1 Department of Biochemistry, Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland, 2 Department of Chemistry, University of Edinburgh, West Mains Road, Edinburgh EH9 3JJ 3 Department of Pure and Applied Chemistry, University of Strathclyde, Thomas Graham Building, 295 Cathedral Street, Glasgow G1 1XL, UK
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: endothelin-1/ETB agonist/molecular modelling/NMR/solution conformation
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Peptides of the endothelin family are widely distributed throughout a variety of tissues and organs such as vascular and non-vascular smooth muscle, nervous tissue, heart, kidney and mesangial cells and adrenal glands. The pharmacological evaluations and structureactivity relationship studies of endothelins have been extensively reviewed (Doherty, 1995; Huggins et al., 1993
). There have been many reports consisting of a combination of high resolution NMR studies and molecular modelling techniques deployed to determine the tertiary structures of peptides belonging to the endothelin family (Reily and Dunbar, 1991
; Mills et al., 1992
; Atkins et al., 1995
; Hewage et al., 1997
).
Increasing evidence for the involvement of endothelin peptides in a variety of human diseases has prompted a major effort in structureactivity relationship studies and drug design based on peptides from the endothelin family. Our previous reports of linear, modified, synthetic endothelin analogues (Hewage et al., 1998a,b
) showed that the disulfide bridges associated with maintaining the structural integrity of the natural peptide are neither important for the formation of helical secondary structure nor essential for the biological activity of ETB receptor. In this study, we present the solution structure of the ETB receptor selective agonist, the peptide ET721[Leu7, Aib11, Cys(Acm)15] (designated here as LJP33), by NMR-based molecular modelling.
ET-1: CSCSSLMDKECVYFCHLDIIW
peptide LJP2: CSASSLLDKEXVYFCHLDIIW
peptide LJP33: LDKEXVYFCHLDIIW
The disulfide bridges of ET-1 are between C1C15 and C3C11. The Cys1 and Cys15 of peptide LJP2 and Cys15 of peptide LJP33 are protected by the acetamidomethyl group (Acm) and X denotes -aminoisobutyric acid.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
NMR spectroscopy
All NMR experiments were performed using a 5 mm probe head on a Varian VXR 600S NMR spectrometer operating at a 1H resonance frequency of 599.945 MHz. Peptide LJP33(5 mg), was dissolved in d3-methanol/H2O co-solvent (1:1) except for amide proton exchange studies when d4-methanol/D2O (1:1) was used as solvent. All NMR experiments were performed on the sample at a pH of 3.6 and a temperature of 298 K. One-dimensional NMR data were recorded with a pre-saturation delay of 1.5 s. A total of 256 transients were acquired over a 7 kHz spectral width into 35K data points. The data were zero filled to 65 536 data points prior to apodization using an optimized shifted squared sinebell function followed by Fourier transformation and phase correction. Two-dimensional phase-sensitive TOCSY (Bax and Davis, 1985) and NOESY (Kumar et al., 1980
) data sets were acquired with a pre-saturation delay of 1.5 s. The 2D-TOCSY pulse sequence was used with an 80 ms mixing time and two 2 ms trim pulses. A series of 2D-NOESY data sets, with mixing times of 40, 80, 150, 200 and 300 ms, were collected and evaluated for the effects of spin-diffusion. The residual water resonance was saturated during preparation and mixing periods. A total of 16 and 32 transients were acquired for each of 2x256 t1 increments (hypercomplex acquisition; States et al., 1982
) into 2048 complex data points for the 2D-TOCSY and 2D-NOESY data sets, respectively. Other acquisition parameters for both data sets were an acquisition time of 0.146 s and a spectral width of 7 kHz. Fourier transformation in F2 was carried out without zero filling whereas data in F1 were zero filled to 1024 data points prior to Fourier transformation. All time domain data were apodized in both dimensions using an optimized shifted squared sinebell window function. The real Fourier transformation was carried out on 1024x2048 data points. All the two-dimensional spectra were acquired on a non-spinning sample.
