Biochimica et Biophysica Acta, 393 (1975) 31-36
© Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands BBA 37027 CIRCULAR
DICHROISM
AND
CONFORMATIONAL
T R A N S I T I O N OF
DOL1CHOS BIFLORUS AND ROBINIA PSEUDOACACIA LECTINS
MAURICE PI~REa, ROLAND BOURRILLON" and BRUNO JIRGENSONS b "Centre de Recherches sur les Prot~ines, Facultd de M~decine Saint Louis-LariboisiOre, 45 rue des Saints POres 75006 Paris (France) and bDepartment of Biochemistry, the University of Texas, System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77025 (U.S.A.)
(Received September 24th, 1974) (Revised manuscript received January 24th, 1975")
SUMMARY The conformation of the lectins from Dolichos biflorus and Robinia pseudoacacia was studied by means of circular dichroism (CD). It was found that N-acetylD-galactosamine induced significant changes in the near-ultraviolet CD spectrum of Dolichos lectin but was ineffective with the lectin from Robinia. Tyrosine and tryp-
tophan chromophores were chiefly involved in this saccharide-lectin interaction. The far-ultraviolet CD spectra indicated that both lectins have a significant content of the pleated sheet conformation, but not much, if any, a-helix. The predominant conformation in these lectins is the aperiodic bend structure which is stabilised chiefly by hydrophobic interactions. This was ascertained by the effect of sodium dodecylsulfate on these proteins.
INTRODUCTION Saccharide-binding proteins, called lectins, have become powerful molecular probes of membrane structure and topology [1]. In certain cases lectins binding to a saccharidic receptor site of the cell membrane induce specific cytoagglutination or stimulate mitosis; consequently the molecular basis of the protein-saccharide interaction deserves investigation. Such studies should include the investigation of the effect of saccharide binding on protein conformation in solution, and circular dichroism is one of the most suitable methods for this purpose. Thus far very little has been done in this field [2]. One of the major aims of this communication is to report new results on saccharide-lectin interaction as tested by CD. Also it was interesting to compare the polypeptide main chain ("backbone") conformation of various lectins, as only Concanavalin A thus far has been extensively studied in this respect by various methods [3-10].
* Publication delayed due to French postal strike.
32 MATERIALS AND METHODS
The lectins The Dolichos biflorus lectin was prepared according to Font et al. [1 l]. The Robinia pseudoacacia lectin was purified by Font according to Bourrillon and Font [12]. The lyophilized specimens contained salts that were removed by dialysis against the appropriate buffer solutions. Chemical reagents The saccharides and alkyl sulfates were reagent grade substances from Mann Research Laboratories. All other chemicals were reagent grade. Circular dichroism measurements CD was measured with a Durrum-Jasco model CD-SP discograph, improved by Donald P. Sproul of Sproul Scientific Instruments, as described previously [10]. The effects of the additives were tested between 0.5 to 24 h after mixing the solutions and the reproducibility was tested by repeating the recording several times. The concentrations of the lectins were determined from known extinction coefficients [11, 12]. The data are expressed as mean residue ellipticities in degrees, cmz. drool-1 taking a mean residue weight of 110 for both lectins. RESULTS
