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The predicted unfolding order of the β-strands in the starch binding domain from Aspergillus niger glucoamylase
Chemical Physics Letters 366 (2002) 284–290 www.elsevier.com/locate/cplett
The predicted unfolding order of the b-strands in the starch binding domain from Aspergillus niger glucoamylase Hsuan-Liang Liu *, Wen-Chi Wang Department of Chemical Engineering, National Taipei University of Technology, No. 1 Sec. 3 Chung-Hsiao E. Rd., Taipei 106, Taiwan Received 11 July 2002; in final form 20 August 2002
Abstract The unfolding of the b-strands in the starch binding domain from Aspergillus niger glucoamylase was predicted to follow the order of b3 ! b2 ! b6 ! b5 ! b4 ! b1 ! b7 by 600 ps molecular dynamics simulations at 300, 400, and 600 K. The interior region around b-strands 2 and 3 acts as the initiation site for unfolding. b-Strands 1 and 7 are probably stabilized by the disulfide bond formed between Cys509 and Cys604. b-Strand 4 is stabilized by forming an antiparallel b-sheet with b-strand 1. Hydrophobic and electrostatic interactions between side chains instead of the hydrogen bonds are important in stabilizing these b-strands, thus the entire starch binding domain. Ó 2002 Elsevier Science B.V. All rights reserved.
1. Introduction Glucoamylase (1,4-a-D -glucan glucohydrolase, EC 3.2.1.3, GA) is one of the most widely used enzyme in industry [1]. Aspergillus niger GA is composed of three functional domains: (1) a globular N-terminal catalytic domain (CD) from residue 1 to 470; (2) a linear and extended linker domain from residue 471 to 508; and (3) a globular C-terminal starch binding domain (SBD) from residue 509–616 [2]. While the 3D structure of the CD from A. awamori var. X100 GA has been determined by X-ray crystallography in its native state [3], the solution structure of the SBD from A. niger GA in its native state has been
*
Corresponding author. Fax: +886-2-2731-7117. E-mail address: [email protected] (H.-L. Liu).
solved by NMR spectroscopy [4]. The SBD is a well defined b-sheet structure consisting of 7 b-strands and 1 a-helix according to the Kabsch– Sander algorithm [5] (Fig. 1), which form one parallel and five antiparallel pairs of b-sheets [4]. The SBD allows GA to adsorb to and digest raw starch [6] and some other substrates [7] without affecting the catalytic behavior of the CD toward soluble substrates [8]. The presence of this domain in a range of hydrolases clearly highlights its functional importance. Although many efforts have been made to improve the substrate selectivity [9–12] and thermostability [13–19] of GA for better industrial applications, not too much attention has been paid to investigate the functional and structural features of the SBD so far. GA undergoes irreversible thermoinactivation at moderately acidic pH and 70 °C [19]. Neither deamidation [20,21] nor Asp-X
0009-2614/02/$ - see front matter Ó 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 6 1 4 ( 0 2 ) 0 1 4 2 5 - 2
H.-L. Liu, W.-C. Wang / Chemical Physics Letters 366 (2002) 284–290
285
Fig. 1. The NMR structure of the SBD binding to b-cyclodextrins (ball and stick representation) obtained from the PDB (PDB entry: 1KUM). b-strands 1 to 7 predicted by Kabsch–Sander algorithm [5] are shown in arrows pointing from N- to C-terminus. The starch binding sites 1 and 2 are indicated as S1 and S2, respectively.
peptide bond hydrolysis [14] determines the thermoinactivation rate. In addition, mismatched disulfide bonds are not observed at 70 °C and pH 3.5 and 4.5 [19]. These results indicate that some other mechanism must account for the thermoinactivation of the GA at high temperatures. The mechanism appears to be a change in the GAÕs secondary or tertiary structure, such as unfolding that destroys the integrity of the CD and the SBD [19,22]. In the present study, 600 ps molecular dynamics (MD) simulations were conducted to predict the unfolding order of the 7 b-strands in the SBD from A. niger GA with temperature jump technique [23,24].
