1,8
1 Division of Biological Science, Graduate School of Science, NaGya University, Furo-cho, Chikusa, NaG
ya 464-8602,
3 Department of Bioapplied Chemistry, Faculty of Engineering, Osaka City University, 3-3-138 Sugimoto, Sumiyoshi, Osaka 558-8585,
5 Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita 565-0871 and
6 Japan Advanced Institute of Science and Technology, Hokuriku, 15 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: barnase/mini-protein/molecular modeling/NMR
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The ancestors of present-day proteins were produced during the course of pre- and early biological evolution. Studies of putative mechanisms of protein evolution should provide insights into protein design. The globular domain of a protein consists of structural units, termed modules, which are defined as compact and contiguous segments of 1030 amino acid residues (G, 1981
, 1983
). There are many cases where the boundaries of a module correlate with intron positions within the gene; for example, hemoglobin (G
, 1981
; Jensen et al., 1981
), lysozyme (G
, 1983
; Isaacs et al., 1985
), triose phosphate isomerase (Gilbert et al., 1986
; G
and Nosaka, 1987
; Gilbert and Glynias, 1993
; Tittiger et al., 1993
) and so forth (G
, 1985
). The observed correlation suggests that each module of a given ancestral protein was encoded by an exon, and the proteins evolved by assembling modules through exon shuffling mediated by introns (G
and Nosaka, 1987
).
Barnase, a bacterial RNase from Bacillus amyloliquefaciens, is a single domain protein consisting of 110 amino acid residues (Nishimura and Nomura, 1959; Hartley and Barker, 1972
). Its conformation has been elucidated by X-ray crystallography (Mauguen et al., 1982
) and by NMR spectroscopy (Bycroft et al., 1991
) and shows three
-helices, a two-stranded parallel ß-sheet and a five-stranded anti-parallel ß-sheet. The boundaries of the modules were assigned to residues 24, 52, 73, 88 and 98 by Noguti et al. (1993). The six modules are M1, 124; M2, 2552; M3, 5373; M4, 7488; M5, 8998 and M6, 99110 (Figure 1a
). Modules M1 and M2 have one and two
-helices, respectively. Module M2 forms a parallel ß-sheet near the N- and C-terminal ends.
|
If this view of protein evolution is valid, one could acquire an artificial protein by removing one or more modules from a globular domain of a present-day protein; the polypeptide chain consisting of the remaining modules will be able to fold into a stable conformation similar to that of the intact protein, except for the absent parts. Design of such a mini-protein is one step in acquiring a new functional protein by module assembly.
Among the six modules of barnase, M2 forms the least number of hydrogen bonds and hydrophobic contacts with the remaining protein (Noguti et al., 1993; Noguti and G
, 1995
). The influence of the excision of one module on conformational stability of the remaining portion of barnase was expected to be the least when M2 is removed. We designed a mini-protein by removing the M2 module from barnase. This mini-barnase was chemically synthesized and its structure was examined by CD and NMR. NMR revealed that the mini-barnase folded into a stable conformation with a similar hydrophobic core structure to that of barnase.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
When we removed module M2 (segment 2552) from barnase two chains, M1 and M3M6, remained. The carbonyl carbon atom at the C-terminus of M1 in barnase is 6.5 Å away from the amide nitrogen atom at the N-terminus of M3. If the two atoms are connected chemically, much stress will be placed on the conformation of the M1 and M3M6 chains and the conformation will be unstable.
Because a module has a compact conformation, the ends are relatively close in proximity. When we calculated the distance between a carbonyl carbon atom in the vicinity of the boundary between M1 and M2, and an amide nitrogen atom in the vicinity of the boundary between M2 and M3, the distance between the carbonyl carbon atom of Ile25 and the amide nitrogen atom of Gly52 was 3.9 Å, the shortest. To acquire a smooth join between the C-terminal end of M1 and the N-terminal end of M3, terminal residues 25 and 52 were included in the design of the mini-barnase.
