Raman spectroscopic evidence for a disulfide bridge in calf γII crystallin

ARCHIVES

AND BIOPHYSICS 15, pp. 250-255,1989

OF BIOCHEMISTRY

Vol. 269, No. 1, February

Raman Spectroscopic JAYANTI

Evidence

PANDE,*p’ MARTIN AND

for a Disulfide

Bridge in Calf yII Crystallin’

J. MCDERMOTT,* ROBERT H. CALLENDER,? ABRAHAM SPECTOR*,”

*Biochemistry and Molecular Biology Labratory, Department of Ophthalmology, College of Physicians Surgeons, Columbia University, New York, New York 10082, and TDepartment of Physics, City College of New York, New York, New York 100.31

and

Received August 8,1988, and in revised form October 18,1988

Laser Raman spectroscopy has been applied to native and dithiothreitol-treated bovine cortical yn crystallin to examine the state of the thiol groups and the presence of a putative disulfide bridge. The data reveal significant differences in two key spectral regions. In the thiol stretching region (2500-2600 cm-‘), the dithiothreitol-reduced form shows a 25% increase in the integrated Raman signal as compared to the native form. The magnitude of this increase corresponds to the presence of 1 mol of disulfide/mol of yn as determined both by the Raman data and the previous biochemical analysis from this laboratory. In the disulfide stretching region (500-540 cm-‘), the native form shows a line near 511 cm-’ which is absent in the reduced form. Both native and reduced forms show a triple-banded thiol signal with one or more distinct shoulders, suggesting at least three and perhaps five different environments for the cysteine residues. The difference spectrum, obtained by a 1:l computer subtraction of the native from the reduced form, indicates that the increase in thiol signal is centered around 2572 cm-‘. In every other spectral region, both native and reduced yn forms are closely similar. These results strongly support the biochemical data reported earlier and indicate that the reduction of the single disulfide bridge is accompanied by minimal changes in secondary structure in solution. 0 1989AcademicPress,Inc

Until recently, the lens specific proteins or crystallins were believed to contribute only to the structural characteristics of the tissue and thus to lens transparency. In higher mammals, only three types of structural lens proteins, CY,p, and y crystallins have been found whereas the lower species contain other types such as 6, C,and 7 crystallins. There is mounting evidence in the last few years that crystallins may be metabolically active. This conclusion is based on observations that (i) (Yand /3 crys-

tallins undergo CAMP-dependent phosphorylation (1,2) and (ii) some crystallins in lower vertebrates have enzymatic activity (3). y crystallins are monomeric, cysteinerich proteins. It has been suggested that bovine y crystallin may contain an intramolecular disulfide bond, but there are conflicting reports in the literature regarding its presence in the protein. X-Ray crystal data of the major bovine y polypeptide, yn at 1.9 A resolution, suggested a disulfide linkage involving Cys” and Cy? (4). In a later study performed with freshly prepared crystals, no disulfide linkage was observed and the sulfur atoms of Cys” and CysZ2 were seen to point away from each other (5). More recent X-ray crystal data seem to indicate an equal probability of finding Cys” and Cys ” in either the free

1 A preliminary report was presented at the Association for Research in Vision and Ophthalmology meeting, Sarasota, FL, May l-6,1988. ‘Present address: Department of Physics, MIT, Camhridge, MA 02139. 3 To whom correspondence should be addressed. 0003-9861/89

$3.00

CopyrightQ 1989by AcademicPress,Inc. All rights of reproductionin any form reserved.

250

RAMAN

SPECTROSCOPY

OF A DISULFIDE

thiol form or linked by a disulfide (S. Najmuddin and C. Slingsby, personal communication). A careful examination of the five polypeptides of calf y crystallin in this laboratory, using the DTNB assay, revealed that only yII, the major polypeptide, contains 1 mol of disulfide (6). The presence of such a linkage in yn may imply its involvement in oxidation-reduction (4, ‘7) and possibly in free radical quenching reactions (8, 9). Preliminary results indicate that y crystallin is a more efficient scavenger of hydrated electrons than ol crystallin (J. Dillon, personal communication). It is possible that the putative disulfide in yII crystallin influences this property. Raman spectroscopy affords a direct means of observing the cystine disulfide group since the S-S stretching frequency normally appears as a sharp line near 500 cm-l. In this report, Raman spectroscopic results are presented which corroborate the biochemical data (6) and argue for the presence of a disulfide linkage in bovine yII in agreement with the previously reported X-ray crystal data (4). The results also indicate that the two forms of the protein, with and without the disulfide linkage, maintain a close structural similarity in solution. MATERIALS

