20 Biochimica et Biophysica A cta, 705 (1982) 20-25 Elsevier Biomedical Press

BBA 31216

LASER RAMAN STUDY ON CROTAMINE

YOSHIO K A W A N O a CARLOS J. L AUR E b and JOSl~ R. GIGLIO b

" lnstituto de Quimica, Universidade de S~o Paulo, Caixa Postal 20780, CEP 01000, Sdo Paulo and ~' Faculdade de Medicina, Universidade de Sdo Paulo, Departamento de Bioquimica, 14100, Ribeir~o Preto, S~o Paulo (Brazil)

(Received December 15 th, 1981 )

Key words: Laser Raman; Snake venom; Crotamine conformation

Crotamine is a toxin of low molecular weight, about 4880, basic protein isolated from snake venom of the South Brazilian rattlesnake, Crotalus durissus terrificus. The Raman spectra in the 400-1800 cm- 1 region of native crotamine in the lyophylized state and in aqueous solution were investigated. In the amide I region, two bands were observed at 1670 and 1650 cm- i in the solid and at 1670 and 1645 cm i in aqueous solution spectra, suggesting that crotamine may contain t - sheet and a-helix structures, with slight predominance of the first. This is supported by the presence of two lines at 1240 and 1278 c m - i in the amide III region, which are characteristic of j0-sheet and a-helix structures, respectively. There is also evidence of random coil structure in the amide III region. The three disulfide bridges take the gauche-gauche-gauche conformation about the CCSSCC linkage, as shown by the presence of the 510 cm-1 Raman line. From the intensity ratio of the tyrosine doublet at 830 and 858 cm- l , it can be concluded that the single N-terminal tyrosine residue is buried. The lack of a peak at 1361 cm i indicates that the two tryptophan residues are exposed to the solvent.

Introduction

The neurotoxin crotamine is a low molecular weight (4880), and very basic protein (isoelectric point 10.3), isolated from a South Brazilian ratt- lesnake, Crotalus durissus terrificus venom [1]. The primary structure was determined recently by Laure [2]. It is a single polypeptide chain of 42 amino acid residues with N-terminal tyrosine and C-terminal glycine, crosslinked by three disulfide bridges. The native crotamine is highly resistant to thermal denaturation [3].

Crotamine is a toxin quite dissimilar to other snake neurotoxins [4,5] and has a very specific toxic activity [6].

Recent optical rotatory dispersion results [3,7] suggest the presence of B-sheet, but the results are not enough to postulate significant a-helical con- tent.

0167-4838/82/0000-0000/$02.75 © 1982 Elsevier Biomedical Press

In recent years, laser Raman spectroscopy has been applied successfully to conformational analy- sis of many snake toxins [8-19]. This technique is very useful for obtaining structural information on proteins in a variety of phases, particularly regard- ing the secondary structure of protein backbone, local geometry of the disulfide crosslinks, specific interaction of tryptophan residues, specific H- bonding of buried tyrosine residues, the configura- tion of the methionine side-chain and the skeletal stretching vibration characteristic of the protein.

Crotamine has, among others, six half-cystine, two tryptophan, one tyrosine and one methionine residues, whose conformations can be inferred from the Raman spectrum.

In this paper, we report the Raman spectra of crotamine in lyophilized form and in aqueous solution, and the structure conformations sug- gested by the spectra are discussed.

Materials and Methods

Crotamine was isolated from Crotalus durissus terrificus snake venom as previously described [20].

The Raman spectra were obtained using a Jar- rell-Ash model 25-300 laser Raman spectrometer with a Spectra Physics model 165 argon ion laser as the excitation source.

The solution of crotamine used for the measure- ments was prepared by dissolving 1 or 2 mg of sample in 20/~1 of twice distilled water, in a small capillary quartz tube cell of 3 mm outer diameter sealed at one end with a glass window. The pH 5.6 was measured with a Expandomatic SS-2 Beckman pH meter. The lyophilized powder was packed into a shallow hole on the front end of a brass rod.

The Raman spectra over the range of 400 to 1800 cm -1 and 2500 to 2700 cm-~ (not shown) were recorded using 514.5 nm excitation line with a laser power at the sample of about 300 mW for aqueous solution and about 150 mW for the

21

lyophilized powder. A 10 c m - I spectral slit-width resolution for both samples was used at 20°C.

Results and Discussion

Comparison of Raman spectra in lyophilized state and in aqueous solution.

The Raman spectra of native crotamine in the lyophilized state and in aqueous solution at pH 5.6 and 20°C are shown, respectively, in Figs. 1 and 2.