Variable temperature studies of peptide LJP33 were carried out on a solution of 1 mg of the peptide using a standard one-dimensional 1H NMR pulse sequence. Spectra were recorded at temperature intervals of 4° from 290 to 298 K and 5° from 298 to 318 K continuously. Amide protondeuterium exchange experiments were carried out soon after dissolution of 1 mg of the peptide and then at regular time intervals. For standard 1D 1H NMR spectra a relaxation delay of 1.5 s was used only during the preparation period to eliminate the residual HOD solvent resonance. All data were internally referenced to the residual 1H NMR resonance of CD2HOH defined as 3.30 p.p.m. Varian's VNMR software (version 4.1) was used for all data acquisition and preliminary data processing.
Molecular modelling
Structure calculation studies were carried out using the Tripos molecular modelling software SYBYL version 6.1. (Tripos Software, 1994). 2D-NOESY data at 150 ms were converted and processed using the TRIAD module of SYBYL. Peak picking was carried out manually. Volume integrals were calculated for all cross-peaks within the TRIAD module which were then used for generating lower and upper distance constraints. The lower distance bound was in all cases set to the van der Waals radius of 1.8 Å. Upper distance bounds were defined by volume integral values of 2.8 (strong), 3.6 (medium) and 5.0 Å (weak). The threshold values of upper distance bounds were established using the known sequential distances of dNN and dN (Billeter et al., 1982
). Pseudoatoms with appropriate distance corrections were employed for protons which could not be stereospecifically assigned (Wüthrich et al., 1983
). Partially overlapped signals were ignored in the structure calculation. Slow amide proton exchange rates were observed for some NHs. Constraints between these protons and associated backbone carbonyl oxygen atoms were introduced after the first phase of the structure calculations, when the fold of the peptide chain emerged. Hydrogen bonding partners were identified using the standard model for polypeptide
-helicies. These inter-atomic distance constraints were set in the range 1.82.0 and 2.73.0 Å for the HO distance and NO distances, respectively (Williamson et al., 1985
). Torsion angle (
) constraints were calculated using the expression 3JHN
= 6.4cos2
1.4cos
+ 1.9 (Pardi et al., 1984
). 3JHN
values were measured directly from the high resolution 1H NMR spectrum of peptide LJP33.
The DIANA package (distance geometry algorithm for NMR applications; Güntert et al., 1991) was used to generate random starting structures based on an initial structure built within SYBYL. The 169 distance constraints obtained from NOESY cross-peak volumes were used as input for the DIANA calculations. Six constraints for hydrogen bonds and 13 torsional angle constraints were also incorporated. All distance and torsional constraints were included in the DIANA calculations. Atomic distances were constrained using the force constant kNOE = 1 kcal mol1 Å2 and torsions were constrained using kDihed_c = 0.01 kcal mol1 deg2. DIANA was used to calculate 300 structures from random starting conformations and the calculation produced 31 acceptable structures dependent on the final target function value. Distances, which were predetermined by the covalent geometry of the molecule or by conformations which violated the constraints, were regarded as irrelevant by the DIANA program. These constraints were eliminated during the calculation to yield 112 structurally important constraints defined as modified bounds.
Structures from DIANA were subsequently constrained by these modified distance bounds. Inter-atomic distances were then constrained using a higher force constant of kNOE = 10 kcal mol1 Å2 and torsions were constrained using the same force constant of kDihed_c = 0.01 kcal mol1 deg2. Structures were then subjected to 200 steps of conjugate-gradient energy minimization. Higher energy structures were initially minimized using an atom-by-atom Simplex minimization. The Tripos 5.2 force field with the energy minimizer MAXIMIN 2 was used in the minimization procedure. Other parameters for constraining covalent geometry were kBond = 600 kcal mol1 Å2, kAngle = 0.02 kcal mol1 deg2 and kTor = 0.2 kcal mol1 deg2. At this stage, 10 structures which showed least violated constraints and lowest energy were chosen for further calculations.
Energy minimized structures were then refined using a dynamical simulated annealing method (DSA), this being a process by which local energy minima are overcome by successive heating and cooling cycles of the molecular model. DSA calculations began by holding the temperature constant at 1000 K for a period of 5400 fs. During the annealing time of 900 fs, the temperature was reduced `stepwise' until a minimum temperature of 100 K was reached. The Boltzmann scaling of atomic velocities was chosen from a random number. This process completed the first cycle. Ten cycles of DSA were run for each starting structure. The conformations obtained by DSA were further minimized with 200 steps of conjugate-gradient energy minimization. The minimization parameters used were the same as those described for the DIANA calculated structures.
