Solution Structure, Backbone Dynamics, and Stability of a Double
Mutant Single-chain Monellin
STRUCTURAL ORIGIN OF SWEETNESS*
Yoon-Hui
Sung
,
Joon
Shin
,
Ho-Jin
Chang§,
Joong Myung
Cho§¶, and
Weontae
Lee
From the
Department of Biochemistry and Protein
Network Research Center, College of Science, Yonsei University, Seoul
120-740 and the § Biotech Research Institute, LG Chemicals,
Research Park, P.O. Box 61, Yu-Sung, Science Town,
Taejon 305-380, Korea
Received for publication, January 31, 2001, and in revised form, March 5, 2001
 |
ABSTRACT |
Single-chain monellin (SCM), which is an
engineered 94-residue polypeptide, has been characterized as being as
sweet as native two-chain monellin. Data from gel-filtration high
performance liquid chromatography and NMR has proven that SCM exists as
a monomer in aqueous solution. In order to determine the structural origin of the taste of sweetness, we engineered several mutant SCM
proteins by mutating Glu2, Asp7, and
Arg39 residues, which are responsible for sweetness. In
this study, we present the solution structure, backbone dynamics, and
stability of mutant SCM proteins using circular dichroism,
fluorescence, and NMR spectroscopy. Based on the NMR data, a stable
-helix and five-stranded antiparallel
-sheet were identified for
double mutant SCM. Strands
1 and
2 are connected by a small
bulge, and the disruption of the first
-strand were observed with
SCMDR comprising residues of
Ile38-Cys41. The dynamical and folding
characteristics from circular dichroism, fluorescence, and backbone
dynamics studies revealed that both wild type and mutant proteins
showed distinct dynamical as well as stability differences, suggesting
the important role of mutated residues in the sweet taste of SCM. Our
results will provide an insight into the structural origin of sweet
taste as well as the mutational effect in the stability of the
engineered sweet protein SCM.
 |
INTRODUCTION |
The native sweet protein, monellin, which was originally isolated
from the berries of the West African plant Dioscoreophyllum cumminsii (1, 2), consists of two separate polypeptide chains: an
A chain of 45 residues, and a B chain of 50 residues. Native two-chain
monellin is ~70,000 times sweeter than sucrose and about 300 times
sweeter than the dipeptide sweetener aspartame (3, 4). Other sweet
taste proteins, such as thaumatin, pentadin, and mabinlin, are also
known (5-9). Among these sweet proteins, a curculin protein has
demonstrated a sweet taste and shown taste-modifying activity (10). The
crystal structure of native two-chain monellin has been determined as
showing a
-sheet comprising five antiparallel strands and a single
17-residue long
-helix. The two chains were packed closely by
hydrogen bonds and hydrophobic interactions (11). In addition, the
crystal structure showed that the amino terminus of the A chain was
connected to the carboxyl terminus of the B chain through
intermolecular hydrogen bond networks.
Recent biochemical studies have reported that the A chain of the
alcohol-denatured state of native monellin performed a structural reorganization from
-sheet to
-helix conversion in 50% ethanol and 50% trifluoroethanol environments (12). In addition, the conformational study for both native and mutated non-sweet analog two-chain monellin have been studied by two-dimensional nuclear magnetic resonance spectroscopy; these studies have shown that the
three-dimensional structures of native monellin and two thiol proteinase inhibitors, cystatin and stefin B, are very similar (13).
These structural homology data indicated that monellin might play some
other biological role in addition to its sweetness.