AND

METHODS

Isolation of y crystallin fractions. The isolation of total y crystallin (rt) from the soluble protein fraction of calf cortex by gel filtration chromatography, has been detailed elsewhere (6). Isolation of the individual polypeptide components (yi-yiv) was performed using sulfopropyl Sephadex C-25 (Pharmacia), a cation exchange resin, by a modification of the procedures of Bjiirk (10,ll) and Slingsby and Miller (12). Briefly, total y crystallin (300-400 mg; 2-3 mg/ml) dissolved in the starting buffer, 10 mM sodium acetate, pH 5, was loaded onto a column (2.5 X 40 cm) previously equilibrated with the same buffer. The column was washed with 150 ml of the starting buffer and the protein eluted with 3 liters of a linear gradient from 10 to 500 mM sodium acetate, pH 5. Five-milliliter fractions were collected at a flow rate of 30-35 ml/hr and the major fractions yi-yiv were pooled, concentrated by ultrafiltration to i their volume, dialyzed against water, lyophilized, and stored at -20°C. The purity of each fraction was confirmed using isoelectric focussing.

BRIDGE

IN CRYSTALLIN

251

Reductimz of yI1. yn (15 mg), at a protein concentration of I mg/ml, was incubated in a buffer containing 0.3 M N-ethylmorpholine, pH 8.2, 2 mM EDTA, 2 mM DETAPAC: and 2 mM DTT for 5 h at room temperature. An equivalent amount of yn incubated similarly in a buffer lacking DTT served as the unreduced control. Sulfhydryl determinations were performed as follows: aliquots of the native control and DTTtreated yn were dialyzed at 4°C against a buffer containing 10 mM sodium acetate, 2 mM EDTA, pH 4.7, saturated with nitrogen. A constant flow of nitrogen was maintained in the dialysis buffer. Aliquots containing 50 yg of protein were dissolved in 700 ~1 of 100 mM Tris, 5.35 M guanidine, 2 mM EDTA, 2 mM DETAPAC, pH 8.2. Thirty-five microliters of 20 mM DTNB in methanol were added and incubated for 20-30 min, and absorbance at 412 nm measured against DTNB/ buffer blanks. Reduced glutathione was used as the standard. The total sulfhydryl content of native and DTT-treated yn crystallin was 5.0 and 7.0 ma1 SH/ ma1 protein, respectively (6). The remaining aliquots of yn control and DTTtreated samples were passed through a gel filtration column, Sephadex G-25 (20 X 30 cm; flow rate, 35-40 ml/h), equilibrated with 10 mM sodium acetate buffer, pH 4.8, containing 2 mM EDTA. Two-milliliter fractions were collected and pooled. Overnight dialysis of pooled control and DTT-treated samples was carried out against 40 liters of nitrogen-saturated water. Dialyzed samples were concentrated to 100200 ~1 using centriconconcentrators (Amicon). The final protein concentration most suitable for Raman experiments was at least 35-45 mg protein/ml. Raman spectra. Raman spectra were obtained on a Spex Model 187’7 triplemate spectrometer, with 48% nm excitation from Spectra Physics argon ion lasers, Models 164 and 165. Power levels at sample typically ranged from 150 to 170 mW. Aliquots of sample (80100 ~1) at a concentration of 1-2 mM based on an extinction Eiz; = 2.1(6), in water at pH 5, containing 20 mM NaCl, were placed in a 3 X 3-mm glass cuvette, flushed with argon, and sealed. The cuvette was placed in a holder thermostatted at 8°C. Radiation scattered at 90” to the incident beam was detected by an OMA system consisting of a thermoelectrically cooled solid-state detector/controller assembly (EG & G Princeton Applied Research Model 1420-2/1218). Data were accumulated and analyzed on an LSI-11 microcomputer (Digital Equipment Corp.), interfaced to the multichannel detection system, at a spectral resolution of 6 cm-.‘. All spectra were corrected for the nonidentical spectral response of the multi-

4 Abbreviations used: DETAPAC, diethylenetriaminepentaacetic acid; DTT, dithiothreitol; DTNB, 5,5’-dithiobis(2-nitrobenzoic acid).