The energy minimized conformations were subjected to a final molecular dynamics (MD) quenching calculation without changing either distance or torsional force constants. The MD calculations were performed in the gas phase. The initial atomic velocities were chosen from a random distribution at 1000 K and the dynamic trajectory (100 fs) was followed for 20 ps in 1 fs steps. These calculations were carried out under NTV ensemble conditions with a 10 fs coupling factor for the temperature. The MD calculations were repeated three times for each conformation using different initial values for random number seed and these three different conformers were then averaged to obtain the averaged conformation. After removing all the distance and torsional experimental energy barriers, these averaged conformers were finally subjected to 500 steps of conjugate-gradient energy minimization as described previously.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The sequence of 15 residue synthetic linear peptide, peptide LJP33, includes nine unique residues, Lys9, Glu10, Aib11, Val12, Tyr13, Phe14, Cys15, His16 and Trp21. The fingerprint region of the 2D-TOCSY spectrum (Figure 1) provided a basis for the identification of individual residue spin-system resonances and showed 13 resonances for NH/
H excluding N-terminal Leu7 and Aib11 resonances. An additional resonance was observed for the side chain protecting group (Acm) of Cys15. The magnetization transfer from NH through the entire spin-systems were observed where possible for all amino acid residues in the 2D-TOCSY spectrum.
|
|
|
Sequential connectivity from the unique residue Val12 to Tyr13 was clearly identifiable. The strong clear resonance of Tyr13ßH/Phe14NH helped to distinguish the Phe14 residue. The backbone connectivities of C-terminal residues Asp18/Ile19, Ile19/Ile20 and Ile20/Trp21 were also clearly identifiable. The backbone connectivities of two adjacent residues, Cys15 and His16, thence Leu17 were clearly distinguished in the 2D-NOESY fingerprint region discriminating the remaining leucine residue, Leu7 thus completing the sequence-specific assignment of the peptide LJP33.
The remainder of the 2D-NOESY spectrum was then searched for secondary structure correlations. The clear, long-range iH/Ni + 3H connectivities of Lys9/Val12, Val12/Cys15 and Tyr13/His16 were identified in the fingerprint region of the 2D-NOESY spectrum. Correlations between Glu10/Tyr13 could not be distinguished due to the presence of overlapping cross-peaks (Figure 2
). Strong NiH/Ni + 1H backbone connectivities and weak
iH/Ni + 1H connectivities confirmed the
-helical pattern between Lys9 and His16. The clear
iH/ßi + 3H connectivities between residues Lys9/Val12, Glu10/Tyr13, Val12/Cys15 and Tyr13/His16 appeared in the
HßH cross-peak region of the 2D-NOESY spectrum, which also confirmed the
-helical nature of the structure. Smaller 3JNH
coupling constants (Table I
) were observed for residues Lys9His16. Figure 3
summarizes the inter-residue nOe connectivity patterns of peptide LJP33.
|
The temperature dependence of backbone amide proton chemical shifts was measured over the temperature range 290313 K. A structured state was apparent from the very small temperature coefficients of Aib11 /
T = 1.4 p.p.b. K1 and Phe14
/
T = 2.1 p.p.b. K1. This suggested that amide protons in the helical region were involved in hydrogen bonding.
The final structures obtained from the structure calculation of peptide LJP33 by DG, DSA followed by MD were in good agreement with the experimentally derived constraints. The calculated structures remain within a single family of conformations. The major structural feature of peptide LJP33 was a highly-ordered right-handed -helix from Lys9His16. The structure calculations reveal that the C-terminal tail region of residues Leu17Trp21 appears to have no well-defined conformation in aqueous co-solvent, consistent with the fewer distance constraints obtained from this region.