An engineered 94-residue single-chain monellin
(SCM),1 which was recently
constructed by fusing the two chains to retain the topology of monellin
(14), has proven to be as sweet as native monellin. Interestingly, SCM
was more stable than the native two-chain monellin for both heat and
acidic environments (14). Very recently, two-dimensional 1H
NMR (15) and heteronuclear three-dimensional NMR studies (16) for
recombinant SCM have been performed as a monomer conformation in
solution state. The solution structure of SCM revealed that the long
-helix was folded into the concave side of a six-stranded antiparallel
-sheet and the common residues for all sweet peptides were mostly solvent-exposed. Interestingly, most of the residues involved in the sweet taste of monellin are found on the same surface
of the molecule. In addition, the solution structure suggested that the
relative orientation of the single
-helix might be responsible for
the global topology of the molecule. The flexibilities of the side
chains were also important for both sweet taste and receptor binding
(16). It is still not clear, however, whether SCM requires conformational preference for sweet taste, even though there is no
doubt that the solution structure is responsible for binding as well as
for sweet taste. Presented here are the solution structure, dynamical
properties, and protein stability of single-chain monellins by
heteronuclear NMR, circular dichroism, and fluorescence spectroscopy.
 |
EXPERIMENTAL PROCEDURES |
Expression and Purification of Wild Type and Mutant SCM--
The
recombinant SCM proteins were expressed in Escherichia coli
strain BL21 (DE3) containing the plasmid pET21. Transformed cells were
propagated in E. coli nitrogen base containing 5% glucose and 0.5% ammonium sulfate at 30 °C for 2 h and grown in M9
media containing 2% glucose and 0.1% ammonium sulfate at 30 °C for
48 h.15N-Labeled ammonium sulfate was used as the sole
source of the nitrogen for uniformly 15N-labeled
SCMDR and wild type SCM. The cells were harvested by
centrifugation at 3500 rpm for 25 min. Cells were stored at
80 °C
and used for purification procedures. Cell pastes were disrupted by a
bead beater in 25 mM sodium phosphate, 5 mM
EDTA, 150 mM NaCl, and 1 mM
phenylmethylsulfonyl fluoride at pH 7.0. The cell lysates were
collected by centrifugation at 12,000 rpm for 215 min. After pH
adjustment and centrifugation, the supernatants were diluted with 10 mM sodium phosphate and loaded onto a CM-Sepharose column. The bound SCM was eluted with a salt gradient. The collected protein solution was dialyzed and dried with a freeze-dryer for spectroscopic measurements. The protein concentration was determined using the Bradford method.
Fluorescence Spectroscopy--
Fluorescence spectra were
measured in 50 mM potassium phosphate buffer, at pH 7.0 and
25 °C, on F-4500 fluorescence spectrophotometer. Fluorescence
emission spectra were recorded from 270 to 450 nm at each GdnHCl
concentration using two different excitation wavelengths, 280 and 295 nm. The protein concentration in the cuvette was 30 µM,
and a path length of 1 cm was used. GdnHCl-unfolding experiments were
carried out after the protein was incubated in solutions containing
different concentrations of the denaturant for 24 h, at 25 °C.
The refolding reaction of SCM was carried out under various conditions
by diluting the denaturant concentration.
Data from the equilibrium denaturation were converted to plots of
fU versus denaturant concentration
using Equation 1.
|
(Eq. 1)
|
YO is the observed signal at a particular
GdnHCl concentration. YF and
YU represent the intercepts, and
mF and mU are the slopes
of the native protein and the unfolded base lines. They were obtained
by extrapolation of linear least-squares fits of the base lines.
To determine whether the two-state unfolding model was appropriate for
analyzing the GdnHCl-induced denaturation data,
fU values were fitted to Equation 2.
|
(Eq. 2)
|
In Equation 2, fU is related to
GU by a transformation of the Gibbs-Helmholtz
equation in which the equilibrium constant for unfolding in the folding
transition zone, KU, is given by KU = fU/(1
fU), for a two-state transition. It is also
implicit in Equation 2 that the free energy of unfolding is dependent
linearly on denaturant concentration (17).