252

PANDE

2550

2650 Wavenumber (cm - ‘)

2750

FIG. 1. Comparison of the Raman spectra of native (a) and DTT-reduced (b) yn crystallin in the -SH stretching region, in 20 mM NaCl, pH 5.4. Laser excitation at 488 nm; spectral resolution, 6 cm-‘; temperature, 8°C. Sample concentration l-2 mM. Spectra are normalized with respect to the invariant protein peak at 2728 cm-‘.

channel detector and calibrated against the Raman lines of toluene. At the conclusion of the Raman experiment, the laser-irradiated samples were reanalyzed with the DTNB assay. No significant difference in sulfhydryl content was observed in the samples before and after the experiment. RESULTS

AND

ET AL.

are apparent, with two shoulders on either side of the triplet. Since the native protein contains five free thiol groups (6), the observed spectral profile (a) probably would correspond to the -SH stretching frequencies of the five individual cysteine residues. Upon treatment with DTT, this profile is essentially maintained (b), but there is a striking intensity enhancement in the components scattering near 2563, 2569, and 2577 cm-‘. The total number of thiols in the reduced form being seven (4,6), the enhancement in the Raman signal thus corresponds to an increase from five free thiols in the native to seven in the DTTtreated form. Since DTT specifically reduces disulfide linkages (17), the changes described are most likely to result from the reduction of 1 mol of disulfide in the native protein. Furthermore, the 25% enhancement in the integrated area of the thiol band is in excellent agreement with the f or 28% increase expected from the reduction of a single disulfide bridge. The increased scattering in the -SH stretching region due to reduction is better illustrated by difference spectra (Fig. 2). Curve d is the difference spectrum obtained by a 1:l computer subtraction of the native form (a) from the reduced form (b). Undersubtraction (b - 0.5a) and oversub-

DISCUSSION

The cysteine side chains of proteins scatter in a relatively narrow and isolated region of the vibrational Raman spectrum (13, 14). For lens proteins, lines in the 2550-2600 cm-’ region have been assigned to the -SH stretching frequency, based on its downshift to about 1900 cm-’ by isotopic exchange in D20 (1516). Figure 1 compares the Raman spectrum of native5 yII (profile a) with that of the DTT-treated form (profile b) between 2500 and 2800 cm-‘. The peak at 2728 cm-’ was found to be essentially unaltered by DTT treatment and was, therefore, used to normalize data in this region. In the native form three distinct lines near 2562, 2568, and 2577 cm-’ 5 Native 7rr refers to the untreated yIr fraction from total y crystallin, as opposed to the DTT-reduced form.

I 2550

I 2650 Wavenumber (cm -‘)

I 2750

FIG. 2. Difference Raman spectra of native (a) and DTT-reduced (b) yn crystallin in the -SH stretching region. c, b - 0.5 a; d, b - a; e, b - 1.5 a. Curved represents the increased thiol scattering due to DDT reduction of native yn.

RAMAN

I 500

SPECTROSCOPY

1 550 Wavenumber

I 600 (cm-‘)

OF A DISULFIDE

1 650

FIG. 3. Comparison of the Raman spectra of native (a) and DTT-treated (b) yrr crystallin in the region showing S-S scattering. Experimental conditions are as in Fig. 1.

traction (b - 1.5a) of the native from the DTT-treated form, result in curves c and e, respectively. Curve d, given by b - a, thus represents the net increase in thiol scattering due to reduction. It is evident that the contribution to curve d from the components scattering near 2563 cm-’ and below, which are prominent in the native protein (a), is minimal. It is also evident from the oversubtracted curve (e) that the only positive peak due to residual scattering occurs near 2572 cm-‘. Direct evidence for a disulfide linkage in the native form is obtained from the data shown in Fig. 3. In the lower energy end of the Raman spectrum, curves a and b represent the native and DTT-treated forms of the protein, respectively (Fig. 3). Both curves have been arbitrarily normalized with respect to the tyrosine line at 641 cm-‘. Despite large variations in background scattering in this region, both forms exhibit bands of comparable intensity at 569,586,622, and 641 cm-‘. Two low intensity lines at 511 and 521 cm-l are present in native yn (Fig. 3, curve a), both of which could be attributed to the symmetrical stretching vibration of a disulfide bridge (13, 18-21). However, it is evident from Fig. 3 that only the line at 511 cm-l (curve a) is sensitive to DTT treatment and disappears in the reduced form of the protein (curve b). Therefore, only this line is

BRIDGE

253

IN CRYSTALLIN

likely to arise from an S-S bridge in the native protein. Clearly, there is a correlated loss in intensity at 511 cm-’ and gain between 2500 and 2600 cm-‘, in the reduced form relative to the native form. X-Ray crystallographic data reveal that Cys” and Cysz2 in the N-terminal domain of -yn are located within 4-5 A of each other and can be bridged by an S-S bond (4,5). Interestingly, the totally buried CysT8 is also within S-S bond forming distance of cys 22. Thus, the most likely candidates for a cysteine bridge in native yn are either Cys” and CYSTSor CYSTSand CYS’~. It is to be noted that the three other y polypeptides,

