Solution Structure, Backbone Dynamics, and Stability of a Double Mutant Single-chain Monellin

STRUCTURAL ORIGIN OF SWEETNESS*

Yoon-Hui SungDagger , Joon ShinDagger , Ho-Jin Chang§, Joong Myung Cho§, and Weontae LeeDagger ||

From the Dagger  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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 alpha -helix and five-stranded antiparallel beta -sheet were identified for double mutant SCM. Strands beta 1 and beta 2 are connected by a small bulge, and the disruption of the first beta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 beta -sheet comprising five antiparallel strands and a single 17-residue long alpha -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 beta -sheet to alpha -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 alpha -helix was folded into the concave side of a six-stranded antiparallel beta -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 alpha -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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.
f<SUB><UP>O</UP></SUB>=<FR><NU><UP>Y<SUB>O</SUB></UP>−(<UP>Y<SUB>F</SUB></UP>+<UP>m<SUB>F</SUB></UP>[<UP>D</UP>])</NU><DE>(<UP>Y<SUB>U</SUB></UP>+<UP>m<SUB>U</SUB></UP>[<UP>D</UP>])−(<UP>Y<SUB>F</SUB></UP>+<UP>m<SUB>F</SUB></UP>[<UP>D</UP>])</DE></FR> (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.
f<SUB><UP>U</UP></SUB><UP>=</UP><FR><NU><UP>exp</UP>[<UP>−</UP>(<UP>&Dgr;G<SUB>U</SUB></UP>+<UP>m<SUB>G</SUB></UP>[<UP>D</UP>])<UP>/RT</UP>]</NU><DE><UP>1</UP>+<UP>exp</UP>[<UP>−</UP>(<UP>&Dgr;G<SUB>U</SUB></UP>+<UP>m<SUB>G</SUB></UP>[<UP>D</UP>]<UP>/RT</UP>]</DE></FR> (Eq. 2)
In Equation 2, fU is related to Delta 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.


&thgr;<SUB>&lgr;</SUB>=<FR><NU>&thgr;<SUB>&lgr;</SUB> · <UP>Mar</UP></NU><DE><UP>c</UP> · 1</DE></FR> (Eq. 3)
c is the protein concentration (in g/ml), l the light path length in the cell (in mm), theta lambda the measured ellipticity (in degrees) at wavelength lambda , 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.
f<SUB><UP>U</UP></SUB><UP>=</UP><FR><NU><UP>exp</UP>[<UP>−&Dgr;H</UP><SUP><UP>van</UP></SUP><SUB><UP>m</UP></SUB><UP>/R</UP>(<UP>1/T</UP>−1/<UP>T<SUB>m</SUB></UP>)]</NU><DE><UP>1</UP>+<UP>exp</UP>[<UP>−&Dgr;H</UP><SUP><UP>van</UP></SUP><SUB><UP>m</UP></SUB><UP>/R</UP>(<UP>1/T</UP>−1/<UP>T<SUB>m</SUB></UP>)]</DE></FR> (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 (pi /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 Phi -angles within elements of secondary structure based on 3JHNalpha coupling-constants (3JHNalpha  > 8, 120 (± 50)°, 3JHNalpha  < 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
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 beta -strand and minor alpha -helical contents. However, a small difference at 217 nm was clearly detected, indicating that the structural change of the beta -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 (open circle ), 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 (< <OVL>SA</OVL>> 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 < <OVL>SA</OVL>> k structures of SCMDR. The deviations from idealized geometry are also very small and satisfy ideal geometry. The < <OVL>SA</OVL>> kr structure clearly demonstrates the relative orientations of its major secondary structures, showing that the twisted beta -sheet partially wrapped the beginning of the alpha -helix. Especially, the bend of three strands (beta 2, beta 3, and beta 4) enable to have a close contacts with the alpha -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 Phi , Psi , which indicates the degree of dihedral heterogeneity of structures, showed that the Phi , Psi  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 Phi , Psi  for the < <OVL>SA</OVL>> kr ensured that all of the Phi , Psi  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 alpha -helix and a five-stranded antiparallel beta -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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Table I
Structural statistics for the 20 final simulated annealing structures of SCMDR

                              
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Table II
Atomic root mean square deviations for the final simulated annealing structures of SCMDR (91 residues)


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Fig. 3.   Steroview of the backbone superposition of the energy-minimized average structure (< <OVL>SA</OVL>> 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 beta -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.


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Fig. 5.   Comparison of the backbone conformation for the SCMDR (red) and wild type SCM (yellow).

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 (open circle ) 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, Dperp /D||, were also calculated. The starting values of the parameters tau m and Dperp /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 beta -strands and above 0.98 for alpha -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 tau 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, tau e, derived from model-free fits; F, generalized order parameter S2 of wild type SCM. The alpha -helical region is represented by a black bar, the beta -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 beta -strands. The first short beta -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 Calpha H chemical shift indices did not provide any evidence of support beta -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 beta -strand. The main structural differences are the following; each beta -strand encompasses 6 residues from Cys41 to Ile46 for a second beta -strand, 9 residues from Gly55 to Tyr63 for a third, and 6 residues from Arg70 to Glu75 for a fourth beta -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 beta -strand in wild type SCM. Several reports have already proposed that a sulfhydryl group of Cys41 in the beginning of beta 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 beta -sheet and alpha -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. 8.   Relative sweet activities of various mutant SCM proteins.


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Fig. 9.   The thermal unfolding transitions of wild type SCM (Tm = 359 K, ) and SCMDR (Tm = 348 K, open circle ) 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.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

1. van der Wel, H. (1976) Biochem. Sensory Funct. 197, 235-242
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3. Brouwer, J. N., Hellekant, G., Kasahara, Y., van der Wel, H., and Zotterman, Y. (1973) Acta Physiol. Scand. 89, 550-557[Medline] [Order article via Infotrieve]
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