CD Spectroscopy--
CD spectra were measured in 50 mM potassium phosphate buffer, at pH 7.0 and 25 °C on a
Jasco 720 spectropolarimeter. Far-UV CD spectra were monitored from 190 to 250 nm using a protein concentration of 30 µM with a
path length of 0.1 mm, 20-millidegree sensitivity, response time of
1 s, and scan speed of 50 nm/min. The spectra were recorded as a
six-scan average value. The molar ellipticity was determined as shown
in Equation 3.
|
(Eq. 3)
|
c is the protein concentration (in g/ml),
l the light path length in the cell (in mm),

the measured ellipticity (in degrees) at
wavelength
, and Mar the mean molecular mass
of amino acid of the protein determined from its amino acid sequence.
The temperature scanning CD measurements were carried out with a Jasco
715 spectropolarimeter from 25 °C to 100 °C using a cuvette of
0.2 mm for 222 nm wavelength. The heating rates were 30 °C/h with a
step interval of 0.1 °C. Full CD spectra were collected at 20 °C,
40 °C, 60 °C, 70 °C, and 80 °C for far-UV region. Noise reduction was applied to thermal scan profiles of CD spectra and for
determination of the transition midpoint temperatures
(Tm), transitions analyzed on the basis of the
two-state approximation were fitted to the following relation derived
from the van't Hoff equation.
|
(Eq. 4)
|
Reversibility was examined by comparing the transition curves of
a sample that was briefly heated to a temperature where the protein was
completely unfolded.
NMR Spectroscopy--
All NMR spectra were acquired on a
Bruker DRX-500 spectrometer in quadrature detection mode, equipped with
a triple-resonance probe with an actively shielded pulsed field
gradient coil. All two-dimensional experiments were performed at 298 K. Pulsed-field gradient techniques were used for all H2O
experiments, resulting in good suppression of the solvent signal.
15N-1H HSQC (18, 19) spectra were recorded with
a uniformly 15N-labeled sample with 2048 complex data
points in t2 and 256 t1 increments. Two-dimensional nuclear Overhauser effect spectroscopy (NOESY) (20, 21) spectra were recorded with mixing times with 100 ms
and 150 ms in H2O and D2O solution.
Two-dimensional total correlation spectroscopy (TOCSY) (22) spectrum
was acquired in H2O solution with a mixing time of 69.668 ms using MLEV17 spin lock pulses. 15N-edited NOESY-HSQC
(23) with a mixing time of 100 ms and 15N-edited TOCSY-HSQC
(24) with a mixing time of 69.668 ms spectra for uniformly
15N-labeled SCMDR were recorded.
15N-Edited HNHA (25) and double quantum-filtered (DQF) COSY
(26) spectra were collected to get vicinal coupling constant values. The 1H spectra were referenced to the water resonance at
4.76 ppm. Longitudinal (R1) and transversal
(R2) relaxation data for the backbone
15N nuclei of wild type SCM were recorded as 2048 × 64 data sets with 64 scans/point using 1 s of a recovery delay.
Seven different values for the relaxation time were used:
t1 = 5, 65, 145, 246, 366, 527, and 757 ms and
t2 = 8.3, 25.1, 41.8, 58.6, 75.3, 108.8, and
142.3 ms. To permit estimation of noise levels, duplicate spectra were
recorded for t = 246 ms (t1
spectra) and t = 56.8 ms (t2
spectra). In order to eliminate the effects of cross correlation between 15N-1H dipolar and 15N
chemical shift anisotropy relaxation mechanisms, 1H
180° pulses were inserted during the relaxation time according to the
published methods (27, 28). 15N-(1H)
steady-state heteronuclear NOE (XNOE) (29, 30) data were also obtained
using a relaxation delay of 5 s.