Comparison of the spectra of crotamine in lyophilized form and in aqueous solution shows that there is an apparent change in relative inten- sity of the conformationally sensitive amide modes and some other bands of the protein. On the other hand, there are no changes in the frequencies of Raman lines of the amide I and III and the S-S stretching frequency, which are the main confor- mationally sensitive bands. The amide I band of solution spectrum is probably masked by the water line at 1645 cm -1. Therefore, it suggests that

B

Z

Z

'w

~E

1 4 4 2 1 3 4 2 Solid

"

LS'

I I I I I I I I I I I I l , I I

1 8 0 0 1 6 O 0 14 O 0 1 9 0 0 1 0 0 0 8 O 0 6 O 0 4 O 0

Fig. 1. Raman spectrum of native crotamine in the lyophilized state, at 20°C.

c m '

22

i.- m

Z Ma

Z m

Z 'K

=E

Amide I Amide Ill 1670 1 6 4 5

N ~ ~ 1550 1442 13j~1~ 78J 245 A'8~e63 S o l u t i o n

100, J ' W

" ~ 8 0 Tyr

I I 1 I I I I I 1 , i I 1 I I I

1800 1600 1400 1200 1 000 800 600 400

Fig. 2. Raman spectrum of native crotamine in aqueous solution, at pH 5.6 and 20°C.

¢ m-1

crotamine has identical conformat ions in both states. The change in relative intensity observed could be due to the change in the side-chain conformation. So, the lyophilization appears to have no significant effect on the secondary struc- ture of crotamine. For many snake neurotoxins, the Raman spectrum of solid and solution states has shown identical amide I and amide III bands, confirming that the peptide backbone conforma- tion is the same in both states [8-10,13,16,17]. For neurotoxins, which are small globular protein molecules with high stability originating from the many disulfide bonds holding the peptide back- bone tightly together, this is a reasonable finding.

Polypeptide backbone conformation It is well-known that the amide I and amide III

modes in the Raman spectrum are correlated with the structural properties of the protein molecules. Usually the amide I mode is found at 1655-1659 cm i, with strong intensity and sharp peak for

a-helical structure, a strong but broad band at 1665-1666 cm -1 for random coil, and a strong and sharp line at 1667-1672 cm i for B-sheet structure [21]. By using the amide I mode one can easily differentiate between the a-helical and non- a-helical structure, but not easily between the non- a-helical conformations, excepting when X-ray dif- fraction results are available.

The amide III band which occurs in the range 1200-1300 cm -~ in the Raman spectrum of a protein is indicative of or-helical structure when it appears at 1250-1280 cm 1/?-sheet when a strong band at 1229-1240 cm ], r andom coil when a medium band at 1243-1265 cm i [21], and B-turn for a band at 1290-1330 cm 1 [22] or at 1230-1300 cm ] [23-25].

The Raman spectra of both the solid and aque- ous samples of crotamine have a broad amide I band. In this region, we observe clearly the pres- ence of two bands, centred at 1670 cm ~ and 1650 c m - I in the solid, and at 1670 and 1645 c m ~ in

solution. In the solid sample a weak shoulder at about 1690 cm-1 is discernible. Despite the inter- ference of the water line near 1645 cm ~ in the amide I band of aqueous sample, the presence is clear in the solid spectrum of a band at 1650 cm 1 which is a clear indication of the a-helical back- bone conformation. The band at 1670 cm ~ is indicative of fl-sheet, apart from a small contribu- tion of random coil. The shoulder at 1690 cm could be due to the fl-turn structure [22-25].

The amide III region of the lyophilized spec- trum presents bands at 1240 and 1278 cm i, which are characteristic of /~-sheet and a-helical structures, respectively. In the aqueous solution spectrum, in this region, we observe a medium broad band in the range 1230-1290 cm i which can be the overlapping of the amide III of a-heli- cal, fl-sheet and also of random coil secondary structure. The lack of any strong Raman line in the 1200-1300 cm-~ region is strong evidence that /~-sheet structure is not the predominant secondary structure. The presence of the band at 1278 cm - I certainly suggests the existence of a-helical struc- ture. The observed broad band in this region could suggest the coexistence of random coil beside the /~-sheet and a-helical structures.

Another band which is used to characterize the a-helical conformation is a line appearing near 900 cm ~ in the Raman spectrum, attributable to skeletal stretching vibration of the peptide back- bone. We observe a band at 930 cm ~ which can be assigned to this vibration, indicating the ex- istence of a-helical structure. This result agrees with the Raman spectrum of many rattlesnake toxins which contain a-helix [11,13,16] in their secondary structures.