CD and conformational transitions of Dolichos and Robinia lectins The Dolichos biflorus lectin, specifically agglutinates A1 type red cells [11, 13] and binds N-acetyl-D-galactosamine, the specific antigenic determinant of this group. Thus, the effects of this saccharide on the conformation of this lectin are of interest. The near-ultraviolet CD spectra of the lectin in the presence or absence of N-acetyl-Dgalactosamine are shown in Fig. 1. Significant effects of the saccharide were observed between 265-300 nm; especially at 280-286 nm and at 290 nm. Since the reproductibility of the mean residue ellipticity values, in this spectral zone was :L 1.0-1.6, the differences between the side-chain chromophore effects in the absence and presence of the saccharide are significant. Testing the p H effects, it was found that the saccharide affected the tertiary structure of the lectin at p H 6.8-7.5 but was ineffective at pH 8.25. The near-ultraviolet CD spectrum of the Robinia pseudoaeacia lectin is shown in Fig. 2. Since this lectin shows no specificity in agglutinating red cells of the ABO system, it was of interest to check the N-acetyI-D-galactosamine effect. As expected, no saccharide effect could be detected under conditions similar to those of the Dolichos lectin. Fig. 3 demonstrates the CD of the Dolichos and Robinia lectins in the far ultraviolet. For these determinations, the salts-containing specimens were dialyzed against 0.025 M sodium phosphate buffer in order to avoid the strongly absorbing CI in the far ultraviolet at 185-200 nm. The Dolichos lectin displayed weak negative bands at 217 and 230 nm and a positive band at 197 nm. The Robinia agglutinin produced a broad negative band at 216-230 nm and a positive band at 195-198 rim. The effect of sodium dodecylsulfate on the conformation of Dolichos and
33 50 ¸
40
30 o E
~E u x
20
10
240
260
280
300
,~,nm
Fig. 1. Effect of N-acetyl-D-galactosamine on the CD of Doliehos biflorus lectin in the near-ultraviolet spectral zone. Curve 1, the 0.0967 % lectin in a buffer composed of 0.025 M sodium phosphate buffer, 0 . 1 5 M NaCI, pH 6.8, without saccharide. Curve 2, same as in Curve 1 but with N-acetyl-ogalactosamine added in the amount of 50 mol of saccharide per tool of lectin assuming a molecular weight 120 000 for the lectin [22]. Curve 3, same as in Curve 2 but with 100 tool of saccharide per tool of lectin. Curve 4, CD of the lectin at pH 8.25 in 0.025 M phosphate buffer. Curve 5, same as in Curve 4 but in the presence of N-acetyl-D-galactosamine. The optical path through the solution was 2.0 cm. The recordings were made immediately after mixing the saccharide and no changes 1o were observed on standing. Lectin concentrations were determined from E 1 ~°m of 11.3 at 280 nm [11 ].
Robinia l e c t i n s a l s o w a s t e s t e d . 0 . 0 5 - 0 . 2 1 % l e c t i n s o l u t i o n s w e r e t e s t e d in t h e p r e s e n c e o f 0 . 0 2 M d o d e c y l s u l f a t e in t h e 2 5 0 - 3 2 0 n m s p e c t r a l z o n e , a n d it w a s f o u n d t h a t t h e CD bands were reduced. Five times diluted solutions at the same lectin/detergent r a t i o s , in t h e f a r u l t r a v i o l e t p r o d u c e d C D s p e c t r a w i t h n e g a t i v e b a n d m a x i m a a t 270 280 290 301 20t I
~
o4
% -io ~
20!
~'- 2s4
) X,nm
250 260 270
Fig. 2. CD of Robinia pseudoacacia lectin in the near ultraviolet. 0.11% solutions of the lectin in 0.025 M sodium phosphate buffer, 0.15 M NaCl, pH 6.8, were used in 2.0-cm cuvettes. Circles, lectin without saccharide; triangles, lectin with added N-acetyl-o-galactosamine (100 rnol per mol of lectin, tool. wt 72 000). Lectin concentrations were determined from ~rq% l cm of 9.65 at 280rim [12].