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Previous NMR studies of endothelin peptides under different solution conditions have also revealed helical structural features. However, these have usually involved fewer residues. Our previous work on ET-1 (Hewage et al., 1997) and modified, synthetic endothelin peptide analogues (Hewage et al., 1998a
,b
) studied under identical solution conditions as those reported here have also shown
-helical conformations in the same residue region of the polypeptide sequence. The nOe patterns observed at both the N- and C-termini regions were also in good agreement with the previously published work. The NMR and molecular modelling studies of the parent linear ET-1 analogue, peptide LJP2, revealed a structure characterized by an
-helix lying between residues Ser5 and His16. Biological activity studies have revealed that peptide LJP2 displays a potency against the ETB receptor which is a factor of 10 greater than that of the parent peptide ET-1 (Jiang, 1996
). For this reason truncated analogues of LJP2 were synthesized in a bid to determine which features of the sequence and structure were responsible for this enhanced potency. The truncation proceeded from the N-terminus of the peptide, since it has been shown that most of the C-terminal residues were essential for activity (Leonard et al., 1995
). Peptide LJP33, described in this report, retained the potency found for peptide LJP2, but was without the same level of receptor selectivity. Therefore, it was of interest to us to know whether the solution structure of this material differed in any way from that of peptide LJP2.
Therefore, we have solved the solution structure of the truncated ETB selective agonist, ET721[Leu7, Aib11, Cys (Acm)15] (LJP33) in an aqueous methanol co-solvent by means of a combination of 1H NMR and molecular modelling studies. Although peptide LJP33 has a rather low molecular weight, good 2D-NOESY spectra, with strong negative nOes, were obtained which is consistent with a slowly tumbling macromolecule of defined tertiary structure. Linear correlation of nOe cross-peak intensities up to 150 ms of the NOESY mixing time was observed. This behaviour may be due to the fact that the effective volume of the hydrated molecule led to a longer correlation time. Evidence for the prevalence of a secondary helical conformation was revealed by the observation of iH/Ni + 3H and strong
iH/ßi + 3H interactions found in the 2D-NOESY spectrum. The slow exchange of Tyr13, Phe14 amide protons suggested that the helix was stabilized by hydrogen bonds, occurring between residues Lys9/Tyr13 and Glu10/Phe14. Small temperature coefficients of the amide protons of Aib11 and Phe14 indicated shielding from solvent exchange.
All NMR spectroscopic studies and molecular modelling calculations of peptide LJP33 yielded a well defined -helical conformation between residues Lys9 and His16. The mode of convergence of the calculated structures is demonstrated by the RMSD values calculated for all atoms in the helical region, which was found to be 1.84 ± 0.26, compared with backbone only atoms, for which the RMSD figure was found to be 0.54 ± 0.16 (Table II
). The C-terminal region of the polypeptide chain beyond residue His16 may exist as an extended random structure as indicated by random coil 3JNH
coupling constants (>7 Hz) and a series of strong
iH/Ni + 1H nOes.
|
|
|
Structureactivity relationship studies of this peptide can be considered in terms of the structural features revealed by our model. Changing Met7 of ET-1 to Leu and also replacing Cys11 by the Aib residue did not alter the formation of an -helical conformation. According to general peptide chemistry, Aib is known to introduce
-helical characteristics into a peptide backbone structure. Previous studies, where Met7 was replaced by alanine (Dalgarno et al., 1992
) or norleucine (Aumelas et al., 1991
), induced very little conformational changes relative to ET-1. Most of the biologically active amino acid residues reported in the literature for these peptides lie in the helical region of the peptide (De Castiglione et al., 1992
; Galantino et al., 1992
; Hunt et al., 1991
). Residues 10 (Glu) and 14 (Phe), which are positioned on the same side of the calculated model, are important residues for biological activity in rat pulmonary artery (Nakajima et al., 1989
). Thus, aromaticity at position 14 plays an essential role for biological activity and also for folding of the peptide.
Although the C-terminal region of the calculated structures of ET-1 showed no preferred conformation, most of the residues present in this region are known to be important for biological activity (Doherty et al., 1991; Leonard et al., 1995
). The N-terminal residues of ET-1, namely Ser2, Ser4, Ser5 and Leu6, have been shown to be relatively tolerant of substitutions. Pharmacological evaluation of this region found fewer biologically important residues compared with the other regions of the peptide (Saeki et al., 1991
; Watanabe et al., 1991
). Although the N-terminal residues are not critical for agonist activity, they may be responsible for receptor selectivity, as displayed by the differences between peptides LJP2 and LJP33. We speculate that it is the extended
-helical structure of peptide LJP2 compared with peptide LJP33 and not the specific peptide sequence, which is responsible for this observed selectivity.