The conformation of mini-barnase was modeled in the following procedures using the program BIOGRAF (Version 3.21, Molecular Simulations, Inc., Burlington, MA, 1992). First, segment 2651 was removed from the crystal structure of barnase [Baudet and Janin, 1991; Brookhaven Protein Data Bank (Bernstein et al., 1977), entry 1RNB]. Secondly, residues 25 and 52 were connected. Then energy minimization was performed, relaxing residues 2325 and 5254. Irregular geometry of chemical bonds and collision of atoms were never observed in the modeled conformation. Thirdly, conformation of the first residue and that of the second side chain, which were lacking in the crystal structure of barnase, were modeled using atomic coordinates of the crystal structure of the complex of barnase and barstar (Guillet et al., 1993
; Brookhaven Protein Data Bank, entry 1BGS) after superimposing the
carbon, carbonyl carbon and amide nitrogen atoms of Glu2 in the two crystal structures of barnase (1RNB and 1BGS). To increase thermodynamic conformational stability and solubility of mini-barnase, Ile25 and Phe82 were replaced by Thr and Ala, respectively (see Results). Hereafter we refer to mini-barnase prior to the replacement of these residues as `mini-barnase (unmodified)' and that after the replacements as `mini-barnase (modified)', in order to distinguish between the two versions.
Four layers of water molecules were put on the modeled mini-barnase (unmodified) and energy related to water and protein system was minimized until the root mean square (r.m.s.) of atomic forces was less than 0.01 kcal mol1 Å1. Molecular dynamics simulation was performed on this system for 115 ps. The temperature was maintained at 298 K by the heat-bath-coupling method ( = 0.2 ps) (Berendsen et al., 1984
); the time step was 1 fs. In minimization and molecular dynamics simulation a linear distance dependent dielectric function,
= r, was applied and nonbonded interactions were truncated at 9 Å distance. Hydrogen atoms that can form hydrogen bonds were treated explicitly but other hydrogen atoms were united with heavy atoms. Side chains of Lys, Arg, Asp and Glu, and both ends of the main chain were ionized. During the simulation some water molecules vapored out of the water shell. After execution of the first 25 ps simulation, we filled defects of the four-layered water shell with water molecules. The water shell was equilibrated with 0.8 ps molecular dynamics simulation after 200 cycles of conjugate gradient minimization, then the simulation was continued. Supplement of water molecules was repeated in this manner every 10 ps after the first 25 ps. We used the program and energy parameters of BIOGRAF. Molecular dynamic simulation of mini-barnase (modified) was carried out in the same manner as that of mini-barnase (unmodified), except for a simulation time length of 75 ps.
Oobatake and Ooi (1993) developed a method for predicting thermodynamics of protein unfolding, whereby Gibbs free energy change in protein unfolding, GU, was calculated as a linear function of changes in accessible surface areas (ASAs) from those of the folded conformation to those of the extended conformation. Using this method, we estimated the contribution of residues to
GU for mini-barnase. Contribution of residue i to
GU was calculated using the following equation
|
|
Atomic ASA was calculated by the method of Shrake and Rupley (1973). The ASA value obtained contains an error caused by incompleteness of uniform distribution of 92 points over the surface of a sphere. The error is not negligible in the calculation of free energies (Oobatake and Ooi, 1993). We improved the accuracy of atomic ASA calculation using the following procedure. We generated a hundred different sets of atomic coordinates by rotating a molecule randomly in the same conformation, and calculated atomic ASAs for each set of the coordinates by the method of Shrake and Rupley (1973). Values of the respective atomic ASAs that were sufficiently precise for the calculation of the free energies were obtained by averaging the 100 sets of calculated ASAs.
Accessibility of a residue was defined as the ratio between the sum of the ASAs of atoms of the residue in the folded conformation and that in the extended form (G and Miyazawa, 1980
), a value which is indicative of whether the residue is located on the surface or in the interior of the molecule.
Chemical synthesis, and NMR and CD measurements of mini-barnase
Boc-amino acid derivatives, reagents for solid-phase peptide synthesis and 15N-labeled amino acids were purchased from Peptide Institute, Inc. (Osaka, Japan), Watanabe Chemical Ind., Ltd (Hiroshima, Japan) and Nihon Shoji Co., Ltd (Osaka, Japan), respectively. Chemical synthesis of the designed mini-barnase (modified) was carried out as previously described (Hojo and Aimoto, 1993). Briefly, partially protected peptide thioesters corresponding to mini-barnase (125+52), (5381) and partially protected peptide (82110) were prepared via a solid-phase method, using Boc-amino acids. For introduction of the residues Ala11, Tyr13, Leu14, Leu89, Tyr90, Ser91, Ser92, Asp93, Leu95 and Tyr97, 15N-labeled amino acid derivatives were used. The peptide segments were successively condensed by activation of thioester groups with silver ions to give a protected form of mini-barnase. After deprotection, mini-barnase was highly purified by HPLC. The purity was confirmed by analytical HPLC, amino acid analysis and matrix-assisted laser desorption ionization (MALDI) mass spectrometry.