34 r 10
i
°
2," ",,
i
E
X,nm 0
hgo 2&o
225
'
240
~
5
L
22O 215
Fig. 3. CD of the Dolichos biflorus (Curve 1) and Robinia pseudoacacia lectins (Curve 2) in the farultraviolet spectral zone. The ellipticity curves were calculated from several CD recordings. The optical path through the solutions was 0.1 cm, the concentration of the proteins was 0.01-0.02%. The experimental error at 200-240 nm was ±50-80 degrees.cm2-dmol -', and it was approx. 3 100200 below 200 nm. The lectins were disolved in 0.005 M sodium phosphate, pH 7.0. 206-208 nm and 220-222 nm and a positive band centered at 190-192 nm, and the strength of the bands was enhanced by lowering the pH from 7.0 to 1.8-2.2. Thus the dodecylsulfate disorganized the tertiary structure and induced the formation of the a-helix in some parts of the main chain, as it was observed earlier with Concanavaline A [3, 10]. DISCUSSION The major objectives of this study were: (l) to test the interacting saccharide effect on lectins conformation; and (2) compare the main-chain conformation of various lectins. The effect of N-acetyl-o-galactosamine on the Dolichos lectin appeared chiefly in the 280-3130-nm spectral zone that expresses the chiral characteristics of the tyrosine and tryptophan chromophores [14]. The changes at 260-275 nm were mostly within the limits of experimental error. Since the band amplitude was slightly diminished by the saccharide, it is possible that in this case the interaction resulted in a slight disorganisation of the tertiary structure. The increase in intensity of the CD bands at p H 8.25 (Fig. 1, Curve 4) occurring in the 280-290-nm spectral zone is probably due to the ionization of some tyrosine. Furthermore, increasing the pH from 6.8 to 8.25 led to a loss of the spectral modification in the presence of the saccharide (Fig. 1, Curve 5). This indicates that some ionizable side-chain groups could be involved in the binding site. Similar observations were made with Concanavalin A on the basis of circular dichroism measurements [2] and chemical modifications [15, 16]. The Robinia lectin was not affected by this saccharide at all which is in accord with the specificity of N-acetyl-D-galactosamine with respect to agglutination inhibition. Since the Dolichos and Robinia lectins are devoid of cystine [11-13], all of the CD bands in the near ultraviolet arise from vicinal effects of the aromatic chromophores.
35
Interpretation of the far-ultraviolet spectra in terms of conformation is possible on the basis of several criteria [17-20]. One of these, probably the most suitable in our case, is to compare the CD of proteins of unknown conformation with those of known structure [19]. The complete three-dimensional structure of Concanavalin A is known [6, 7] from high-resolution X-ray structural analysis, hence the CD curves of this lectin [2-5, 10] should be of decisive help in elucidating the main-chain conformation of the other lectins. It is shown that about 4 0 ~ of the main chain in Concanavalin A, has the hydrogen-bonded /3-pleated sheet conformation, the rest being folded in the aperiodic bends stabilized chiefly by hydrophobic interactions. The far-ultraviolet spectrum of Concanavalin A has a broad negative band at 208240 nm with a maximum at 223-224 nm and a shoulder at 210-212 nm; and a positive band centered at 196-197 nm [3, 10]. The CD spectra of Dolichos and Robinia lectins at 190-240 nm are similar to the spectrum of Concanavalin A in so far as none of the curves have the typical shape of curves exhibited by proteins of high a-helix content (negative band at 221-222 nm and 206-209 nm and a positive band at 190-191 nm) but they have weak and broad negative band at 200-240 nm and a weak positive band at 192-198 nm. While the experimental error is relatively high in the positive zone below 200 nm (approx. ~ 200 degrees.cm2.dmol-1), a much higher precision (approx. dz 50-80 degrees.cm 2 .dmol -~) in the negative band regions