(Received for publication, March 4, 1997, and in revised form, May 2, 1997)
From the Department of Molecular and Cell Biology and
¶ Department of Chemistry, University of California, Berkeley,
California 94720 and the ¶ Division of Structural Biology,
Lawrence Berkeley Laboratory, Berkeley, California 94720
Binding of ligands to the maltose-binding protein
(MBP) of Escherichia coli often causes a global
conformational change involving the closure of its two lobes. We have
introduced a cysteine residue onto each of these lobes by site-directed
mutagenesis and modified these residues with spin labels. Using EPR
spectroscopy, we examined the changes, caused by the ligand binding, in
distance between the two spin labels, hence between the two lobes. The
binding of both maltose and maltotetraose induced a considerable
closure of the N- and C-terminal lobes of MBP. Little closure occurred upon the binding of maltotetraitol or -cyclodextrin. Previous study
by fluorescence and UV differential absorbance spectroscopy (Hall, J. A., Gehring, K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17605-17609) showed that maltose and a large
portion of maltotetraose bound to MBP via one mode (R mode or
"end-on" mode), which is physiologically active and leads to the
subsequent transport of the ligands across the cytoplasmic membrane. In
contrast, maltotetraitol and
-cyclodextrin bound to MBP via a
different mode (B mode or "middle" mode), which is
physiologically inactive. The present work suggests that the B mode is
nonproductive because ligands binding in this manner prevent the
closure of the two domains of MBP, and, as a result, the resulting
ligand-MBP complex is incapable of interacting properly with the inner
membrane-associated transporter complex.
The maltose-binding protein (MBP)1 of
Escherichia coli is absolutely required for maltose and
maltodextrin transport across the cytoplasmic membrane (see Ref. 1 and
references therein). Various ligands bind tightly to MBP. However, the
specific binding to MBP does not necessarily lead to the subsequent
transport of the ligand via the inner membrane-associated transporter
complex, which is composed of one copy of MalF, one copy of MalG, and
two copies of MalK (2). In the preceding paper (1) we used fluorescence and UV differential absorbance spectroscopy to characterize the modes
of binding of various ligands to MBP. Maltose bound exclusively via one
mode that involved a red shift of the intrinsic fluorescence emission
spectrum of MBP as well as other characteristic changes in UV
absorption spectrum (R mode, for red shift). Maltodextrins were also shown to bind largely through the R mode when added in
excess. In contrast, -cyclodextrin as well as reduced or oxidized maltodextrins bound to MBP exclusively through another mode, which caused a blue shift of the fluorescence emission spectrum and other
signature alterations in UV absorption spectrum (B mode, for
blue shift). A mutant MBP, MalE254, bound even unmodified maltodextrins exclusively via the B mode. We found that whenever a
ligand was bound to MBP via the R mode, it was transported. In
contrast, when it bound to MBP purely through the B mode, it failed to
be transported through a wild type transporter complex, even when the
binding to MBP occurred with very high affinity. We have also argued
(1) that the R and B modes most likely correspond to the "end-on"
and "middle" modes of binding, previously defined on the basis of
NMR chemical shift of the 3H atom on the anomeric carbon of
the ligand molecules (3).
The binding of R mode ligands, such as maltose and maltotriose, was
earlier shown to produce a large scale conformational change in MBP (4)
involving the closing of its two lobes (5, 6). In contrast, one of the
B mode ligands, -cyclodextrin, binds to MBP without causing the
closing (6). This correlation suggests that the R and B modes may
produce closing and nonclosing of the two lobes of MBP, respectively.
However, because fluorescence, UV absorption, and chemical shift of the
anomeric 3H are all influenced mainly by the local
environment surrounding the ligand, these results did not provide
concrete data on the global conformation of MBP.
In the present work we have used electron paramagnetic resonance (EPR)
spectroscopy to examine the global conformational changes of MBP,
caused by the R and B modes of binding. EPR spectroscopy is a sensitive
method for examining protein structure and dynamics and has become
widely applicable due to the development of site-directed spin labeling
(for reviews see Refs. 7 and 8). Spin labeling EPR has previously been
used to estimate interspin separations in proteins (9, 10). Recently,
this approach has been systematically extended to measure distances
between two site-specifically placed spin labels and calibrated using
well known -helical peptides (11). Using this approach, we have
spin-labeled two residues of MBP, one in the N-terminal lobe and one in
the C-terminal lobe, and examined the extent of closure of these lobes
occurring upon the binding of various ligands. Our results indicated
that those ligands that bind to MBP solely by the B mode caused little
change in the global conformation of MBP upon binding. In contrast,
those ligands that complex with MBP primarily via the R mode caused the
two domains of MBP to come together considerably.