As the computer power has been increased in recent years, MD simulation has been intensively employed to gain insight into protein folding/unfolding processes [25]. The large number of interatomic interactions in force fields that have been taken into account for a realistic description of a protein in solution has made MD technique a promising tool in examining the early events in protein folding/unfolding. Unfortunately, the fs time-step for the integration of the equation of motion limits the MD simulations to an extremely small time-span (e.g., ns or ls), which is relatively short comparing to the second time-scale of the real protein folding/unfolding processes. Thus,
286
H.-L. Liu, W.-C. Wang / Chemical Physics Letters 366 (2002) 284–290
instead of predicting protein folding processes directly, a lot of attention has been paid on the unfolding simulations, in which the unfolding of a protein in its native-like structure is usually initiated by raising the temperature or induced by changing the solvent [26]. Instead of simulating the full-length SBD using an all-atom model, which may be limited by the current computer power, the present MD simulations were performed in a mean external field with dielectric constant (DC) of 80 [27].
2. Methods The initial NMR structure of the SBD, residues 509–616 from A. niger GA, was taken from the Protein Data Bank (PDB entry 1KUM) [4]. The energy minimization and MD simulations were performed by the Insight II program (Accelyrs, San Diego, CA, USA) with the force field Discover CVFF (consistent valence force field) [28] in the SGI O200 workstation (Silicon Graphics, Mountain View, CA, USA) with 64-bit HIPS
RISC R12000 270 MHz CPU and PMC-Sierra RM7000A 350 MHz processor. The NMR structure of the SBD was subjected to energy minimization calculations in an external field with DC ¼ 80 by steepest descent method with 5000 iterations to be used as the lowest energy starting structure for further structural analysis. The starting structure was then subjected to 600 ps MD simulations at DC ¼ 80 after equilibrating for about 10 ps at pH 5.2 and 300, 400, and 600 K using the Discover module. The temperature and pressure were maintained for each MD simulation by weak coupling the system to a heat bath at the simulation temperature and an external pressure bath at one atmosphere, respectively, according to themethod described by Berendsen et al. [29]. The time-step of the MD simulations was 1 fs. The trajectories and coordinates of the SBD were saved every 5 ps for further analysis. For each MD simulation, the root-mean-square deviations (RMSDs) of the trajectories recorded every 5 ps interval were calculated for the backbone Ca atom of the entire SBD and the 7 b-strands at different temperature with reference to
Fig. 2. The RMSDs for the entire SBD during the 600 MD simulation at different temperatures.
H.-L. Liu, W.-C. Wang / Chemical Physics Letters 366 (2002) 284–290
the starting structure according to KoehI [30]. The RMSDs were obtained after optimal superimposition of the coordinates to remove translational and rotational motion [31]. The secondary structure (a-helix, b-strand, turn, and random coil) was predicted according to the Kabsch–Sander algorithm [5], in which pattern recognition of hydrogen-bonded was correlated to the geometrical features. The residual b-strand content, BðtÞ, for each b-strand was defined as the ratio of the number of the residual cooperative H-bonds in the b-strands at time t to the number of the cooperative H-bonds in the NMR structure. The averaged
287
b-residual strand content, hBi, was calculated by averaging the sampled 120 BðtÞ values for each b-strand in each run. The melting temperature, tm , for each b-strand, calculated according to Arrhenius equation, was defined as the temperature at which the b-strand remains 50% of its hBi.
3. Results and discussion In this study, the starting structure was obtained from the NMR solution structure of the SBD from A. niger GA followed by energy mini-
Table 1 The mean RMSDs for each b-strand and the entire SBD in the 600 ps MD simulations b1
b2
b3
b4
b5
b6
b7
SBD
300 K
) RMSD (A hBi (%)
0.98 68
1.22 44
1.08 25
0.98 60
1.21 61
1.17 45
0.81 73
3.43 60
400 K
) RMSD (A hBi (%)
1.13 54
1.45 34
1.41 12
1.24 59
1.56 44
1.54 49
0.93 55
4.50 48
600 K
) RMSD (A hBi (%)
1.24 23
2.27 16
1.65 12
1.45 28
1.82 27
1.70 43
0.95 31
6.74 25
Fig. 3. The residual b-strand contents, defined in the text, for the entire SBD during the 600 MD simulation at different temperatures.