permits an analysis from which we conclude that both lectins we studied are practically devoid of a-helix. Resolution into gaussian bands yields a negative component centered at 210-220 nm indicative of the pleated sheet structure. The positive band at 195-198 nm of the lectins is more typical for the fl-pleated sheet, as is the negative band at 217 nm for the Dolichos case, and it is noteworthy that a remarkably similar CD curve has been observed by Bures et al. [21] for the pea lectin. The negative band near 230 nm (Fig. 3, Curve 2) probably expresses side-chain, most likely tyrosine, effects. The ability of the sodium dodecylsulfate to disorganize the lectins (which are not prohibitively rigid due to extensive disulfide cross-links) and reconstruct parts of the chain in the a-helix form, is in accord with the predominance of the aperiodic bend conformation in them [10]. ACKNOWLEDGMENTS This work was supported by C.N.R.S. (E.R.A. 321), I . N . S . E . R . M . (Action T h 6 m a t i q u e No. 711.4232), D . G . R . S . T . ( C o n v e n t i o n No. 71-7-3183) a n d by the research g r a n t G-051 from the R o b e r t A. Welch F o u n d a t i o n , H o u s t o n , Texas. REFERENCES 1 2 3 4 5 6 7 8
Lis, H. and Sharon, N. (1973) Annu. Rev. Biochem. 42, 541-574 Pflumm, M. N., Wang, J. L. and Edelman, G. M. (1971) J. Biol. Chem. 246, 4369-4370 Kay, C. M. (1970) FEBS Lett. 9, 78-80 McCubbin, W. D., Oikawa, K. and Kay, C. M. (1971) Biochem. Biophys. Res. Commun. 43, 666-674 Zand, R., Agrawal, B. B. L. and Goldstein, I. J. (1971) Proc. Natl. Acad. Sci. U.S. 68, 2173-2176 Edelman, G. M., Cunninggham, B. A., Reeke, G. N., Becker, J. W., Waxdal, M. J. and Wang, J. L. (1972) Proc. Natl. Acad. Sci. U.S. 69, 2580-2584 Hardman, K. D. and Ainsworth, C. F. (1972) Biochemistry 11, 4910-4919 Hardman, K. D. and Ainsworth, C. F. (1973) Biochemistry 12, 4442~,448
36 9 Brewer, C. F., Sternlight, H., Marcus, D. M. and Grollman, A. P. (1973) Biochemistry 12, 44484457 10 Jirgensons, B. (1973) Biochim. Biophys. Acta 328, 314-322 11 Font, J., Leseney, A. M. and Bourrillon, R. (1971) Biochim. Biophys. Acta 243, 434-446 12 Bourrillon, R. and Font, J. (1968) Biochim. Biophys. Acta 154, 28-39 13 Etzler, M. E. and Kabat, E. A. (1970) Biochemistry 9, 869-877 14 Strickland, E. H. (1974) CRC Crit. Rev. Biochem. 2, 113-175 15 Hassing, G. S. and Goldstein, 1. J. (1972) Biochim. Biophys. Acta 271, 388-399 16 Hassing, G. S. and Goldstein, I. J. and Marini, M. (1971) Biochim. Biophys. Acta 243, 90-97 17 Bush, C. A. (1971) in Physical Technics in Biological Research (Oster, G., ed.), 2nd edn, Part A of Vol. I, pp. 347408, Academic Press, New York 18 Adler, A, J., Greenfield, N. J. and Fasman, G. D. (1973) in Methods in Enzymology (Hirs, C. H. W. and Timasheff, S. N., eds), Vol. 27, Part D, pp. 675-735, Academic Press, New York 19 Jirgensons, B. (1973) in Optical Activity of Proteins and Others Macromolecules (Vol. 5 in the series Molecular Biology, and Biophysics, Kleinzeller, A., Springer, G. F. and Wittmann, H. G., eds), 2nd edn, pp. 77-123, Springer-Verlag, Berlin 20 Sears, D. W. and Beychok, S. (1973) in Physical Principles and Technics in Protein Chemistry, (Leach, S. J,, ed.), Part C, pp. 445-593, Academic Press, New York 21 Bures, L., Entlicher, G. and Kocourek, J. (1972) Biochim. Biophys. Acta 285, 235-242 22 Pere, M., Font, J. and Bourrillon, R. (1974) Biochim. Biophys. Acta 365, 40-46
© Elsevier Scientific Publishing Company, Amsterdam - - Printed in The Netherlands BBA 37027 CIRCULAR
DICHROISM
AND
CONFORMATIONAL
T R A N S I T I O N OF
DOL1CHOS BIFLORUS AND ROBINIA PSEUDOACACIA LECTINS
MAURICE PI~REa, ROLAND BOURRILLON" and BRUNO JIRGENSONS b "Centre de Recherches sur les Prot~ines, Facultd de M~decine Saint Louis-LariboisiOre, 45 rue des Saints POres 75006 Paris (France) and bDepartment of Biochemistry, the University of Texas, System Cancer Center, M.D. Anderson Hospital and Tumor Institute, Houston, Texas 77025 (U.S.A.)