Lyophilized mini-barnase was dissolved in 90% H2O/10% D2O. Acids remaining after the purification were excluded by ultrafiltration until the pH of solution was 5.1; simultaneously, mini-barnase was concentrated to 0.1 mM. Two-dimensional 1H-15N HSQC spectrum (Boyd et al., 1992) was measured at 25°C on a Varian Unity-plus 750 spectrometer. Resonance of the solvent water was suppressed by presaturation during the relaxation delay. The spectrum consisted of 128 complex t1 points with each free induction decay having 2048 complex t2 points. 160 transients were acquired per t1 value to gain a signal to noise ratio. The spectral width for 1H observation was 12 000 Hz and that for 15N was 3000 Hz. The spectrum was zero-filled to 2048 x 512 real points and was processed with shifted sinebell window function, in both dimensions. Linear prediction was applied to raise the spectral resolution of the 15N dimension. 1H and 15N chemical shifts were referenced to external TSP (0 p.p.m.) and NH4Cl (24.93 p.p.m.), respectively.
CD spectra were measured using a Jasco J-600 spectropolarimeter at room temperature in a quartz cell with a light path length of 1.0 cm. The concentration of mini-barnase was 0.6 µM, determined by measurement of UV absorbance at 280 nm. The extinction coefficient of the mini-barnase was estimated to be 21 750 M1 cm1 on the basis of the number of tryptophan and tyrosine residues in the peptide (Kuramitsu et al., 1990). The pH was adjusted with NaOH or HCl.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
To acquire a mini-barnase we chose to remove module M2. By removing the segment 2651 and connecting residues 25 and 52 of the remaining protein, we prepared a molecular model of mini-barnase lacking module M2 (Figure 1b). In this report, the same numbering of residues and modules have been used for mini-barnase as those for barnase, to facilitate correspondence. We considered the following points: (i) a conformationally smooth connection of polypeptide chains of both ends of the module M2; (ii) mechanical stability of the designed conformation; and (iii) thermodynamic stability of the conformation and solubility of the acquired model.
Mechanical stability of modeled mini-barnase
The conformation of a native protein is mechanically stable as the balance of all forces on its atoms is maintained at small amplitude thermal perturbations. The absence of 26 amino acid residues might perhaps disturb the balance of atomic forces and the mini-barnase would be unable to maintain conformation. We examined the mechanical stability of the modeled mini-barnase conformation by studying the molecular dynamics. The conformation of mini-barnase was retained during 115 ps in a water environment, except for the loop of module M4 (Figure 1). C
atoms in the conformation on the trajectory of the simulation at 115 ps were superimposed on the corresponding atoms in the 3D structure of barnase, except for those in the loop formed by residues 7983. The root mean square deviations (r.m.s.d.) of the superimposed C
atoms was 1.3 Å. Thus, conformation of the modeled mini-barnase was essentially mechanically stable. For C
atoms in the loop, the r.m.s.d. value was 4.7 Å. Movement of the loop narrowed the cleft between modules M1 and M4 (Figure 1
).
Increase of thermodynamic conformational stability and solubility of mini-barnase by amino acid replacement
An appropriate method for the prediction of thermodynamic stability of a protein conformation has yet to be established. Whether or not the designed mini-barnase takes on the expected conformation has to be examined experimentally. By doing so, one can test the prediction and improve methods used for prediction. In the process of designing a mini-barnase, we directed attention to the amino acid residues that might contribute to the decrease in thermodynamic conformational stability or solubility of the molecule, and to substitute suitable residues in their place. High solubility in aqueous solution is a sine qua non for NMR measurements.