288
H.-L. Liu, W.-C. Wang / Chemical Physics Letters 366 (2002) 284–290
Fig. 4. Secondary structure, as analyzed according to Kabsch–Sander algorithm [5], as a function of simulation time at (A) 300 and (B) 400 K.
H.-L. Liu, W.-C. Wang / Chemical Physics Letters 366 (2002) 284–290
mization in an external mean field with DC of 80. Fig. 2 shows the RMSDs of the backbone Ca for the entire SBD with reference to the starting structure in the course of 600 ps MD simulations at different temperatures. The RMSDs increased at 300, 400, and from 0 to about 4, 5.5, and 8 A 600 K, respectively, for the entire SBD. In contrast, the RMSD for each b-strand fluctuated at 300, 400, and 600 K (data within 1.6, 2, and 3 A not shown). The mean RMSDs for each b-strand and the entire SBD are listed in Table 1. b-Strands 1, 4, and 7 usually had smaller mean RMSD values than the others at all simulation temperatures, indicating that they were more thermostable. The increased kinetic energy obtained by raising temperature compensated for the increased entropy, leading to spontaneous structural fluctuations. Higher temperature provides more kinetic energy to overcome the energy barrier between the folded and the transition states, resulting in faster unfolding rate. To determine the stability of the b-strands, the residual b-strand content, BðtÞ, were calculated during the 600 ps MD simulations. The results are given in Fig. 3. The values of BðtÞ for the entire SBD fluctuated between 40% and 70% with reference to the starting structure at 300 K, whereas it fluctuated around 50% at 400 K and dropped rapidly to about 20–25% at 600 K. The values of BðtÞ for each b-strand fluctuated dramatically in all simulations (data not shown). It indicates that the stability of the entire SBD is not only attributed to the H-bonds only. There should be some other forces such as hydrophobic and electrostatic interactions which are responsible for the stabilization of these b-strands, thus the entire SBD. Fig. 4 shows the secondary structure propensity for each residue in the SBD using DSSP [5]. b-Strands 1, 4, and 7 seemed to maintain higher b-strand content than the others at 300 and 400 K. b-Strands 1 and 7 are located very close to the intrinsic disulfide bond formed between Cys509 and Cys604. Disulfide bond has been shown to play an important role in stabilizing the tertiary structures of proteins, leading to enhancing the thermostability. In addition, b-strands 1 and 4 and b-strands 1 and 7 form antiparallel and parallel b-sheets, respectively. The H-bonding network
289
amongst these b-strands contributes to the secondary structure stabilization. Furthermore, the numbers of residues in b-strands 1, 4, and 7 are 11, 11, and 9, which are the three of the four longest b-strands in the SBD. From our observation, we found that the longer the b-strand is, the higher is the stability. Here, we also estimate that the putative starch binding site 2, located near b-strands 1 and 4, is more thermostable than site 1, which is located close to b-strands 2, 5, and 6. In order to determine the unfolding order of the 7 b-strands in the SBD, the averaged residual bstrand content, hBi, were calculated by averaging BðtÞ over 600 ps and the results were given in Table 1. According to the Arrhenius equation where the higher the Tm of the b-strand is, the slower is the unfolding rate, the unfolding of the b-strands in the SBD was predicted to follow the order of b3 ! b2 ! b6 ! b5 ! b4 ! b1 ! b7 according to the calculated Tm (data not shown). The region around b-strands 2 and 3 seemed to act as the initiation site during the unfolding of the SBD. Acknowledgements The authors thank National Science Council of Taiwan for financial support (project number NSC-89-2311-B-027-001). We also thank ChiaMing Hsu, Chia-Yuen Hu, Ming-Yi Hwang and Yen-Chi Su of NTUT for data analysis.