(Received September 24th, 1974) (Revised manuscript received January 24th, 1975")
SUMMARY The conformation of the lectins from Dolichos biflorus and Robinia pseudoacacia was studied by means of circular dichroism (CD). It was found that N-acetylD-galactosamine induced significant changes in the near-ultraviolet CD spectrum of Dolichos lectin but was ineffective with the lectin from Robinia. Tyrosine and tryp-
tophan chromophores were chiefly involved in this saccharide-lectin interaction. The far-ultraviolet CD spectra indicated that both lectins have a significant content of the pleated sheet conformation, but not much, if any, a-helix. The predominant conformation in these lectins is the aperiodic bend structure which is stabilised chiefly by hydrophobic interactions. This was ascertained by the effect of sodium dodecylsulfate on these proteins.
INTRODUCTION Saccharide-binding proteins, called lectins, have become powerful molecular probes of membrane structure and topology [1]. In certain cases lectins binding to a saccharidic receptor site of the cell membrane induce specific cytoagglutination or stimulate mitosis; consequently the molecular basis of the protein-saccharide interaction deserves investigation. Such studies should include the investigation of the effect of saccharide binding on protein conformation in solution, and circular dichroism is one of the most suitable methods for this purpose. Thus far very little has been done in this field [2]. One of the major aims of this communication is to report new results on saccharide-lectin interaction as tested by CD. Also it was interesting to compare the polypeptide main chain ("backbone") conformation of various lectins, as only Concanavalin A thus far has been extensively studied in this respect by various methods [3-10].
* Publication delayed due to French postal strike.
32 MATERIALS AND METHODS
The lectins The Dolichos biflorus lectin was prepared according to Font et al. [1 l]. The Robinia pseudoacacia lectin was purified by Font according to Bourrillon and Font [12]. The lyophilized specimens contained salts that were removed by dialysis against the appropriate buffer solutions. Chemical reagents The saccharides and alkyl sulfates were reagent grade substances from Mann Research Laboratories. All other chemicals were reagent grade. Circular dichroism measurements CD was measured with a Durrum-Jasco model CD-SP discograph, improved by Donald P. Sproul of Sproul Scientific Instruments, as described previously [10]. The effects of the additives were tested between 0.5 to 24 h after mixing the solutions and the reproducibility was tested by repeating the recording several times. The concentrations of the lectins were determined from known extinction coefficients [11, 12]. The data are expressed as mean residue ellipticities in degrees, cmz. drool-1 taking a mean residue weight of 110 for both lectins. RESULTS
CD and conformational transitions of Dolichos and Robinia lectins The Dolichos biflorus lectin, specifically agglutinates A1 type red cells [11, 13] and binds N-acetyl-D-galactosamine, the specific antigenic determinant of this group. Thus, the effects of this saccharide on the conformation of this lectin are of interest. The near-ultraviolet CD spectra of the lectin in the presence or absence of N-acetyl-Dgalactosamine are shown in Fig. 1. Significant effects of the saccharide were observed between 265-300 nm; especially at 280-286 nm and at 290 nm. Since the reproductibility of the mean residue ellipticity values, in this spectral zone was :L 1.0-1.6, the differences between the side-chain chromophore effects in the absence and presence of the saccharide are significant. Testing the p H effects, it was found that the saccharide affected the tertiary structure of the lectin at p H 6.8-7.5 but was ineffective at pH 8.25. The near-ultraviolet CD spectrum of the Robinia pseudoaeacia lectin is shown in Fig. 2. Since this lectin shows no specificity in agglutinating red cells of the ABO system, it was of interest to check the N-acetyI-D-galactosamine effect. As expected, no saccharide effect could be detected under conditions similar to those of the Dolichos lectin. Fig. 3 demonstrates the CD of the Dolichos and Robinia lectins in the far ultraviolet. For these determinations, the salts-containing specimens were dialyzed against 0.025 M sodium phosphate buffer in order to avoid the strongly absorbing CI in the far ultraviolet at 185-200 nm. The Dolichos lectin displayed weak negative bands at 217 and 230 nm and a positive band at 197 nm. The Robinia agglutinin produced a broad negative band at 216-230 nm and a positive band at 195-198 rim. The effect of sodium dodecylsulfate on the conformation of Dolichos and
33 50 ¸
40
30 o E
~E u x
20
10
240
260
280
300
,~,nm
Fig. 1. Effect of N-acetyl-D-galactosamine on the CD of Doliehos biflorus lectin in the near-ultraviolet spectral zone. Curve 1, the 0.0967 % lectin in a buffer composed of 0.025 M sodium phosphate buffer, 0 . 1 5 M NaCI, pH 6.8, without saccharide. Curve 2, same as in Curve 1 but with N-acetyl-ogalactosamine added in the amount of 50 mol of saccharide per tool of lectin assuming a molecular weight 120 000 for the lectin [22]. Curve 3, same as in Curve 2 but with 100 tool of saccharide per tool of lectin. Curve 4, CD of the lectin at pH 8.25 in 0.025 M phosphate buffer. Curve 5, same as in Curve 4 but in the presence of N-acetyl-D-galactosamine. The optical path through the solution was 2.0 cm. The recordings were made immediately after mixing the saccharide and no changes 1o were observed on standing. Lectin concentrations were determined from E 1 ~°m of 11.3 at 280 nm [11 ].