Strain XL1-Blue MRF
(
(mcrA)183
(mcrCB-hsdSMR-mrr)173 endA1 supE44
thi-1 recA1 gyrA96 relA1 lac [F
proAB
lacIqZ
M15 Tn10]) (Stratagene
Cloning Systems) was used for all cloning steps, and plasmid pLWY
(malE+ bla) was utilized as the
template for all mutagenesis reactions. This plasmid was constructed by
ligating the EcoRI-NdeI fragment of
malE+ allele from the plasmid pPD1
(malE+ bla) (12) with the large
EcoRI-NdeI fragment of plasmid pUC19. The ligated
DNA was transformed into strain XL1-Blue MRF
. DNA from these
transformants was analyzed, and a plasmid that could be cut with both
HindIII and BglII was retained as pLWY.
Site-directed mutagenesis was performed by using sequential PCR steps
(13). To produce the S211C mutation the mutant primer 5-GATTACTGCATCGCAGAAGCT-3
and an external primer
immediately outside the BlpI site unique to pLWY, and the
mutant primer 5
-TGCGATGCAGTAATCGGTGTC-3
and an external
primer immediately outside the BglII site unique to pLWY
were used in the first PCR amplification. The resulting blunt-end
fragments were isolated on a 0.8% agarose gel, purified using the
GenecleanII kit (BIO 101), and used in the second PCR amplification.
The two blunt-ended fragments were here amplified with the two
aforementioned external primers to generate the full-length fragment
containing the S211C mutation. This full-length product was then
digested with both BlpI and BglII. The resultant
fragment was isolated and purified as above and finally ligated into
BlpI- and BglII-digested pLWY to produce pJH1.
This procedure was also used to produce the D41C mutation except that,
in the first PCR amplification, the mutant primers
5
-CATCCGTGTAAACTGGAAGAG-3
and
5
-CAGTTTACACGGATGCTCAAC-3
were used with external primers immediately outside the unique BsiWI and PvuII
sites, respectively. The full-length product was digested with
BsiWI and PvuII, and the resultant fragment
ligated into pLWY to produce plasmid pJH2. Both pJH1 and pJH2 were
sequenced across their respective PCR-amplified regions using the
Sequenase version 2.0 DNA sequencing kit (U.S. Biochemical Corp.) to
ensure that no other mutations occurred.
A plasmid that contains both the S211C and D41C mutations in the
malE gene was constructed by ligating the small fragment derived from digestion of pJH2 with BglII and
EcoRI with the large fragment derived from digestion of pJH1
with the same two restriction enzymes. After ligation, and subsequent
transformation of strain XL1-Blue MRF, plasmid DNA was isolated, and
was digested with both BglII and EcoRI to ensure
that they exhibited the correct restriction patterns. The plasmid DNA
was further sequenced across both mutated regions. One plasmid, pJH3,
was used for subsequent expression of D41C/S211C MBP.
These were described in the preceding paper (1).
Purification of MBPWild type MBP and D41C, S211C, and
D41C/S211C mutant MBPs were prepared from strain HS2019 (K-12
F araD139
lacU169 rpsL thi
malE444) (14)
containing the appropriate plasmid and purified as described (1),
except for the following changes. All mutant MBP proteins were eluted
from cross-linked amylose column with 100 mM maltose, and
bound maltose was removed from all MBP proteins by denaturation in 6 M guanidine HCl followed by renaturation through dialysis.
Purified MBP proteins were shown to be at least 95% pure by staining
of SDS-polyacrylamide gels.
The binding of various ligands to wild type and mutant MBP proteins, unlabeled or labeled with spin label, was determined using fluorescence emission spectroscopy (1). For the determination of the mode of binding, ligand was added to MBP such that greater than 90% of all MBP present would be in the ligand-complexed form.