NMR Data Processing and Analysis--
The NMR data were
processed using the nmrPipe/NMRDraw software packages (Biosym/Molecular
Simulations, Inc.) and analyzed using Sparky 3.60 software. The
experimental data were extended by linear prediction and zero-filled to
give 2048 × 512 data matrices and processed using gaussian
multiplication and a shifted (
/3) sine bell function prior to
Fourier transformation. The peak intensities in the two-dimensional
spectra were measured by peak heights using the Sparky program. The
XNOE value for a given residue was calculated as the intensity ratio
(I/I0) of the
15N-1H correlation peak in the presence
(I) and absence (I0) of 3-s proton
saturation. The standard deviations of these values were measured
background noise levels. Relaxation rates were determined by nonlinear
fits of the time dependence of the peak intensities. In addition, Monte
Carlo simulations were performed to estimate the uncertainty of the
relaxation parameters.
Experimental Constraints and Structure
Calculations--
Structures were generated using hybrid distance
geometry and dynamical simulated annealing protocol with the CNS 1.0 program on a SGI Indigo2 work station. Our methodology was
similar to that used by Clore and Gronenborn (31, 32) and their
co-workers. Distance geometry substructures were generated using a
subset of atoms in the peptide and followed a refinement protocol
described by Lee et al. (33). The target function for
molecular dynamics and energy minimization consisted of a covalent
structure, van der Waals repulsion, NOE, and torsion angle constraints
(34). The torsion angle and NOE constraints were represented by
square-well potentials. Based on cross-peak intensities in the NOESY
spectra with mixing times of 100 and 150 ms, the distance restraints
were then classified as strong, medium, or weak corresponding to upper
distance bounds of 2.7, 3.3, and 5.0 Å, respectively. An additional 1 Å was added to upper distance bounds for pseudoatom involving
non-stereospecifically assigned methylene protons, methyl groups, and
the ring protons of phenylalanine residue (35). A lower distance bound
of 1.8 Å was used for all NOE-derived distance restraints. Structures were calculated using 296 intraresidues, 245 sequential, 124 medium range, and 331 long range NOE restraints. A total of 76 hydrogen bond
restraints were also included in the calculations. Potential hydrogen
bond donors were assigned from a 1H-15N HSQC
spectrum recorded immediately after dissolving lyophilized H2O sample to 100% D2O solution. Hydrogen
bonds were further identified from characteristic NOE patterns that
were observed for residues in regular secondary structure, together
with the solvent exchange data. From two-dimensional DQF-COSY (36, 37)
and 15N-edited HNHA spectra, 65 torsion angle restraints
were also derived for backbone
-angles within elements of secondary
structure based on 3JHN
coupling-constants (3JHN
> 8, 120 (± 50)°, 3JHN
< 6, 60 (±45)°). Distance geometry substructures were generated using a
subset of atoms in the peptide, and followed a refinement protocol
described by Lee et al. (33). All modeling calculations were
performed within the InsightII program (Biosym/Molecular Simulations,
Inc.) on a SGI Indigo2 work station.
 |
RESULTS AND DISCUSSION |
Circular Dichroism--
Circular dichroic spectra of both wild
type and double mutant proteins in the far-UV region were collected in
50 mM sodium phosphate buffer solution at pH 7.0. The
spectra suggest that the global folding of the two proteins are
similar, showing a major
-strand and minor
-helical contents.
However, a small difference at 217 nm was clearly detected, indicating
that the structural change of the
-sheet region was due to double
mutation of SCM. The additional minima observed at 206-208 nm have
been ascribed from the contributions of aromatic side chains (38, 39)
(Fig. 1).

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Fig. 1.
CD spectra of mutant SCMDR and
wild type SCM at far-UV region from 190 to 250 nm at pH 7.0. The
symbols represent SCMDR ( ), and wild type SCM
( ).
|
|
Solution Structures and Sweet Taste--
Spin system
assignments were easily made by homonuclear two-dimensional TOCSY and
15N-edited 3D TOCSY-HSQC spectra. All identified spin
systems served as a starting point for complete sequence-specific
resonance assignment procedure. Fig. 2
shows two-dimensional 1H-15N HSQC spectrum with
the assignments. A total of 50 substructures generated from distance
geometry algorithms were used as starting structures in the simulated
annealing stage. After simulated annealing calculations, the 20 structures (
SA
k) showed no constraint
violations greater than 0.5 Å for distances and 5° for torsional
angles. These structures were used for detailed structural analysis.