References [1] H.-L. Liu, Recent Res. Devel. Protein Eng. 1 (2001) 75. [2] P.M. Coutinho, P.J. Reilly, Proteins 29 (1997) 334. [3] A. Aleshin, A. Golubev, L.M. Firsov, R.B. Honzatko, J. Biol. Chem. 267 (1992) 19291. [4] K. Sorimachi, A.J. Jacks, M.F. Le Gal-coeffet, G. Williamson, D.B. Archer, M.P. Williamson, J. Mol. Biol. 259 (1996) 970. [5] W. Kabsch, C. Sander, Biopolymers 22 (1983) 2577. [6] B. Svensson, T.G. Pedersen, I. Svendsen, T. Sakai, M. Ottesen, Carlsberg Res. Commun. 47 (1982) 55. [7] C. Apparu, H. Driguez, G. Williamson, B. Svensson, Carbohydr. Res. 277 (1995) 313. [8] T. Watanabe, Y. Ito, T. Yamada, M. Hashitomo, S. Sekine, H. Tanaka, J. Bacteriol. 176 (1994) 4465. [9] T.-Y. Fang, P.M. Coutinho, P.J. Reilly, C. Ford, C. Protein, Eng. 11 (1998) 119.
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[10] T.-Y. Fang, R.B. Honzatko, P.J. Reilly, C. Ford, Protein Eng. 11 (1998) 127. [11] H.-L. Liu, P.M. Coutinho, C. Ford, P.J. Reilly, Protein Eng. 11 (1998) 389. [12] H.-L. Liu, C. Ford, P.J. Reilly, Protein Eng. 12 (1999) 163. [13] H.-M. Chen, C. Ford, P.J. Reilly, Protein Eng. 8 (1995) 575. [14] H.-M. Chen, P.M. Coutinho, Z. Nikolov, C. Ford, Protein Eng. 8 (1995) 1049. [15] H.-M. Chen, Y. Li, T. Panda, F.U. Buehler, C. Ford, P.J. Reilly, Protein Eng. 9 (1996) 499. [16] Y. Li, P.J. Reilly, C. Ford, Protein Eng. 10 (1997) 1199. [17] Y. Li, P.M. Coutinho, C. Ford, Protein Eng. 11 (1998) 661. [18] M.J. Allen, P.M. Coutinho, C.F. Ford, Protein Eng. 11 (1998) 783. [19] H.-L. Liu, Y. Doleyres, P.M. Coutinho, C. Ford, P.J. Reilly, Protein Eng. 13 (2000) 655. [20] H.-M. Chen, C. Ford, P.J. Reilly, Biochem. J. 301 (1994) 275.
[21] H.-M. Chen, U. Bakir, P.J. Reilly, C. Ford, Biotechnol. Bioeng. 43 (1994) 101. [22] G. Williamson, N.J. Belshaw, T.R. Noel, S.G. Ring, M.P. Williamson, Eur. J. Biochem. 207 (1992) 661. [23] P.A. Thompson, W.A. Eaton, J. Hofrichter, J. Biochem. 36 (1997) 9200. [24] V. Mu~ noz, E.R. Henry, J. Hofrichter, W.A. Eaton, Proc Natl. Acad. Sci. USA 95 (1998) 5872. [25] M. Karplus, A. Sali, Curr. Opin. Struct. Biol. 5 (1995) 58. [26] A. Li, V. Daggett, J. Mol. Biol. 257 (1996) 412. [27] P. Biggin, J. Breed, H.S. Son, M.S.P. Sansom, Biophys. J. 72 (1997) 627. [28] M.-J. Hwang, X. Ni, M. Waldman, C.S. Ewig, A.T. Hagler, Biopolymers 45 (1998) 435. [29] H.J.C. Berendsen, J.P.M. Postma, W.F. van Gunsteren, H. DiNola Jr., J. Comp. Phys. 81 (1984) 3684. [30] P. KoehI, Curr. Opin. Struct. Biol. 11 (2001) 348. [31] W. Kabsch, Acta Cryst. A 32 (1976) 922.
The predicted unfolding order of the b-strands in the starch binding domain from Aspergillus niger glucoamylase Hsuan-Liang Liu *, Wen-Chi Wang Department of Chemical Engineering, National Taipei University of Technology, No. 1 Sec. 3 Chung-Hsiao E. Rd., Taipei 106, Taiwan Received 11 July 2002; in final form 20 August 2002
Abstract The unfolding of the b-strands in the starch binding domain from Aspergillus niger glucoamylase was predicted to follow the order of b3 ! b2 ! b6 ! b5 ! b4 ! b1 ! b7 by 600 ps molecular dynamics simulations at 300, 400, and 600 K. The interior region around b-strands 2 and 3 acts as the initiation site for unfolding. b-Strands 1 and 7 are probably stabilized by the disulfide bond formed between Cys509 and Cys604. b-Strand 4 is stabilized by forming an antiparallel b-sheet with b-strand 1. Hydrophobic and electrostatic interactions between side chains instead of the hydrogen bonds are important in stabilizing these b-strands, thus the entire starch binding domain. Ó 2002 Elsevier Science B.V. All rights reserved.