After excision of module M2, the interface between M2 and the other modules in barnase becomes part of the surface of the mini-barnase. Twelve residues of the mini-barnase gained solvent accessibility by more than 0.1 from those of barnase. Of the 12, four were hydrophobic: Val3, Ile25, Tyr78 and Phe82 (Figure 2). Exposure of a hydrophobic residue on the protein surface usually decreases thermodynamic conformational stability and/or solubility of the protein in water. We focused attention on four hydrophobic residues and estimated their contribution in decreasing the conformational stability and the solubility of mini-barnase. Considering conformational changes in the vicinities of the four residues during the 115 ps simulation, we calculated two sets of averaged atomic ASAs in the folded state; one was a set of averages of respective atomic ASAs in conformations in the period 8085 ps (set 1) and the other in the period 110115 ps (set 2). Then we calculated, for each set, contributions from the four residues to the Gibbs free energy change of unfolding,
giU, and those to Gibbs free energy change of aggregation,
giA (Table I
).
|
|
In the final process of design, the molecular dynamics of mini-barnase (modified) was carried out for 75 ps to examine the mechanical stability of the molecule and to predict the effect of the amino acid replacements on the thermodynamic properties. The replacements of Ile25Thr and Phe82Ala did not alter the mechanical stability of modeled mini-barnase. Conformation was retained during the 75 ps simulation. The r.m.s.d. value of the corresponding C atoms, except for the loop in M4, was 1.3 Å between the simulated mini-barnase conformation at 75 ps and the X-ray structure of barnase. For C
atoms in the loop, the value was 5.1 Å. Contributions of Thr25 and Ala82 to
GU and
GA were calculated from the simulated conformations in the periods 4045 ps and 7075 ps (Table II
). Because the predicted values are all positive, the amino acid replacements Ile25Thr and Phe82Ala were expected to increase thermodynamic stability and solubility of mini-barnase.
|
The CD spectrum of mini-barnase at pH 2.5 exhibited a deep minimum at 200 nm, a finding indicative of a random coil conformation (Figure 3). Above pH 3.5, however, this minimum disappeared and a positive maximum near 190 nm appeared, which implies that the mini-barnase folded into a non-random conformation above pH 3.5.
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Module M2 had the most independent conformation among the six modules of barnase (Noguti et al., 1993; Noguti and G
, 1995
). Twenty-six residues of M2 were removed from barnase; deletion of such a fragment from a globular protein would appear difficult in molecular modeling. We attained success in modeling the conformation of mini-barnase which proved to be mechanically stable, presumably for the following reasons. First, N- and C-terminal residues of a module are often located close in space because of a compact and small conformation of a module. This characteristic of modules facilitates the connection of two chains that remain after removal of a module by shifting the excision points one or two residues from module boundaries. Secondly, most of the module conformations in barnase are mechanically stable (Takahashi et al., 1997
). Removal of a module from a globular protein seems to maintain the conformation of the remaining parts and mechanical stability is retained.
De Sanctis et al. (1988) made a mini-myoglobin of 108 amino acid residues, a peptide excised from myoglobin and which closely corresponds to the peptide encoded by the central exon but contains an additional 30 residues at the C-terminal end. The peptide binds heme stoichiometrically, forms a helical structure and exhibits the reversible oxygen-binding function of myoglobin itself. Pessi et al. (1993) designed a mini-protein of 61 amino acid residues, referred to as a minibody, using the central portion of the heavy chain variable domain of an immunoglobulin as a template, which constitutes a compact sub-domain consisting of a ß-sandwich of two triple-stranded ß-sheets and contains the hypervariable loops H1 and H2. Hydrophobic residues buried in the parent molecule, but exposed in the minibody, were suitably replaced and a metal-binding site was engineered by introducing one histidine in H1 and two in H2. The minibody exhibited all-ß structural features following CD spectra, cooperative transition in urea-induced unfolding and Zn-binding activity. The design of mini-proteins will become an important technology in protein engineering, because the small size of designed proteins facilitates theoretical and experimental treatments such as molecular dynamics simulation and structure determination by NMR.
