulolyticum cohesin would exhibit a more
hydrophobic microenvironment at a given, yet unidentified locale, which
may account for the observed change of specificity in the mutated dockerin.
No less remarkable, perhaps, is the fact that the dockerin could be
subjected to such a broad number of mutations without abolishing
self-recognition of its own cohesin. Thus, the C. thermocellum dockerin could be mutated at positions 10, 11, 18, and 22 in both duplicated segments, encompassing a series of one to
eight mutations of the 67-residue dockerin domain, and the entire
spectrum of mutated constructs recognized the cohesin of C. cellulolyticum as well as its own. This suggests that the two
species of cohesin-dockerin pairs are, perhaps, much more similar than
originally presumed (21).
Ultimately, the precise molecular determinants that dictate binding
specificity of the cohesin-dockerin interaction will probably be
resolved by three-dimensional structure determination of
cohesin-dockerin complexes from different species. To date, however,
both crystallogenesis and NMR spectroscopy of such complexes have
proved elusive. In the interim, indirect approaches, such as
site-directed or random mutagenesis of the different components, should
continue to provide us with clues into the molecular determinants of
the tenacious cohesin-dockerin interaction.
1) by a cohesin from
C. cellulolyticum. This suggests that the presence or
absence of a single decisive hydroxyl group is critical to the observed
biorecognition. This study further implicates additional residues as
secondary determinants in the specificity of interaction, because
interconversion of selected residues reduced intraspecies
self-recognition by at least three orders of magnitude. Nevertheless,
as the latter mutageneses served to reduce but not annul the
cohesin-dockerin interaction within this species, it follows that other
subtle alterations play a comparatively minor role in the recognition
between these two modules.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
DE3) was used as a host for the expression of the C. thermocellum CelS enzyme in pET9d (Novagen, Madison, WI), and the
resulting construct was termed pET9d-CelS (26). The various
CelS-derived mutants were expressed in the same host strain.
Details of mutagenic primers used for site-directed mutagenesis
1 kanamycin. The
cultures were then cooled to 15 °C, and
isopropyl-1-thio-
-D-galactoside was added to a final
concentration of 0.1 mM. After 16 h, the cells were
centrifuged (10 min, 3000 × g), resuspended in 30 ml of 50 mM Tris-HCl (pH 8) containing 4 mM
CaCl2, and broken in a French press. The crude extract was
then loaded onto a column containing 5-6 g of Avicel (Fluka, Buchs,
Switzerland), equilibrated in the same buffer. The cellulose was washed
with the same buffer, and the recombinant protein was eluted with
distilled water. To further purify the protein of interest, the eluted
fraction was supplemented with Tris-HCl, pH 8.5, and CaCl2
at final concentrations of 25 and 2 mM, respectively, and
loaded on 3 ml of DEAE-Trisacryl (Sepracor, Villeneuve-la-Garenne,
France) equilibrated with the same buffer. The elution was performed
using a linear gradient from 0 to 0.5 M NaCl, and fractions
were analyzed by SDS-PAGE. The fractions containing pure recombinant
protein were pooled and dialyzed using ultrafiltration (cutoff of 10 kDa; Millipore, Bedford, MA) by several concentration/dilution steps in
10 mM Tris-HCl (pH 8.0),
1). Biotinylated cohesin-containing constructs (~100
resonance units) were immobilized onto streptavidin-bearing
sensor chips. The cellulases were diluted in the same buffer (final
concentrations ranging from 5 nM to 1 µM) and
allowed to interact with the immobilized receptor by injections of
600 s.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
Sites and mutations of the C. thermocellum CelS and the C. cellulolyticum CelA dockerins
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Fig. 1.
Affinity-blotting of C. thermocellum CelS-dockerin mutants using recombinant
cohesin-containing probes. Enzyme samples were subjected to
SDS-PAGE and blotted onto nitrocellulose membranes (see Table II for
composition of the respective native and mutant enzymes). The separated
proteins were probed using the desired biotinylated cohesin construct:
Blot A, Coh-cc from C. cellulolyticum; Blot
B, Coh-ct from C. thermocellum (17, 32). The blots were
developed using an avidin-peroxidase conjugate.