1. Introduction Glucoamylase (1,4-a-D -glucan glucohydrolase, EC 3.2.1.3, GA) is one of the most widely used enzyme in industry [1]. Aspergillus niger GA is composed of three functional domains: (1) a globular N-terminal catalytic domain (CD) from residue 1 to 470; (2) a linear and extended linker domain from residue 471 to 508; and (3) a globular C-terminal starch binding domain (SBD) from residue 509–616 [2]. While the 3D structure of the CD from A. awamori var. X100 GA has been determined by X-ray crystallography in its native state [3], the solution structure of the SBD from A. niger GA in its native state has been
*
Corresponding author. Fax: +886-2-2731-7117. E-mail address: [email protected] (H.-L. Liu).
solved by NMR spectroscopy [4]. The SBD is a well defined b-sheet structure consisting of 7 b-strands and 1 a-helix according to the Kabsch– Sander algorithm [5] (Fig. 1), which form one parallel and five antiparallel pairs of b-sheets [4]. The SBD allows GA to adsorb to and digest raw starch [6] and some other substrates [7] without affecting the catalytic behavior of the CD toward soluble substrates [8]. The presence of this domain in a range of hydrolases clearly highlights its functional importance. Although many efforts have been made to improve the substrate selectivity [9–12] and thermostability [13–19] of GA for better industrial applications, not too much attention has been paid to investigate the functional and structural features of the SBD so far. GA undergoes irreversible thermoinactivation at moderately acidic pH and 70 °C [19]. Neither deamidation [20,21] nor Asp-X
0009-2614/02/$ - see front matter Ó 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 9 - 2 6 1 4 ( 0 2 ) 0 1 4 2 5 - 2
H.-L. Liu, W.-C. Wang / Chemical Physics Letters 366 (2002) 284–290
285
Fig. 1. The NMR structure of the SBD binding to b-cyclodextrins (ball and stick representation) obtained from the PDB (PDB entry: 1KUM). b-strands 1 to 7 predicted by Kabsch–Sander algorithm [5] are shown in arrows pointing from N- to C-terminus. The starch binding sites 1 and 2 are indicated as S1 and S2, respectively.
peptide bond hydrolysis [14] determines the thermoinactivation rate. In addition, mismatched disulfide bonds are not observed at 70 °C and pH 3.5 and 4.5 [19]. These results indicate that some other mechanism must account for the thermoinactivation of the GA at high temperatures. The mechanism appears to be a change in the GAÕs secondary or tertiary structure, such as unfolding that destroys the integrity of the CD and the SBD [19,22]. In the present study, 600 ps molecular dynamics (MD) simulations were conducted to predict the unfolding order of the 7 b-strands in the SBD from A. niger GA with temperature jump technique [23,24].
As the computer power has been increased in recent years, MD simulation has been intensively employed to gain insight into protein folding/unfolding processes [25]. The large number of interatomic interactions in force fields that have been taken into account for a realistic description of a protein in solution has made MD technique a promising tool in examining the early events in protein folding/unfolding. Unfortunately, the fs time-step for the integration of the equation of motion limits the MD simulations to an extremely small time-span (e.g., ns or ls), which is relatively short comparing to the second time-scale of the real protein folding/unfolding processes. Thus,
286
H.-L. Liu, W.-C. Wang / Chemical Physics Letters 366 (2002) 284–290
instead of predicting protein folding processes directly, a lot of attention has been paid on the unfolding simulations, in which the unfolding of a protein in its native-like structure is usually initiated by raising the temperature or induced by changing the solvent [26]. Instead of simulating the full-length SBD using an all-atom model, which may be limited by the current computer power, the present MD simulations were performed in a mean external field with dielectric constant (DC) of 80 [27].