Robinia l e c t i n s a l s o w a s t e s t e d . 0 . 0 5 - 0 . 2 1 % l e c t i n s o l u t i o n s w e r e t e s t e d in t h e p r e s e n c e o f 0 . 0 2 M d o d e c y l s u l f a t e in t h e 2 5 0 - 3 2 0 n m s p e c t r a l z o n e , a n d it w a s f o u n d t h a t t h e CD bands were reduced. Five times diluted solutions at the same lectin/detergent r a t i o s , in t h e f a r u l t r a v i o l e t p r o d u c e d C D s p e c t r a w i t h n e g a t i v e b a n d m a x i m a a t 270 280 290 301 20t I
~
o4
% -io ~
20!
~'- 2s4
) X,nm
250 260 270
Fig. 2. CD of Robinia pseudoacacia lectin in the near ultraviolet. 0.11% solutions of the lectin in 0.025 M sodium phosphate buffer, 0.15 M NaCl, pH 6.8, were used in 2.0-cm cuvettes. Circles, lectin without saccharide; triangles, lectin with added N-acetyl-o-galactosamine (100 rnol per mol of lectin, tool. wt 72 000). Lectin concentrations were determined from ~rq% l cm of 9.65 at 280rim [12].
34 r 10
i
°
2," ",,
i
E
X,nm 0
hgo 2&o
225
'
240
~
5
L
22O 215
Fig. 3. CD of the Dolichos biflorus (Curve 1) and Robinia pseudoacacia lectins (Curve 2) in the farultraviolet spectral zone. The ellipticity curves were calculated from several CD recordings. The optical path through the solutions was 0.1 cm, the concentration of the proteins was 0.01-0.02%. The experimental error at 200-240 nm was ±50-80 degrees.cm2-dmol -', and it was approx. 3 100200 below 200 nm. The lectins were disolved in 0.005 M sodium phosphate, pH 7.0. 206-208 nm and 220-222 nm and a positive band centered at 190-192 nm, and the strength of the bands was enhanced by lowering the pH from 7.0 to 1.8-2.2. Thus the dodecylsulfate disorganized the tertiary structure and induced the formation of the a-helix in some parts of the main chain, as it was observed earlier with Concanavaline A [3, 10]. DISCUSSION The major objectives of this study were: (l) to test the interacting saccharide effect on lectins conformation; and (2) compare the main-chain conformation of various lectins. The effect of N-acetyl-o-galactosamine on the Dolichos lectin appeared chiefly in the 280-3130-nm spectral zone that expresses the chiral characteristics of the tyrosine and tryptophan chromophores [14]. The changes at 260-275 nm were mostly within the limits of experimental error. Since the band amplitude was slightly diminished by the saccharide, it is possible that in this case the interaction resulted in a slight disorganisation of the tertiary structure. The increase in intensity of the CD bands at p H 8.25 (Fig. 1, Curve 4) occurring in the 280-290-nm spectral zone is probably due to the ionization of some tyrosine. Furthermore, increasing the pH from 6.8 to 8.25 led to a loss of the spectral modification in the presence of the saccharide (Fig. 1, Curve 5). This indicates that some ionizable side-chain groups could be involved in the binding site. Similar observations were made with Concanavalin A on the basis of circular dichroism measurements [2] and chemical modifications [15, 16]. The Robinia lectin was not affected by this saccharide at all which is in accord with the specificity of N-acetyl-D-galactosamine with respect to agglutination inhibition. Since the Dolichos and Robinia lectins are devoid of cystine [11-13], all of the CD bands in the near ultraviolet arise from vicinal effects of the aromatic chromophores.