Exchange of modules or exons between homoloGus proteins has been previously reported: Kumagai et al. (1992) succeeded in functional conversion of
-lactalbumin and the c-type lysozyme by exon exchange. These two proteins are functionally different but structurally homoloG
us and their genes have the same exonintron organization. Exchange of the second exon of
-lactalbumin with that of lysozyme produced a chimeric
-lactalbumin with lysozyme activity, whereas the replacement of 10 amino acids, which are directly involved in substrate-binding, did not produce
-lactalbumin with lysozyme activity. Yaoi et al. (1996) successfully converted the coenzyme specificity of isocitrate dehydrogenase (ICDH) from NADP-dependent to NAD-dependent by replacing a module involved in coenzyme binding with the corresponding module of NAD-dependent enzyme 3-isopropylmalate dehydrogenase (IPMDH). Replacement of a few residues interacting with the 2'-phosphate group of NADP did not result in conversion of coenzyme specificity and seemed to destabilize the structure of ICDH. The module-replaced ICDH was as stable as the wild-type ICDH. Wakasugi et al. (1994) in making a chimeric globin replaced the fourth module of the hemoglobin ß-subunit by the fourth module of the hemoglobin
-subunit. The chimeric globin associated with the hemoglobin ß-subunit through interactions between the fourth module of the chimera and that of the ß-subunit. These studies demonstrate designs of functionally chimeric proteins by exchanging modules. Indeed modules are functional units as well as structural units of proteins.
Fersht's group has studied and obtained valuable information on the folding processes and conformational stability of barnase, mainly using systematic amino acid replacements (Matouschek et al., 1989). They also studied truncated barnase in which the N-terminal 22 or 36 residues were excised (Sancho and Fersht, 1992
; Kippen et al., 1994
). The truncated barnases did not appear to form stable conformations in the isolated state. Our approach to designing a mini-protein was to remove a single module of the protein. To confirm the success of the design of mini-barnase, we are in the process of determining its 3D structure.
![]() |
Acknowledgments |
---|
![]() |
Notes |
---|
4 Present address: Department of Industrial Chemistry, Tokai University, 1117 Kitakaname, Hiratsuka 259-1292, Japan
7 Present address: Faculty of Pharmaceutical Science, Osaka University, 1-6 Yamadaoka, Suita 565-0871, Japan
8 To whom correspondence should be addressed
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Berendsen,H.J.C., Postma,J.P.M., van Gunsteren,W.F., DiNola,A. and Haak,J.R. (1984) J. Chem. Phys., 81, 36843690.[ISI]
Bernstein,F.C., Koetzle,T.F., Williams,G.J.B., Meyer,E.E.,Jr, Brice,M.D., Rodgers,J.R., Kennard,O., Shimanouchi,T. and Tasumi,M. (1977) J. Mol. Biol., 112, 535542.[ISI][Medline]
Betz,S.F., Raleigh,D.P. and DeGrado,W.F. (1993) Curr. Opin Struct. Biol., 3, 601610.[ISI]
Betz,S.F., Bryson,J.W. and DeGrado,W.F. (1995) Curr. Opin. Struct. Biol., 5, 457463.[ISI][Medline]
Boyd,J., Soffe,N., John,B., Plant,D. and Hurd,R. (1992) J. Magn. Reson., 98, 660664.[ISI]
Bundi,A. and Wüthrich,K. (1979) Biopolymers, 18, 285297.[ISI]
Bycroft,M., Ludvigsen,S., Fersht,A.R. and Poulsen,F.M. (1991) Biochemistry, 30, 86978701.[ISI][Medline]
Dahiyat,B.I. and Mayo,S.L. (1997) Science, 278, 8287.