2. Methods The initial NMR structure of the SBD, residues 509–616 from A. niger GA, was taken from the Protein Data Bank (PDB entry 1KUM) [4]. The energy minimization and MD simulations were performed by the Insight II program (Accelyrs, San Diego, CA, USA) with the force field Discover CVFF (consistent valence force field) [28] in the SGI O200 workstation (Silicon Graphics, Mountain View, CA, USA) with 64-bit HIPS
RISC R12000 270 MHz CPU and PMC-Sierra RM7000A 350 MHz processor. The NMR structure of the SBD was subjected to energy minimization calculations in an external field with DC ¼ 80 by steepest descent method with 5000 iterations to be used as the lowest energy starting structure for further structural analysis. The starting structure was then subjected to 600 ps MD simulations at DC ¼ 80 after equilibrating for about 10 ps at pH 5.2 and 300, 400, and 600 K using the Discover module. The temperature and pressure were maintained for each MD simulation by weak coupling the system to a heat bath at the simulation temperature and an external pressure bath at one atmosphere, respectively, according to themethod described by Berendsen et al. [29]. The time-step of the MD simulations was 1 fs. The trajectories and coordinates of the SBD were saved every 5 ps for further analysis. For each MD simulation, the root-mean-square deviations (RMSDs) of the trajectories recorded every 5 ps interval were calculated for the backbone Ca atom of the entire SBD and the 7 b-strands at different temperature with reference to
Fig. 2. The RMSDs for the entire SBD during the 600 MD simulation at different temperatures.
H.-L. Liu, W.-C. Wang / Chemical Physics Letters 366 (2002) 284–290
the starting structure according to KoehI [30]. The RMSDs were obtained after optimal superimposition of the coordinates to remove translational and rotational motion [31]. The secondary structure (a-helix, b-strand, turn, and random coil) was predicted according to the Kabsch–Sander algorithm [5], in which pattern recognition of hydrogen-bonded was correlated to the geometrical features. The residual b-strand content, BðtÞ, for each b-strand was defined as the ratio of the number of the residual cooperative H-bonds in the b-strands at time t to the number of the cooperative H-bonds in the NMR structure. The averaged
287
b-residual strand content, hBi, was calculated by averaging the sampled 120 BðtÞ values for each b-strand in each run. The melting temperature, tm , for each b-strand, calculated according to Arrhenius equation, was defined as the temperature at which the b-strand remains 50% of its hBi.
3. Results and discussion In this study, the starting structure was obtained from the NMR solution structure of the SBD from A. niger GA followed by energy mini-
Table 1 The mean RMSDs for each b-strand and the entire SBD in the 600 ps MD simulations b1
b2
b3
b4
b5
b6
b7
SBD
300 K
) RMSD (A hBi (%)
0.98 68
1.22 44
1.08 25
0.98 60
1.21 61
1.17 45
0.81 73
3.43 60
400 K
) RMSD (A hBi (%)
1.13 54
1.45 34
1.41 12
1.24 59
1.56 44
1.54 49
0.93 55
4.50 48
600 K
) RMSD (A hBi (%)
1.24 23
2.27 16
1.65 12
1.45 28
1.82 27
1.70 43
0.95 31
6.74 25
Fig. 3. The residual b-strand contents, defined in the text, for the entire SBD during the 600 MD simulation at different temperatures.
288
H.-L. Liu, W.-C. Wang / Chemical Physics Letters 366 (2002) 284–290
Fig. 4. Secondary structure, as analyzed according to Kabsch–Sander algorithm [5], as a function of simulation time at (A) 300 and (B) 400 K.