Table I shows the energy and structural
statistics of the final 20 simulated annealing structures related to
experimental constraints. The final
SA
k
structures were well converged with a root mean square deviation of
0.96 Å for all backbone atoms, 0.61 Å for backbone atoms except for
disordered regions (Gly1-Trp3,
Glu48-Ile53, and
Asp76-Arg82), and 0.49 Å for residues
involved in secondary structures. The REM average structure
(

kr) from 20 final structures
exhibited a root mean square deviation of 0.99 Å for backbone
atoms with respect to 20
SA
k
structures and 0.64 Å for backbone atoms excluding unstructured
regions (Gly1-Trp3,
Glu48-Ile53,
Asp76-Arg82). Table
II summarizes the structural statistics
associated with 20 final 

k
structures of SCMDR. The deviations from idealized
geometry are also very small and satisfy ideal geometry. The


kr structure clearly
demonstrates the relative orientations of its major secondary
structures, showing that the twisted
-sheet partially wrapped the
beginning of the
-helix. Especially, the bend of three strands
(
2,
3, and
4) enable to have a close contacts with the
-helix. A best fit backbone superposition of all final
SA
k structures with an average REM structure is displayed in Fig. 3. The
angular order parameter for
,
, which indicates the degree of
dihedral heterogeneity of structures, showed that the
,
values
of all residues except prolines and residues of
Gly30-Thr33 and Ile8 in loop
regions are close to 1, suggesting the well defined backbone angles of
SCMDR (data not shown). The backbone torsion angles
,
for the 

kr ensured that all
of the
,
values of final 20 structures were distributed in
energetically favorable regions. The ribbon diagram of the REM average
structure clearly shows that the general topology consisted of both a
single long
-helix and a five-stranded antiparallel
-sheet (Fig.
4). Five loops including an engineered
one were also characterized. The side chain orientations of
Tyr63 and Asp66, which are common to all sweet
peptides and correspond to Phe and Asp residues of aspartame, were
observed on the opposite side to the H1 helix and those that were
mostly exposed to solvent.

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Fig. 2.
The two-dimensional
1H-15N HSQC spectrum of uniformly
15N-labeled double mutant SCMDR. Two
boxes are indicated as mutated residues. Indole NH
cross-peak from Trp3 residue is marked as W(NH),
and cross-peaks connected by dotted lines
correspond to side chain NH2 groups of Gln and Asn
residues.
|
|

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Fig. 3.
Steroview of the backbone
superposition of the energy-minimized average structure
(  kr, thick
line) over the family of 20 final simulated annealing
structures ( SA k, thin
line).
|
|

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Fig. 4.
Ribbon diagram of the REM average structure
displaying ordered secondary structure elements and relative
orientation of secondary structures. The side chain atoms for
mutated residues are also displayed. The figure was generated with
MOLSCRIPT.
|
|
Fig. 5 displays a best-fit superposition
of REM average solution structure of SCMDR over that of the
wild type. A high flexibility around an engineered loop and structural
rearrangements of
-sheet due to double mutation were observed, even
though the overall three-dimensional structure of the SCMDR
is similar to that of the wild type.
Stability and Folding of SCMDR Based on Fluorescence
Data--
The emission spectra of the SCM at an excitation wavelength
of 280 nm in the various GdnHCl concentrations are shown in Fig. 6A. The emission spectrum of
the SCM was dominated by the tryptophan fluorescence. In the unfolding
process, spectra exhibited two emission peaks, corresponding to the
contribution of tyrosine and tryptophan residues based on the
tyrosine-tryptophan energy transfer mechanism. The efficiency of energy
transfer can be used to estimate the distances between the donor and
acceptor. The fluorescence energy transfer transition curve was
measured using denaturant GdnHCl and the ratio of fluorescence
intensities at 298 nm corresponding to tyrosine fluorescence and at 350 nm to tryptophan fluorescence. It can be seen that GdnHCl-induced
denaturation begins at 2.0 M concentration and fully
denatures at 3.4 M GdnHCl. Fig. 6B shows that
the fractional change of unfolding suggests a two-state model for
GdnHCl-induced denaturation of both SCMDR and wild type
protein. However, the transition midpoint of the double mutant SCM has
shifted slightly toward the left, implying that the double mutation
induces destabilization of the protein.