Proteoliposome Assays of ATP Hydrolysis and Maltose TransportThe inner membrane transport complex
(MalFGK2) was prepared from strain HN741 (K-12 argH
his rplL1 malTc malB13
uncBC
ilv::Tn10/F
lacIq
Tn5) (15) containing pMR11 (malK cat) and pFG23
(malF malG bla) (16, 17). Cells were grown, and envelope
fraction was prepared as described (15). Proteoliposomes were
reconstituted by using octyl
-D-glucoside extract of
cell envelope fraction by dilution into a buffer (2).
To measure the ability of MBP to stimulate ATP hydrolysis by the MalFGK2 complex, this dilution was made into a buffer containing MBP and ligand (15), which were added to give a final concentration of 0.15 and 500 µM, respectively, after dilution. Based on the Kd values of wild type and mutant MBP, this ensures that >90% of all MBP inside the proteoliposomes is in the ligand-complexed form. The hydrolysis of ATP by the MalFGK2 transport complex was measured as described (15), except that all reactions were stopped after 5 min. All values were corrected by subtracting ATP hydrolysis by liposomes prepared without the MalFGK2 transport complex, MBP, and ligand.
When the transport of maltose into proteoliposomes was examined, the dilution mixture contained 20 mM KPO4, pH 6.2, 5 mM ATP, and 1 mM DTT. The proteoliposomes were resuspended to a final concentration of 0.45 mg/ml protein in 20 mM KPO4, pH 6.2, 3 mM MgCl2 with or without 1.0 µM MBP, and incubated with 10 µM [14C]maltose at room temperature for 5 min. The mixture was then diluted 1:10 with 20 mM KPO4, pH 6.2, 3 mM MgCl2, filtered through a Millipore filter (0.22-µm GSWP), and washed with 5 ml of 50 mM LiCl. Filters were then air-dried and counted in a liquid scintillation counter using Ecolume (ICN) as scintillant.
Spin LabelingMBPs were labeled with
(1-oxy-2,2,5,5-tetramethylpyrrolidin-3-yl) methyl methanethiosulfonate
spin label (MTSSL), obtained from Renal (Budapest, Hungary). MBP (15 µM) was first incubated for 1 h at room temperature
in 20 mM DTT, 10 mM KPO4, pH 8.0. DTT was then removed via extensive dialysis against 10 mM
KPO4, pH 8.0, at 4 °C. Spin labeling of MBPs was
accomplished by incubating 15 µM protein with 150 µM MTSSL (for D41C and S211C mutants) or 300 µM MTSSL (for D41C/S211C and wild type MBP proteins) for
2 h at room temperature in 10 mM KPO4, pH
8.0. Wild type MBP was "mock" spin-labeled to ensure that the spin
labeling procedure had no significant effect on the activity of the
MBP. After the MTSSL labeling, the solutions were dialyzed extensively
against 10 mM KPO4, pH 8.0, to remove excess
free MTSSL. After dialysis, MBP proteins were placed in 10 mM KPO4, pH 8.0, 15% glycerol, concentrated
using Microcon 10 filter units (Amicon, Inc.), and used immediately for
EPR experiments or substrate uptake assays. Final protein
concentrations were calculated using the UV extinction coefficient of
MBP of 1.7 (1 cm0.1%) at 280 nm (3)
and were between 250 and 400 µM.
EPR spectra were collected using a Bruker ESP 300 E spectrometer equipped with a loop-gap resonator (Medical Advances, Milwaukee, WI) and a low noise amplifier (Miteq, Hauppauge, NY). Room temperature spectra were collected using 1-milliwatt microwave power and a modulation amplitude of 1 gauss. Low temperature spectra were collected at 140 K using 8-milliwatt microwave power and a modulation amplitude of 2 gauss. Spin concentrations were determined by comparing doubly integrated spectra to that of a TEMPO standard solution. The labeling percentages were 90 ± 10% for both of the single mutants and 80 ± 10% for the double mutant.
Electron spin-spin interactions between two spin labels lead to EPR
spectral broadening in the case of interspin distances less than about
25 Å. However, quantitative analysis of data is difficult.
Alternatively, use of motionally frozen, unoriented samples near liquid
nitrogen temperature allows us to obtain interspin distances directly
from spectral analysis (11). This technique provides accurate estimates
for interspin distances in the range of 8-25 Å, and its application
to a model system (a series of spin-labeled -helical peptides) has
been described (11).