De Sanctis,G. Falcioni,G., Giardina,B., Ascoli,F. and Brunori,M. (1988) J. Mol. Biol., 200, 725733.[ISI][Medline]
Gilbert,W. and Glynias,M. (1993) Gene, 135, 137144.[ISI][Medline]
Gilbert,W., Marchionni,M. and McKnight,G. (1986) Cell, 46, 151153.[ISI][Medline]
G,M. (1981) Nature, 291, 9092.[ISI][Medline]
G,M. (1983) Proc. Natl Acad. Sci. USA, 80, 19641968.[Abstract]
G,M. (1985) Adv. Biophys., 19, 91131.[Medline]
G,M. and Miyazawa,S. (1980) Int. J. Peptide Protein Res., 15, 211224.[ISI][Medline]
G,M. and Nosaka,M. (1987) Cold Spring Harb. Symp. Quant. Biol., 52, 915924.[ISI][Medline]
Guillet,V., Lapthorn,A., Hartley,R.W. and Mauguen,Y. (1993) Structure, 1, 165176.[ISI]
Hartley,R.W. and Barker,E.A. (1972) Nature New Biol., 235, 1516.[ISI][Medline]
Hojo,H. and Aimoto,S. (1993) Bull. Chem. Soc. Jpn, 66, 30043008.[ISI]
Ikura,T., G,N. Kohda,D., Inagaki,F., Yanagawa,H., Kawabata,M., Kawabata,S., Iwanaga,S., Noguti,T. and G
,M. (1993) Proteins, 16, 341356.[ISI][Medline]
Isaacs,N.W., Machin,K.J. and Masakuni,M. (1985) Aust. J. Biol. Sci., 38, 1322.[ISI][Medline]
Jensen,E.Ø., Paludan,K., Hyldig-Nielsen,J.J., Jørgensen,P. and Marcker,K.A. (1981) Nature, 291, 677679.[ISI]
Jones,D.N.M., Bycroft,M., Lubienski,M.J. and Fersht,A.R. (1993) FEBS Lett., 331, 165172.[ISI][Medline]
Kippen,A.D., Sancho,J. and Fersht,A.R. (1994) Biochemistry, 33, 37783786.[ISI][Medline]
Kumagai,I., Takeda,S. and Miura,K. (1992) Proc. Natl Acad. Sci. USA, 89, 58875891.[Abstract]
Kuramitsu,S., Hiromi,K., Hayashi,H., Morino,Y. and Kagamiyama,H. (1990) Biochemistry, 29, 54695476.[ISI][Medline]
Matouschek,A., Kellis,J.T., Jr, Serrano,L. and Fersht,A.R. (1989) Nature, 340, 122126.[ISI][Medline]
Mauguen,Y., Hartley,R.W., Dodson,E.J., Dodson,G.G., Bricogne,G., Chothia,C. and Jack,A. (1982) Nature, 297, 162164.[ISI][Medline]
Nishimura,S. and Nomura,M. (1959) J. Biochem., 46, 161167.[ISI]
Noguti,T., Sakakibara,H. and G,M. (1993) Proteins, 16, 357363.[ISI][Medline]
Noguti,T. and G,M. (1995) In G
,M. and Schimmel,P. (eds), Tracing Biological Evolution in Protein and Gene Structures. Elsevier, Amsterdam, pp. 161174.
Oobatake,M. and Ooi,T. (1993) Prog. Biophys Mol. Biol., 59, 237284.[ISI][Medline]
Pessi,A., Bianchi,E., Crameri,A., Venturini,S., Tramontano,A. and Sollazzo,M. (1993) Nature, 362, 367369.[ISI][Medline]
Ramírez-Alvarado,M., Blanco,F.J. and Serrano,L. (1996) Nature Struct. Biol., 3, 604612.[ISI][Medline]
Sancho,J. and Fersht,A.R. (1992) J. Mol. Biol., 224, 741747.[ISI][Medline]
Shao,Z. and Arnold,F.H. (1996) Curr. Opin. Struct. Biol., 6, 513518.[ISI][Medline]
Shrake,A. and Rupley,J.A. (1973) J. Mol. Biol., 79, 351371.[ISI][Medline]
Takahashi,K., Oohashi,M., Noguti,T. and G,M. (1997) FEBS Lett., 405, 4754.[ISI][Medline]
Tittiger,C., Whyard,S. and Walker,V.K. (1993) Nature, 361, 470472.[ISI][Medline]
Wakasugi,K., Ishimori,K., Ima,K., Wada,Y. and Morishima,I. (1994) J. Biol. Chem., 269, 1875018756.
Wishart,D.S., Sykes,B.D. and Richards,F.M. (1991) J. Mol. Biol., 222, 311333.[ISI][Medline]
Yaoi,T., Miyazaki,K., Oshima,T., Komukai,Y. and G,M. (1996) J. Biochem., 119, 10141018.[Abstract]
Yoshida,K., Shibata,T., Masai,J., Sato,K., Noguti,T., G,M. and Yanagawa,H. (1993) Biochemistry, 32, 21622166.[ISI][Medline]
Received March 18, 1999; revised April 26, 1999; accepted April 30, 1999.