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Fig. 6.
A, fluorescence emission spectra of
SCMDR on excitation at 295 nm with various GdnHCl
concentrations. B, equilibrium denaturation curves of
SCMDR ( ) and wild type SCM ( ). All amplitudes are
relative to a value of 0 to fU. The
solid lines are non-linear extrapolation of the
equilibrium unfolding data to two-state F-U transitions.
|
|
In each case, the reversibility of the unfolding reaction was
confirmed by obtaining a refolding curve through dilution of the
protein from high GdnHCl concentration. The denaturation and renaturation curves are determined to be exactly superimposable.
Backbone Dynamics of SCMDR--
The rotational
diffusion anisotropy was estimated for each residue, yielding a ratio
of the principal moments of inertia of SCMDR as
1.00:0.86:0.40. These data suggest that the global shape of SCMDR is a prolate ellipsoid. Using data from
R2/R1 ratios and
structural coordinates of 66 selected residues, axial diffusion
tensors, D
/D
, were also
calculated. The starting values of the parameters
m and
D
/D
were 4.9 ns and
0.65 at 500 MHz, respectively. The standard values of
R1, R2, and
15N-(1H) NOEs were fitted in the model-free
system selected by an F-test. Fig.
7 demonstrates that the regions of the
secondary structures showed higher S2 values
than those of loops, as we expected. The average values of
S2 are above 0.94 for most residues in
-strands and above 0.98 for
-helical region. More than 80% of
the residues in SCMDR have order parameter
S2 values greater than 0.8, indicating that the
protein in general is relatively rigid. Five residues
(Glu4, Ile5, Thr33,
Tyr47, and Arg82) belonged to the loop regions
showed relatively higher
R2/R1 ratios, originated
from a significant chemical exchange contribution (Fig. 7A).
In addition to this exchange contribution, residues of Glu4
and Ile6 exhibited a significant reduction in the
15N-(1H) NOEs and S2
values of 0.7 and 0.69 (Fig. 7B). Thus, it can be supposed
that both Glu4 and Ile5 residues might be
involved in dynamical motions. Four residues of Ile5,
Tyr47, Arg51, and Asp66 located in
the loop regions contained
e contributions, indicating enhanced backbone dynamics on a fast time scale.

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Fig. 7.
15N NMR relaxation parameters and
model-free analysis of SCMDR (A-E) and
wild type SCM (F). A and B,
R2/R1 (A) and XNOE (B)
values plotted against the number of residue; C, generalized
order parameter S2; D,
Rex exchange broadening; E, effective
correlation times, e, derived from model-free fits;
F, generalized order parameter S2 of
wild type SCM. The -helical region is represented by a
black bar, the -strand region by a
gray bar, and the loop region by a
white bar.
|
|
In the previous report, we determined that SCM exists as a monomer
conformation based on NMR and gel-filtration experiments. Solution
structure suggested that Arg70 and Arg86
residues have a close correlation with the degree of sweetness of SCM
protein. A comparison of the secondary structure of
SCMDR with wild type protein indicates that the
structural difference between the two proteins can be observed mainly
in
-strands. The first short
-strand composed of residues
Glu2-Ile5 found in the wild type SCM was not
detected in SCMDR. Amide hydrogen exchange data,
backbone-backbone NOEs, and C
H chemical shift indices
did not provide any evidence of support
-strand in this region.