Our objective was to
examine the global conformational change that MBP undergoes upon
binding various ligands, through alteration of the distance between its
N- and C-terminal domains. We constructed a MBP double mutant that had
both Asp41 and Ser211 mutated to Cys. These two
residues were chosen for the following reasons. The Asp41
and Ser211 residues are located on the N- and C-terminal
lobes of MBP, respectively, and both are in close proximity of the
ligand binding site (Fig. 1). These residues are also on
the surface of MBP and, therefore, are presumably more accessible to
the labeling agent, MTSSL. The distance between these residues changes
from 24 to 15 Å (measured between -carbons) when MBP binds maltose
(Fig. 1). Finally, neither of these two residues interact directly with
maltose or
-cyclodextrin, as judged from the crystal structures of
MBP complexed with these ligands (5, 6).
The properties of MTSSL-modified D41C, S211C, and D41C/S211C mutant MBP proteins were examined to make certain that the site-directed mutations and the introduction of spin labels did not lead to functional alterations. Table I shows that neither the mock labeling of the wild type MBP nor MTSSL modification of mutant MBPs caused a large decrease of affinity when compared with the unmodified, wild type MBP. All MTSSL-modified MBPs were also capable of stimulating maltose transport into proteoliposomes containing the MalFGK2 maltose transport complex. Thus, unmodified wild type MBP, labeled D41C MBP, labeled S211C MBP, and labeled D41C/S211C MBP allowed the accumulation of 3.5, 4.8, 2.1, and 2.6 nmol of maltose in 5 min, under the conditions described under "Experimental Procedures." Background accumulation by proteoliposomes in the absence of either ATP or MBP was around 0.1 nmol in 5 min.
|
We also examined binding and transport activities of the mutant MBPs
without MTSSL modification. The D41C/S211C double mutant MBP seemed to
have slightly decreased affinity for all ligands tested (Table I).
However, the mutant MBP bound these ligands in the same manner as did
the wild type protein; maltose and maltotetraose bound via the R mode,
while maltotetraitol and -cyclodextrin bound by the B mode (data not
shown). We also examined the ability of this mutant MBP, when bound to
various ligands, to stimulate ATP hydrolysis and substrate transport
activities of MalFGK2 transport complex. Maltose, when
complexed with wild type or the double mutant MBP, stimulated ATP
hydrolysis activity in reconstituted proteoliposomes to 14 and 4 nmol/mg of protein/5 min, and maltotetraose produced somewhat higher
levels of stimulation with both types of binding proteins.
Maltotetraitol, which binds to both of these MBPs by the B mode, did
not stimulate ATP hydrolysis. The double mutant MBP was also able
to produce maltose transport in proteoliposomes, although the transport
rate was somewhat lower (1.8 nmol/mg of protein/5 min) in comparison
with that obtained by the addition of the wild type MBP (4.2 nmol/mg of
protein/5 min). Finally, strain HS2019 expressing either wild type or
D41C/S211C MBP grew on both maltose and maltotetraose at approximately
the same rate, but neither strain grew on maltotetraitol (data not
shown).
Fig. 2 (left
column) shows the EPR spectra, normalized to spin concentration,
for unliganded and various liganded forms of spin-labeled D41C/S211C
double mutant MBP. The increased spin-spin interactions in maltose- or
maltotetraose-liganded MBP are seen to have broadened EPR lines,
resulting in a reduction in the amplitudes of the first derivative
spectra in comparison with that of the unliganded MBP (Fig. 2,
a and c). This spectral broadening is more
clearly seen in the integrated spectra, which are shown in the
right column of Fig. 2 (see a and c).
In contrast, the addition of -cyclodextrin produced little effect
(Fig. 2b), and maltotetraitol produced an almost identical
spectra (Fig. 2d). All of the spectra contained a fast
motional component that was less than 5% of the total spin population
and was most likely caused by nonspecific spin labeling of impurities
in the MBP protein preparation.
Ligand binding did not affect mobilities of the nitroxide side chains,
since the addition of maltose, maltotetraose, -cyclodextrin, or
maltotetraitol produced no changes in the EPR spectra for either of the
spin-labeled single mutant MBPs (not shown). Therefore, the spectral
broadening that occurred upon the addition of maltose and maltotetraose
to the spin-labeled double mutant MBP must be the result of an increase
in spin-spin interactions.