Distribution of amino acid residues at critical positions in the known
dockerins of C. thermocellum and C. cellulolyticum
Predicted solvent accessibility for dockerin residues
-helices are also indicated
(21).
1) was in itself indicative of a very high
affinity interaction and in accord with previously reported values
(20). In the thermophilic system, the affinity constants were even
higher and essentially exceeded the upper limits (>1011
M
1) of the BIAcore system. It should be noted
that such a high affinity may be required to maintain tight binding
under thermophilic conditions. In any case, the cohesin-dockerin
complex in the C. thermocellum system represents one of the
highest protein-protein affinity interactions known in nature, rivaling
many other very high affinity associations, e.g.
receptor-effector and enzyme-inhibitor pairs (40).
Affinity constants for binding of wild-type and mutant dockerin-borne
enzymes to immobilized cohesins
9
M. It is interesting to note that all of the
C. thermocellum dockerin mutants that carried the Thr-11
Leu mutation exhibited similarly high rates of association for
Coh-cc (~106 M
1
s
1), resembling that of CelA-cc, i.e. the
native enzyme from C. cellulolyticum (Table V). Also of note
are the consistently higher koff values for the
mutants, compared with CelA-cc, indicating a shorter half-life for
their complexes with the C. cellulolyticum cohesin. In the
complementary system, the four mutations in the dockerin of CelA-cc
gained a similarly high affinity constant (3.3 × 109
M
1) with Coh-ct, whereas the native enzyme
showed no measurable binding with the cross-species cohesin.
View larger version (25K):
[in a new window]
Fig. 2.
Relative binding of mutant dockerin-borne
enzymes to cohesins. The ratio of affinity constants were
calculated as Ka (C. thermocellum)/Ka (C. cellulolyticum). Values greater than 1 indicate a preferred
affinity of the given dockerin for the C. thermocellum-derived cohesin, and values lower than 1 show a
preference for the C. cellulolyticum cohesin. White
bars indicate native and mutant constructs derived from C. thermocellum, and gray bars indicate those from
C. cellulolyticum. Bars broken at the top
indicate minimum values, either due to lack of detectable binding
(Ka < 106) to one of the cohesins or to
extremely strong binding (Ka > 1011) to
Coh-ct. Note, mutants mS8 and mCelA-cc show a preference for the rival
species of cohesin.
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ACKNOWLEDGEMENT |
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We appreciate the preparation of mutants mS3 and mS5 by Dr. Sima Yaron.
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FOOTNOTES |
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* This work was supported by a contract from the European Commission (Fourth Framework, Biotechnology Programme, BIO4-97-2303). Grants from the Israel Science Foundation (administered by the Israel Academy of Sciences and Humanities, Jerusalem) are gratefully acknowledged. Additional support was provided by the Otto Meyerhof Center for Biotechnology, established by the Minerva Foundation, (Munich, Germany).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 should be addressed: Tel.:
972-8-934-2373; Fax: 972-8-946-8256; E-mail:
bfbayer@wicc.weizmann.ac.il.
Published, JBC Papers in Press, January 8, 2001, DOI 10.1074/jbc.M009237200
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ABBREVIATIONS |
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The abbreviations used are: OE PCR, Overlap-extension polymerase chain reaction; CBD, cellulose-binding domain; CelS-ct, recombinant cellulosomal Family-48 cellulase from C. thermocellum; CelA-cc, recombinant cellulosomal Family-5 cellulase from C. cellulolyticum; Coh-ct, recombinant probe from the C. thermocellum scaffoldin subunit that contains the cohesin-2 module and the CBD; Coh-cc, recombinant probe from the C. cellulolyticum scaffoldin subunit that contains the CBD and cohesin-1 module; PAGE, polyacrylamide gel electrophoresis; bp, base pair(s).
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REFERENCES |
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