%IIA,

%IIB,

and

WV,

which

show no evidence of a disulfide, also lack CYSTSin their amino acid sequence (6). This observation supports the argument that in yn, CYSTSis most probably involved in the disulfide bridge. It is noteworthy that the torsion angle of 94.5 around the S-S bond for a cystine bridge between CYSTSand CYS?~(4) approaches the f90” that is generally observed in proteins (14). Thus, a Cy~~~-Cys~~disulfide linkage is conceivable although there is no evidence for this in the X-ray data. One distinguishing feature of the -SH band profile presented here (Fig. 1) is the appearance of a triplet and one or more shoulders in both protein forms. Previous reports on unfractionated y and intact lenses (22, 23) generally show a doublet. Our data indicate that there may be at least three and possibly five different environments for the cysteine residues of yrI in solution. In general, the -SH stretching frequency is shifted to lower energy by hydrogen bonding, the magnitude of the downshift being proportional to H-bonding strength (24). Thus, the strongest nucleophilic acceptors of H-bonding would cause the largest shift to lower wavenumbers. If sulfur containing amino acids are within 5 A of aromatic residues, competing effects due to strong S-r interactions are known to occur (25,26). This would tend to raise the -SH stretching frequency, as observed for the p-93 cysteine of hemoglobin (27). In yu, every one of the seven cysteine residues is in the vicinity of one or more aromatic residues (5), which leads to com-

254

PANDE 7 8 i

E e P 5 5 (E

Wavm~mbcr(crn-~)

FIG. 4. Raman spectrum of native yI1 crystallin in the amide I region. Experimental conditions are as in Fig. 1.

peting S-?r and r-a interactions, the Stryptophan S-a interaction being generally favored over the others (26). Thus, the opposing influences of H-bonding and S-r and/or ?T-?Tinteractions can lead to a complex splitting pattern of the -SH stretch. In every other spectral region besides the ones discussed above, the native and reduced protein forms are strikingly similar. The solution Raman spectrum of native bovine cortical 7rI from 500 to 1700 cm-l is shown in Fig. 4. The reduced protein is identical within signal to noise variation to the native form in this region and has been omitted from Fig. 4 for reasons of clarity. A number of the spectral features in Fig. 4 are of special interest. The characteristic lines due to tryptophan at 569,757,871, and 1546 cm-‘, phenylalanine at 622,1003, and 1030, tyrosine at 641, 830, 856, 1209, and 1617 cm-‘, the C-S stretching mode due to methionine at 727 cm-l, the backbone skeletal C-C stretch near 926 cm-‘, and C-N stretching frequencies at 1069, 1097, and 1124 cm-’ are all clearly evident in this region. Two intense lines at 1239 and 1669 cm-’ due to the amide III and amide I modes of the peptide group, respectively, are immediately obvious. These bands are sensitive to secondary structure and the frequencies of 1239 and 1669 cm-‘, respectively, are indicative of antiparallel p structure in the protein (28). Clearly, the

ET AL.

predominantly 0 type structure observed in the 1.9 A X-ray map (4) is maintained in solution. That both the native and reduced proteins yield essentially identical Raman spectra in the amide III and I regions is evidence that both forms have very close secondary structures. Another feature is the ratio of the doublet near 830-860 cm-‘, indicative of the degree of hydrogen bonding or ionization state of the phenolic hydroxyl groups of tyrosyl residues (29). For yn, the ratio 1ss6/ lssO = 1.13 indicates that most of the tyrosyl residues are moderate hydrogen bond donors and acceptors (29). Applying Craig and Gaber’s method to the Raman data (13,30), 10 of the 15 tyrosyl residues present in the protein appear to be solvent exposed in both forms. This is in good agreement with X-ray crystal data (4) which indicate that 11 of the tyrosyl residues are at least partially solvent exposed. The presence of a sharp line at 1362 cm-’ (Fig. 4) indicates that the tryptophan residues of yrI are buried (31). In addition, the strong shoulder at 871 cm-’ adjoining the 856 cm-l tyrosine line is considered a good monitor of the solvent accessibility of tryptophan residues (32). Its ratio to the methylene scissoring vibration at 1444 cm-* (1871/1L444)approximates to 0.8 aIs suggesting that the tryptophans of yII are in a hydrophobic environment in solution. This is in close agreement with the X-ray data. Yu and East (15) have made numerous assignments in the 600-1700 cm-’ region, for unfractionated y. The Raman lines reported here for yII (Fig. 4) are all within a few wavenumbers of their assignments with the notable exception of the disulfide line at 511 cm-‘. The question of whether the disulfide detected in +yII is an artifact of preparation has been amply addressed in our previous report (6). Reduced 711is stable to handling under a variety of conditions including lyophilization and exposure to alkaline pH (6) and to long exposure to coherent radiation (this report). Furthermore, the disulfide content of yII is constant when isolated both in the presence and absence of Z-mercaptoethanol. These results strongly indi-