At room temperature, broadening due to spin-spin interactions is the result of a complex interplay of exchange interactions, dynamic and static magnetic dipolar interactions, and molecular tumbling and is difficult to analyze quantitatively, although several methods aimed at estimating interspin distances at ambient temperatures have been proposed (10, 18, 19). Analysis of the room temperature EPR spectra was also made difficult by the fact that labeling of the MBP was not quite quantitative. The spin-labeled double mutant MBP contained 1.6 ± 0.1 spin labels/protein, and the observed changes in the EPR spectra were due to changes in spin-spin interactions in the fraction (approximately 60%) that was labeled at both sites.
Because of these complications, we did not attempt to analyze the room
temperature spectra in a quantitative manner, but the data of Fig. 2
nevertheless suggest the following: 1) binding of maltose and
maltotetraose produces an identical EPR change consistent with a
significant reduction in interspin distance; 2) binding of
maltotetraitol and -cyclodextrin produces EPR changes of a different
type, consistent with little reduction in interspin distance.
To determine quantitatively the change in distance between the nitroxides attached to two sites in the D41C/S211C mutant MBP, the EPR spectra at low temperature (140 K) were measured and analyzed using a Fourier deconvolution technique described previously (11). Recording the spectra in frozen solutions eliminates effects resulting from molecular tumbling, and the dipolar interaction becomes the dominant source of spectral broadening, allowing a convenient determination of interspin distances in the range of 8-25 Å. Furthermore, the analysis does not require completely quantitative labeling, and it can be applied in the presence of monoradical impurities.
Fig. 3 shows EPR spectra recorded at 140 K. Comparison
of the spectrum of the spin-labeled double mutant MBP with the average of the spectra for the singly labeled MBPs revealed dipolar broadening that is increased upon the addition of maltose (Fig. 3a).
The effects of the various ligands upon the EPR spectra follow the same
pattern as observed in the room temperature spectra; maltose and
maltotetraose produced the same effect (Fig. 3b), and the addition of either maltotetraitol or -cyclodextrin yielded little or
no additional broadening.
Fourier deconvolution analysis (11) showed that the interspin distance
changed from 16.5 ± 1 Å in the unliganded form to 10.5 ± 1 Å in the maltose-MBP complex, a substantial decrease. The spectra in
Fig. 3 all have a significant monoradical component, but in the Fourier
analysis this fraction is accounted for, since it results in a constant
y axis offset in the ratio of the concentrations of
interacting species to that of noninteracting species at
t = 0 in the Fourier space (Fig. 4). The
monoradical fraction was determined to be 40% by the Fourier analysis,
in good agreement with the spin labeling ratio determined by comparing
the spin count at room temperature to the protein concentration from UV measurements.
Intrinsic fluorescence spectra of MBP suggested the possibility that the conformation of MBP is altered in different ways by different ligands (1, 20). This possibility is supported also by UV differential absorbance spectroscopic studies (1, 21). It has thus become clear that maltose and excess linear maltodextrins bind in a way that produce a significant red shift of fluorescence emission spectra and other signature changes in UV absorbance spectra (R mode), whereas reduced, oxidized, or cyclic maltodextrins bind exclusively in a way that produces large blue shifts in fluorescence emission spectra (B mode) (1). Previous 3H NMR study, using maltose and maltodextrins 3H-labeled on the anomeric carbon of the reducing glucose moiety (3), showed the existence of two ligand binding modes, "end-on" and "middle," and we have indicated already the reasons why these two modes are likely to correspond to the R and B modes, respectively (1). Thus the end-on mode, which involves a tight interaction of the anomeric hydrogen with the binding site (3), is utilized only by ligands with the reducing glucose moiety. In contrast, ligands without reducing glucose residue, and therefore without anomeric hydrogen, such as reduced, oxidized, or cyclic maltodextrins, must use an alternative, middle (or B) binding mode.
These spectroscopic studies clearly showed that ligands can complex
with MBP in different ways, but they could not provide any information
on the global conformation of MBP. On the other hand, x-ray
crystallographic studies have shown that maltose binding causes the N-
and C-terminal domains of MBP to come closer together (5, 22).
-Cyclodextrin, in contrast, does not cause this conformational
change (6). As described in the preceding paper (1), we can hypothesize
that R and B modes of binding may generate global conformational
alterations of the type seen with the binding of maltose and
-cyclodextrin, respectively, a hypothesis substantiated by the
present study using EPR spectroscopy.