H.-L. Liu, W.-C. Wang / Chemical Physics Letters 366 (2002) 284–290
mization in an external mean field with DC of 80. Fig. 2 shows the RMSDs of the backbone Ca for the entire SBD with reference to the starting structure in the course of 600 ps MD simulations at different temperatures. The RMSDs increased at 300, 400, and from 0 to about 4, 5.5, and 8 A 600 K, respectively, for the entire SBD. In contrast, the RMSD for each b-strand fluctuated at 300, 400, and 600 K (data within 1.6, 2, and 3 A not shown). The mean RMSDs for each b-strand and the entire SBD are listed in Table 1. b-Strands 1, 4, and 7 usually had smaller mean RMSD values than the others at all simulation temperatures, indicating that they were more thermostable. The increased kinetic energy obtained by raising temperature compensated for the increased entropy, leading to spontaneous structural fluctuations. Higher temperature provides more kinetic energy to overcome the energy barrier between the folded and the transition states, resulting in faster unfolding rate. To determine the stability of the b-strands, the residual b-strand content, BðtÞ, were calculated during the 600 ps MD simulations. The results are given in Fig. 3. The values of BðtÞ for the entire SBD fluctuated between 40% and 70% with reference to the starting structure at 300 K, whereas it fluctuated around 50% at 400 K and dropped rapidly to about 20–25% at 600 K. The values of BðtÞ for each b-strand fluctuated dramatically in all simulations (data not shown). It indicates that the stability of the entire SBD is not only attributed to the H-bonds only. There should be some other forces such as hydrophobic and electrostatic interactions which are responsible for the stabilization of these b-strands, thus the entire SBD. Fig. 4 shows the secondary structure propensity for each residue in the SBD using DSSP [5]. b-Strands 1, 4, and 7 seemed to maintain higher b-strand content than the others at 300 and 400 K. b-Strands 1 and 7 are located very close to the intrinsic disulfide bond formed between Cys509 and Cys604. Disulfide bond has been shown to play an important role in stabilizing the tertiary structures of proteins, leading to enhancing the thermostability. In addition, b-strands 1 and 4 and b-strands 1 and 7 form antiparallel and parallel b-sheets, respectively. The H-bonding network
289
amongst these b-strands contributes to the secondary structure stabilization. Furthermore, the numbers of residues in b-strands 1, 4, and 7 are 11, 11, and 9, which are the three of the four longest b-strands in the SBD. From our observation, we found that the longer the b-strand is, the higher is the stability. Here, we also estimate that the putative starch binding site 2, located near b-strands 1 and 4, is more thermostable than site 1, which is located close to b-strands 2, 5, and 6. In order to determine the unfolding order of the 7 b-strands in the SBD, the averaged residual bstrand content, hBi, were calculated by averaging BðtÞ over 600 ps and the results were given in Table 1. According to the Arrhenius equation where the higher the Tm of the b-strand is, the slower is the unfolding rate, the unfolding of the b-strands in the SBD was predicted to follow the order of b3 ! b2 ! b6 ! b5 ! b4 ! b1 ! b7 according to the calculated Tm (data not shown). The region around b-strands 2 and 3 seemed to act as the initiation site during the unfolding of the SBD. Acknowledgements The authors thank National Science Council of Taiwan for financial support (project number NSC-89-2311-B-027-001). We also thank ChiaMing Hsu, Chia-Yuen Hu, Ming-Yi Hwang and Yen-Chi Su of NTUT for data analysis.
References [1] H.-L. Liu, Recent Res. Devel. Protein Eng. 1 (2001) 75. [2] P.M. Coutinho, P.J. Reilly, Proteins 29 (1997) 334. [3] A. Aleshin, A. Golubev, L.M. Firsov, R.B. Honzatko, J. Biol. Chem. 267 (1992) 19291. [4] K. Sorimachi, A.J. Jacks, M.F. Le Gal-coeffet, G. Williamson, D.B. Archer, M.P. Williamson, J. Mol. Biol. 259 (1996) 970. [5] W. Kabsch, C. Sander, Biopolymers 22 (1983) 2577. [6] B. Svensson, T.G. Pedersen, I. Svendsen, T. Sakai, M. Ottesen, Carlsberg Res. Commun. 47 (1982) 55. [7] C. Apparu, H. Driguez, G. Williamson, B. Svensson, Carbohydr. Res. 277 (1995) 313. [8] T. Watanabe, Y. Ito, T. Yamada, M. Hashitomo, S. Sekine, H. Tanaka, J. Bacteriol. 176 (1994) 4465. [9] T.-Y. Fang, P.M. Coutinho, P.J. Reilly, C. Ford, C. Protein, Eng. 11 (1998) 119.
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