35
Interpretation of the far-ultraviolet spectra in terms of conformation is possible on the basis of several criteria [17-20]. One of these, probably the most suitable in our case, is to compare the CD of proteins of unknown conformation with those of known structure [19]. The complete three-dimensional structure of Concanavalin A is known [6, 7] from high-resolution X-ray structural analysis, hence the CD curves of this lectin [2-5, 10] should be of decisive help in elucidating the main-chain conformation of the other lectins. It is shown that about 4 0 ~ of the main chain in Concanavalin A, has the hydrogen-bonded /3-pleated sheet conformation, the rest being folded in the aperiodic bends stabilized chiefly by hydrophobic interactions. The far-ultraviolet spectrum of Concanavalin A has a broad negative band at 208240 nm with a maximum at 223-224 nm and a shoulder at 210-212 nm; and a positive band centered at 196-197 nm [3, 10]. The CD spectra of Dolichos and Robinia lectins at 190-240 nm are similar to the spectrum of Concanavalin A in so far as none of the curves have the typical shape of curves exhibited by proteins of high a-helix content (negative band at 221-222 nm and 206-209 nm and a positive band at 190-191 nm) but they have weak and broad negative band at 200-240 nm and a weak positive band at 192-198 nm. While the experimental error is relatively high in the positive zone below 200 nm (approx. ~ 200 degrees.cm2.dmol-1), a much higher precision (approx. dz 50-80 degrees.cm 2 .dmol -~) in the negative band regions permits an analysis from which we conclude that both lectins we studied are practically devoid of a-helix. Resolution into gaussian bands yields a negative component centered at 210-220 nm indicative of the pleated sheet structure. The positive band at 195-198 nm of the lectins is more typical for the fl-pleated sheet, as is the negative band at 217 nm for the Dolichos case, and it is noteworthy that a remarkably similar CD curve has been observed by Bures et al. [21] for the pea lectin. The negative band near 230 nm (Fig. 3, Curve 2) probably expresses side-chain, most likely tyrosine, effects. The ability of the sodium dodecylsulfate to disorganize the lectins (which are not prohibitively rigid due to extensive disulfide cross-links) and reconstruct parts of the chain in the a-helix form, is in accord with the predominance of the aperiodic bend conformation in them [10]. ACKNOWLEDGMENTS This work was supported by C.N.R.S. (E.R.A. 321), I . N . S . E . R . M . (Action T h 6 m a t i q u e No. 711.4232), D . G . R . S . T . ( C o n v e n t i o n No. 71-7-3183) a n d by the research g r a n t G-051 from the R o b e r t A. Welch F o u n d a t i o n , H o u s t o n , Texas. REFERENCES 1 2 3 4 5 6 7 8
Lis, H. and Sharon, N. (1973) Annu. Rev. Biochem. 42, 541-574 Pflumm, M. N., Wang, J. L. and Edelman, G. M. (1971) J. Biol. Chem. 246, 4369-4370 Kay, C. M. (1970) FEBS Lett. 9, 78-80 McCubbin, W. D., Oikawa, K. and Kay, C. M. (1971) Biochem. Biophys. Res. Commun. 43, 666-674 Zand, R., Agrawal, B. B. L. and Goldstein, I. J. (1971) Proc. Natl. Acad. Sci. U.S. 68, 2173-2176 Edelman, G. M., Cunninggham, B. A., Reeke, G. N., Becker, J. W., Waxdal, M. J. and Wang, J. L. (1972) Proc. Natl. Acad. Sci. U.S. 69, 2580-2584 Hardman, K. D. and Ainsworth, C. F. (1972) Biochemistry 11, 4910-4919 Hardman, K. D. and Ainsworth, C. F. (1973) Biochemistry 12, 4442~,448
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