We measured the distance between the two lobes of MBP. Using a doubly
MTSSL-modified D41C/S211C MBP, which was spin-labeled in both the N-
and C-terminal domains, we have found that only those ligands that
complexed via the physiologically active R mode caused a marked
broadening in the EPR spectra (Figs. 2 and 3). This broadening effect
is most likely due to stronger spin-spin interactions between the two
spin labels and indicates that the two spin labels come closer to each
other when MBP binds ligands by this mode. Low temperature EPR measured
the change in distance to be from 16.5 to 10.5 Å. Although the crystal
structures predict the distance to change from 24 to 15 Å, much of the
difference between these two sets of figures is likely to be caused by
the fact that the crystallographic distance was measured between
-carbons, whereas the EPR method measured the distance between
paramagnetic centers of the spin label, which is located several
angstroms away from the
-carbon. We note that in both sets of
numbers the maltose-induced decrease was of a similar relative
magnitude (36 and 38%). One could argue that the observed broadening
and the speculated change in distance are due only to physical
perturbation of the spin labels due to ligand binding. However, the
fact that neither of the singly MTSSL-labeled MBPs (D41C and S211C)
exhibited broadening upon maltose binding argues against this
interpretation. It should also be emphasized that the doubly
spin-labeled MBP exhibited binding and transport properties very
similar to those of the wild type MBP (see Table I and
"Results").
Ligands that bound solely via the physiologically inactive B mode
(maltotetraitol and -cyclodextrin) caused little additional broadening in the EPR spectra of the doubly labeled MBPs (Figs. 2 and
3), a result suggesting that they did not cause the two spin labels
and, therefore, the two domains of MBP to close together. In other
words, maltotetraitol-bound MBP is in a global conformation similar to
the
-cyclodextrin-bound MBP, whose structure has been defined by
x-ray crystallography (6). However, maltotetraitol appeared to cause a
very slight broadening in the EPR spectra in comparison with
-cyclodextrin (Fig. 3c); it may therefore cause the two
lobes of MBP to come together a little. This is consistent with the
observation that maltotetraitol alters the fluorescence emission and UV
differential spectra of MBP in a direction caused by
-cyclodextrin,
but not as extensively (1). However, we cannot exclude the possibility
that these slight changes observed were due to the presence of small
amounts of unmodified maltotetraose in the maltotetraitol
preparation.
This study has made it possible to explain why certain ligands, although they bind to MBP with high affinity, are incapable of being transported across the cytoplasmic membrane. Ligands that bind exclusively by the B or middle mode do not induce the closing of the two lobes of MBP. Possibly only the MBP in the closed conformation can induce, when interacting with the periplasmic surface of the MalFGK2 transporter complex, the signals that allow the MalK ATPase to become active and hydrolyze ATP (15). This possibility is examined in the following paper (23).
The knowledge that the middle mode binding does not cause the closing
of the lobes of MBP explains much of the difference observed by
fluorescence and UV differential absorbance spectroscopy between
various ligands. X-ray crystallographic studies (5, 6, 22) showed that
several Trp and Tyr residues enter into stacking interactions with
pyranose rings of the ligands. Both -cyclodextrin and maltose
interact almost identically with Trp340 and
Tyr155 residues of MBP (6, 24). However, Trp62
and Trp230 interact very differently with ligands,
depending on whether the closure of the two lobes takes place. It
therefore seems likely that differences in fluorescence emission and UV
absorption between the two binding modes are ultimately caused by the
different degrees of quenching of these two Trp residues.
Why does the middle mode binding result in MBP remaining in an
"open" conformation? Decisive answers to this question obviously require crystallographic studies. However, a possible scenario can be
proposed. In the -cyclodextrin-MBP complex, three consecutive glucose residues of the ligand, labeled as g1, g2, and g3 in Fig. 1 of
Ref. 5, interact tightly with the binding site of MBP. It is thus clear
that there are at least three monosaccharide-binding sites, which we
may call sites 1-3. Maltotetraitol, for example, may be expected to
bind so that the three glucose residues of this ligand bind,
respectively, to sites 1-3 to maximize the stabilization. This,
however, will leave the linear, glucitol moiety unbound at the head,
which may produce steric hindrance for the closing of the two lobes,
just as the rest of the
-cyclodextrin molecule, including the g7
residue presumably at the position corresponding to glucitol in
maltotetraitol, may act in preventing the closing of the lobes (6).
We thank Pascale Duplay and Howard Shuman for the gifts of pPD1 and HS2019, respectively.