S-S and C-S stretching vibrations. Crotamine has three disulfide bridges, like cobramine B [18]. A single band at 510 cm ~ assigned to S-S stretch- ing vibration was obtained in the Raman spec- trum, indicating similar geometry for all disulfide bonds. According to the study on model com- pounds by Sugeta et al. [26] which has been ap- plied to the case of snake venom protein mole- cules, the gauche-gauche-gauche conformation of the CCSSCC linkage must be present on crota- mine. The S-S stretching vibration of three dis- ulfide linkages of cobramine was also observed in the Raman spectrum at 510 cm -~ [18].

23

In the course of this work no sulfhydryl vibra- tional band was found in the 2500-2700 cm -1 region (this section of the spectrum is omitted in Figs. 1 and 2). This observation is similar to those previously made on a snake neurotoxin [27] and a tobacco mosaic virus and its components [28].

A single methionine residue in crotamine is at the 28th position in the primary structure. Accord- ing to Nogami et al. [29], the C-S stretching modes from the methionine residue can be correlated to internal rotation angles surrounding the C-C bonds adjacents to the C-S bonds. Two C-S frequencies are expected at 760 and 719 cm -~ for the trans- trans form, at 746 and 697 cm-~ for the trans- gauche form, at 667 cm-~ for the gauche-trans form, at 723 and 645 cm-~ for the gauche-gauche form. In the solid spectrum, we observe a band at 655 cm -~ which can be assigned to the C-S stretching mode of the methionine residue in gauche-gauche form. Another peak expected at 723 cm i for this form was not observed, perhaps due to its very weak intensity being masked by the fluorescence background.

The tyrosine doublet near 858-830 cm i The spectrum of protein presents some bands

due to the vibrations of the aromatic side-chain that are themselves of interest because of their sensitivity to the local environment of the side- chain. In particular the intensity ratio of the two lines of tyrosine which appear at 850-830 cm-1 is correlated with the local environment and interac- tion of tyrosine residue in the protein [30]. This doublet originates from the Fermi ressonance be- tween the ring-breathing vibration and the over- tone of an out-of-plane ring-bending vibration of the para substituted benzene [30].

Crotamine has only one tyrosine residue and in the Raman spectra we observe a weak doublet band at 858-830 c m - I . Despite the superposition of background with a medium band at 880 cm in all spectra, which prevents the intensity ratio of these two lines being accurately estimated, it is clear in the inserted solution spectrum that the intensity ratio for 858-830 cm -~ is lower than unit, indicating that the single tyrosine residue is not readily accessible to water molecules, and is perhaps involved in moderate hydrogen bonding between the phenolic hydroxyl group and other

24

side-chain residues. This result agrees with one obtained by Hampe et al. [3], who, using dif- ference spectrophotometry on crotamine, con- cluded that in native conformation the N-terminal tyrosine residue is buried.

Tryptophan environment Crotamine has two tryptophan residues. Most

of the snake neurotoxins contain one or two tryp- tophan residues and its presence is believed to be essential for biological activity [31]. It is reported that the 1361 cm -1 band of tryptophan is very sensitive to the indole ring environment. Its being buried or exposed within a protein molecule can be correlated with the presence or absence of a sharp peak at 1361 c m - I in the Raman spectrum [321.

The absence of a sharp peak at 1361 cm ~ in the Raman spectra of crotamine indicates that the indole ring becomes accessible to water molecules, i.e., the two tryptophan residues are exposed to the solvent. All neurotoxins studied gave no sharp and strong Raman bands around 1360 cm 1, suggest- ing that all tryptophan residues in the neurotoxins are in similar exposed states. These results differ from that obtained by Hampe et al. [3], using difference spectrophotometry on crotamine, in the sense that the two tryptophan residues at the 32nd and 34th positions are buried in the native confor- mation.

Conclusion Summing up, from the Raman spectroscopy

results, the secondary structure of crotamine is seen to be composed of segments in B-sheet, a-helix and random coil structures, and probably B-turn conformations. It is known that sea snake neuro- toxins usually have the amide I band observed near 1670 cm 1, suggesting a large fraction of B-sheet conformations. On the other hand, toxins isolated from the venoms of rattlesnake present differing structures. For example, the Mojave toxin consists predominantly of the a-helical structure [13], whilst myotoxin a contains large amounts of a mixture of B-sheet and/3-turn structures [33].

The three disulfide bonds in crotamine assume the gauche-gauche-gauche conformation of the CCSSCC linkage. With a few exceptions, all of the

snake neurotoxins present this conformation for the disulfide bonds.

From the intensity ratio of the two lines at 858-830 c m - i it was concluded that the tyrosine residue is buried in the interior of the molecule. From the lack of a distinct band at 1361 cm ~ due to the tryptophan residues it was concluded that the two tryptophan residues are exposed to the solvent.