Therefore, it can be supposed that a mutation of Asp7
disrupts the first short strand, perturbating the stability of the
network of
-strand. The main structural differences are the following; each
-strand encompasses 6 residues from
Cys41 to Ile46 for a second
-strand, 9 residues from Gly55 to Tyr63 for a third, and 6 residues from Arg70 to Glu75 for a fourth
-strand, whereas 7 residues from Cys41 to
Tyr47, 11 residues from Lys54 to
Ala64, and 7 residues from Phe69 to
Glu75 constitute each third, fourth, and fifth
-strand
in wild type SCM. Several reports have already proposed that a
sulfhydryl group of Cys41 in the beginning of
3 strand
could be critical for sweetness. In our solution structures of both
wild type SCM and SCMDR, the side chain of
Cys41 is located on the hydrophobic interface between
-sheet and
-helix. We propose that the side chain of
Cys41 plays a role for sweetness because it maintains a
bulge between Ile38 and Cys41 responsible for
structural organization, especially the H1 helix orientation and,
furthermore, the tertiary structure of single chain monellin. We also
proposed that hydrophobic and/or side chain-side chain interaction
related to tertiary structure of SCM is in part responsible for
sweetness. Mutational studies of SCM proteins have supported our
structural data, showing that the size of Asp7 residue in
loop A is important for sweetness (Fig.
8). Our structure showed that
Asp7 and Arg39 residues, which reside on the
same surface of the protein, are involved in charge-charge interaction,
causing structural instability from mutations of these residues. This
instability is also proven from the data of thermal unfolding
experiments of SCM proteins (Fig. 9). The
midpoint of denaturation temperature (Tm) of
SCMDR was determined to be significantly less than that of
the wild type. In addition, our data suggest that the secondary
structures and stability of tertiary structure were closely correlated
with mutations of Asp7 and Arg39 residues. We
thought that the H1 helix would be responsible for the general topology
as well as the side chain orientations of the key residues involved in
sweetness. Therefore, we can conclude that, even though amino acids
located in the loop regions are mainly involved in biological activity
of monellin, both the relative orientations of those side chains based
on tertiary structure and protein stability are of importance for the
sweet taste of the SCM protein.

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Fig. 9.
The thermal unfolding transitions of wild
type SCM (Tm = 359 K, ) and SCMDR
(Tm = 348 K, )
monitored by the decrease of the CD signal at 222 nm are
displayed. The results of the analysis based on a two-state model
are shown by the solid lines.
|
|
 |
ACKNOWLEDGEMENTS |
We thank TMSI Korea for use of the molecular
simulation programs (Molecular Modeling Tools, Molecular Simulations,
Inc.). We also thank Dr. Dan Garrett (National Institutes of Health, Bethesda, MD) for providing the programs PIPP and CAPP.
 |
FOOTNOTES |
*
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.
The atomic coordinates and the structure factors (code 1FUW) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶
Current address: CrystalGenomics, Inc., Daeduck Biocommunity,
Taejon 305-390, Korea.
To whom correspondence should be addressed: Dept. of
Biochemistry, College of Science, Yonsei University, Seodaemoon-Gu,
Shinchon-Dong, Seoul 120-740, Korea. Tel.: 82-2-2123-2706; Fax:
82-2-362-9897; E-mail: wlee@biochem.yonsei.ac.kr.
Published, JBC Papers in Press, March 7, 2001, DOI 10.1074/jbc.M100930200
 |
ABBREVIATIONS |
The abbreviations used are:
SCM, single-chain
monellin;
NOE, nuclear Overhauser effect;
NOESY, nuclear Overhauser
effect spectroscopy;
TOCSY, total correlated spectroscopy;
DQF-COSY, double quantum-filtered correlated spectroscopy;
HSQC, heteronuclear
single quantum coherence;
SA, simulated annealing;
XNOE, heteronuclear nuclear Overhauser effect;
GdnHCl, guanidine
hydrochloride;
REM, restrained energy minimized.
 |
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