![]() |
Conclusion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Notes |
---|
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Arai,H., Hori,S., Aramori,I., Ohkubo,H. and Nakanishi,S. (1990) Nature, 348, 730732.[CrossRef][ISI][Medline]
Atkins,A.F., Martin,R.C. and Smith,R. (1995) Biochemistry, 34, 20262033.[ISI][Medline]
Aumelas,A., Chiche,L., Mahe,E., Le-Nguyen,D., Sizun,P., Berthault. P. and Perly,B. (1991) Int. J. Pept. Protein Res., 37, 315324.[ISI][Medline]
Bax,A. and Davis,D.G. (1985) J. Magn. Reson., 65, 355360.[ISI]
Bdolah,A., Wollberg,Z., Fleminger,G. and Kochva,E. (1989) FEBS Lett., 256, 13.[CrossRef][ISI]
Becker,A., Dowdle,E.B., Hechler,U., Kauser,K., Donner,P. and Schleuning, W.-D. (1993) FEBS Lett., 315, 100103.[CrossRef][ISI][Medline]
Billeter,M., Braun,W. and Wüthrich,K. (1982) J. Mol. Biol., 155, 321346.[ISI][Medline]
Dalgarno,D.C., Slater,L., Chackalamannil,S. and Senior,M.M. (1992) Int. J. Pept. Protein Res., 40, 515523.[ISI][Medline]
De Castiglione,R., Tam,J.P., Liu,W., Zhang,J.-W., Galantino,M., Bertolero,F. and Vaghi,F. (1992) In Smith,J.A. and Rivier,J.E. (eds), Peptides: Chemistry and Biology; Proceedings of the 12th American Peptide Symposium. ESCOM, Leiden, The Netherlands, pp. 402.
Doherty,A.M., Cody,W.L., Leitz,N.L., DePue,P.L., Taylor,M.D., Rapundalo,S.T., Hingorani,G.P., Major,T.C., Panek,R.L. and Taylor,D.G. (1991) J. Cardiovasc. Pharmacol., 17, S59S61.
Doherty,A.M. et al. (1993) J. Med. Chem., 36, 25852594.[ISI][Medline]
Doherty,A.M. (1995) In Weiner,D.B. and Williams,W.B. (eds), Chemical and Structural Approaches to Rational Drug Design. CRC Press, Ann Arbor, pp. 85123.
Galantino,M., De Castiglione,R., Tam,J.P., Liu,W., Zhang,J.-W., Cristiani,C. and Vaghi,F. (1992) In Smith,J.A. and Rivier,J.E. (eds), Peptides: Chemistry and Biology; Proceedings of the 12th American Peptide Symposium. ESCOM, Leiden, The Netherlands, pp. 404.
Güntert,P., Braun,W. and Wüthrich,K. (1991) J. Mol. Biol., 217, 517530.[ISI][Medline]
Hewage,C.M., Jiang,L., Parkinson,J.A., Ramage,R. and Sadler,I.H. (1997) J. Pept. Sci., 3, 415428.[CrossRef][ISI][Medline]
Hewage,C.M., Jiang,L., Parkinson,J.A., Ramage,R. and Sadler,I.H. (1998a) FEBS Lett., 425, 234238.[CrossRef][ISI][Medline]
Hewage,C.M., Jiang,L., Parkinson,J.A., Ramage,R. and Sadler,I.H. (1998b) J. Biomol. Struct. Dyn., 16, 425435.[ISI][Medline]
Hewage,C.M., Jiang,L., Parkinson,J.A., Ramage,R. and Sadler,I.H. (1999a) J. Pept. Res., 53, 223233.[CrossRef][ISI][Medline]
Hewage,C.M., Jiang,L., Parkinson,J.A., Ramage,R. and Sadler,I.H. (1999b) Neurochem. Int., 35, 3545.[CrossRef][ISI][Medline]
Huggins,J.P., Pelton,J.T. and Miller,R.C. (1993) Pharmacol. Ther., 59, 55123.[CrossRef][ISI][Medline]
Hunt,J.T., Lee,V.G., Stein,P.D., Hedberg,A., Liu,E.C.K., McMullen,D. and Moreland,S. (1991) Bioorg. Med. Chem. Lett., 1, 3338.[CrossRef][ISI]
Inoue,A., Yanagisawa,M., Kimura,S., Kasuya,Y., Miyauchi,T., Goto,K. and Masaki,T. (1989) Proc. Natl Acad. Sci. USA, 86, 28632867.[Abstract]
Janes,R.W., Peapus,D.H. and Wallace,B.A. (1994) Nat. Struct. Biol., 1, 311319.[ISI][Medline]
Jiang,L. (1996) PhD Thesis, University of Edinburgh, UK.