Acknowledgements

This research was supported by The Fundaqio de Amparo A Pesquisa do Estado de Silo Paulo (Project 79.1676). One of us (Y.K.) thanks CNPq for a research fellowship.

References

1 Conqalves, J.M. and Vieira, L.G. (1950) Ann. Acad. Bras. Cienc. 22, 141-150

2 Laure, C.J. (1975) Hoppe-Seyler's Z. Physiol. Chem. 356, 213-215

3 Hampe, O.G., Vozhri-Hampe, M.M. and Gonqalves, J.M., (1978) Toxicon 16, 453-460

4 Yang, C.C. (1974) Toxicon 12, 1-43 5 Tu, A.T. (1973) Annu. Rev. Biochem. 42, 235-258 6 Moussatch6, H., Conqalves, J.M., Vieira, G.D. and Hasson,

A. (1956) in Venoms, (Buckley, E.E. and Porjes, N., eds.), pp. 275, Publication No. 44, AAAS, Washington

7 Hampe, O.G. and Gonqalves, J.M. (1976) Polymer 17, 638-639

8 Takamatsu, T., Harada, I., and Hayashi, K. (1980) Biochim. Biophys. Acta 622, 189-200

9 Tu, A.T. (1979) in Infrared and Raman Spectroscopy of Biological Molecules (Nato Advances Study Institutes Series, Serie C) 43, pp. 139-152, D. Reidel Publishing Co., Holland

10 Hseu, T.H., Chang, H. Hwang, D.M. and Yang, C.C. (1978) Biochim. Biophys. Acta 537, 284-292

11 Bjarnason, J.B. and Tu, A.T. (1978) Biochemistry 17, 3395- 3404

12 Hseu, T.H., Liu, Y.C., Wang, C., Chang, H., Hwang, D.M. and Yang, C.C. (1977) Biochemistry 16, 2999-3006

13 Tu, A.T. (1977) The Spex Speaker 22, 1-6 14 Harada, I., Takamatsu, T., Shimanouchi, T., Miyazawa, T.

and Tamiya, N. (1976) J. Phys. Chem. 80, 1153-1156 15 Takamatsu, T., Harada, I., Shimanouchi, T., Ohta, M. and

Hayashi, K. (1976) FEBS Lett. 72, 291-294 16 Tu, A.T., Prescott, B., Chou, C.H. and Thomas, G.J., Jr.,

(1976) Biochem. Biophys. Res. Commun. 68, 1139-1145. 17 Yu, N.Y., Lin, T.S. and Tu, A.T. (1975) J. Biol. Chem. 250,

1782-1785 18 Yu, N,T., Jo, B.H. and O'shea, D.C. (1973) Arch. Biochem.

Biophys. 156, 71 76

19 Tu, A.T., Jo, B.H. and Yu, N.Y. (1976) Int. J. Peptide Protein Res. 8, 337-345

20 Giglio, J.R. (1975) Anal. Biochem. 69, 207-221 21 Koenig, J.L. (1979) in Infrared and Raman Spectroscopy of

Biological Molecules (Nato Advances Study Institutes Series, Serie C) 43, pp. 109-124, D. Reidel Publishing Co., Holland

22 Bandekar, J. and Krimm, S. (1980) Biopolymers 19, 31-36 23 Ishizaki, H., Balaram, P., Nagaraj, R., Venkatachalapathi,

Y.V. and Tu, A.T. (1981) Biophys. J. 36, 509-517 24 Fox, J.A., Tu, A.T., Hruby, V.J. and Mosberg, H.I. (1981)

Arch. Biochem. Biophys. 211,628-631 25 Hseu, T.H. and Chang, H. (1980) Biochim. Biophys. Acta

624, 340-345 26 Sugeta, H., Go, A. and Miyazawa, T. (1973) Bull. Chem.

Soc. Japan 46, 3407-3411

25

27 Fox, J.W., Elzinka, M., and Tu, A.T. (1977) FEBS Lett. 80, 217-220

28 Fox, J.W., Elzinka, M. and Tu, A.T. (1979) J. Appl. Bio- chem. 1,336-343

29 Nogami, N., Sugeta, H. and Miyazawa, T. (1975) Chem. Lett. 147-150

30 Siamwisa, M.N., Lord, R.C., Chen, M.C., Takamatsu, T., Harada, I., Matsuura, H. and Shimanouchi, T. (1975) Bio- chemistry 14, 4870-4876

31 Tu, A.T., Liu, T.S. and Bieber, A.J. (1975) Biochemistry 14, 3408-3411

32 Yu, N.T. (1974) J. Am. Chem. Soc. 96, 4664-4668 33 Bailay, G.S., Lee, J. and Tu, A.T. (1979) J. Biol. Chem. 254,

8922-8926