Karadi,R., Billeter,M. and Wüthrich,K. (1996) J. Mol. Graph., 14, 5155.[CrossRef][ISI][Medline]
Karne,S., Jayawickreme,C.K. and Lerner,M.R. (1993) J. Biol. Chem., 268, 1912619133.
Kumar,A., Ernst,R.R. and Wüthrich,K. (1980) Biochem. Biophys. Res. Commun., 95, 16.[ISI][Medline]
Leonard,D.M., Reily,M.D., Dunbar,J.B., Holub,K.E., Cody,W.L., Hill,K.E., Welch,K.M., Flynn,M.A., Reynolds,E.E. and Doherty,A.M. (1995) Bioorg. Med. Chem. Lett., 5, 967972.[CrossRef][ISI]
Mills,R.G., O'Donoghue,S.L., Smith,R. and King,G.F. (1992) Biochemistry, 31, 56405645.[ISI][Medline]
Nakajima,K. et al. (1989) Biochem. Biophys. Res. Commun., 163, 424429.[ISI][Medline]
Pardi,A., Billeter,M. and Wüthrich,K. (1984) J. Mol. Biol., 180, 741751.[ISI][Medline]
Ramage,R. et al. (1996) In Epton,R. (ed.), Proceedings of the Fourth International Symposium: Innovation and Perspectives in Solid Phase Synthesis and Combinatorial Libraries. Mayflower Scientific Ltd, Birmingham, UK, pp. 110.
Reily,M.D. and Dunbar,J.B. (1991) Biochem. Biophys. Res. Commun., 178, 570577.[ISI][Medline]
Saeki,T., Ihara,M., Fukuroda,T., Yamagiwa,M. and Yano,M. (1991) Biochem. Biophys. Res. Commun., 179, 286292.[ISI][Medline]
Saeki,T., Ihara,M., Fukuroda,T., Yamagiwa,M. and Yano,M. (1992) Biochem. Int., 28, 305312.[ISI][Medline]
Saida,K., Mitsui,Y. and Ishida,N. (1989) J. Biol. Chem., 264, 1461314616.
Sakurai,T., Yanagisawa,M., Takuwa,Y., Miyazaki,H., Kimura,S., Goto K. and Masaki,T. (1990) Nature, 348, 732735.[CrossRef][ISI][Medline]
Skamoto,A., Yanagisawa,M., Sakurai,T., Takuwa,Y., Yanagisawa,H. and Masaki,T. (1991) Biochem. Biophys. Res. Commun., 178, 656663.[ISI][Medline]
States,D.J., Haberkorn,R.A. and Ruben,D.J. (1982) J. Magn. Reson., 48, 286292.[ISI]
Takasaki,C., Tamiya,N., Bdolah,A., Wollberg,Z. and Kochva,E. (1988) Toxicon, 26, 543548.[CrossRef][ISI][Medline]
Tripos Molecular Modelling Software (1994) SYBYL Version 6.1. Tripos Associates, St Louis, MO, USA.
Wallace,B.A., Janes,R.W., Bassolino,D.A. and Krystek,S.R. (1995) Protein Sci., 4, 7583.
Watanabe,T.X., Itahara,Y., Nakajima,K., Kumagaye,S., Kimura,T. and Sakakibara,S. (1991) J. Cardiovasc. Pharmacol., 17, S5S9.[ISI][Medline]
Williamson,M.P., Havel,T.F. and Wüthrich,K. (1985) J. Mol. Biol., 182, 295315.[ISI][Medline]
Wüthrich,K., Billeter,M. and Braun,W. (1983) J. Mol. Biol., 169, 949961.[ISI][Medline]
Yanagisawa,M., Kurihara,H., Kimura,S., Tomobe,Y., Kobayashi,M., Mitsui,Y., Yazaki,Y., Goto,K. and Masaki,T. (1988) Nature, 332, 411415.[CrossRef][ISI][Medline]
Received August 31, 2001; revised November 29, 2001; accepted December 7, 2001.