RAMAN

SPECTROSCOPY

OF A DISULFIDE

cate that the disulfide bond in yn is not generated during its isolation or handling. Our data ((6), this report) differ from those reported by Mandal et al. (33) in which yn was isolated in the fully reduced form. Since the recent X-ray map (S. Najmuddin and C. Slingsby, personal communication) indicates that either form is equally probable, these two opposing viewpoints may not be irreconcilable. The finding of a single, stable cystine bridge in calf yII crystallin raises many questions about its role in the lens. The earlier suggestion for the possible involvement of yrI in redox reactions (4,7) now assumes greater importance. ACKNOWLEDGMENTS This work was supported by grants from the National Eye Institute to A.S. and R.C. and Grant No. GM35183 to R.C. from the National Institute of General Medical Sciences. J.P. is indebted to Drs. H. Deng, C. Pande, and K. W. Rhee, and Mr. P. Rath and J. Zheng for their willingness to help at all times during the course of this work. We are grateful to Mr. J. Horton for help with the molecular graphics system, Dr. R. Chiesa for many suggestions to improve the manuscript, and E. Bluberg for expert secretarial assistance. REFERENCES 1. SPECTOR, A., CHIESA, R., SREDY, J., AND GARNER, W. H. (1985j Proc. N&l. Acad. Sci. USA 82, 4712-4716. 2. KLEIMAN, N., CHIESA, R., AND SPECTOR, A. (1987) Invest. Ophthd Vis. Sci. Suppl. 28,150. 3. WISTOW, G., PIATIGORSKY, J., WAWROUSEK, E., NICKERSON, J., MULDERS, J., DE JONG, W., AND O’BRIEN, W. (1987) Invest. Ophthal. Vis. Sci. Suppl. 28, 15. 4. WISTOW, G., TURNELL, B., SUMMERS, L. J., SLINGSBY, C., Moss, D., MILLER, L., LINDLEY, P., AND BLUNDELL, T. (1983) J. Mol. BioL 170, 175-202. 5. SIJMMERS, L. J., WISTOW, G. J., NAREBOR, M. E., Moss, D. S., LINDLEY, P. F., SLINGSBY, C., BLUNDELL, T. L., BARTUNIK, H., AND BARTELS, K. (1984) Pept. Protein Rev. 3,147-168. 6. MCDERMOTT, M. J., GAWINOWICZ-KOLKS, M. A., CHIESA, R., AND SPECTOR, A. (1988) Arch,. Biothem. Biophys. 262,609-619. 7. BLUNDELL, T. L., LINDLEY, P., MILLER, L., Moss, D., SLINGSBY, C., TICKEL, I. J., TURNELL, B., AND WISTOW, G. (1981) Nature (London) 289, 771-777.

BRIDGE

IN CRYSTALLIN

255

8. BENT, D. V., AND HAYON, E. (1975) J. Amer. Chem. Sot. 97,2612-2619. 9. ARIAN, S., FEITELSON, J., AND STERN, G. (1980) Photochem. Photobiol. 12,481-487. 10. BJBRK, I. (1964) Exp. Eye Res. 3,254-261. 11. BJBRK, I. (1970) Exp. Eye Res. 9,152-157. 12. SLINGSBY, C., AND MILLER, L. R. (1983) Exp. Eye Res. 37,517-530. 13. Tu, A. T. (1982) Raman Spectroscopy in Biology: Principles and Applications, pp. 65-116. Wiley, New York. 14. Tu, A. T. (1986) in Advances in Spectroscopy (Clark, R. J. H., and Hester, R. E., Eds.), Vol. 13, pp. 47-112, Wiley, Chichester. 15. Yu, N.-T., AND EAST, E. J. (1975) J. Biol. Chem. 250,2196-2202. 16. ITOH, K., OZAKI, Y., MIZUNO, A., AND IRIYAMA, K. (1983) Biochemistry 22,1773-1778. 17. CLELAND, W. W. (1964) Biochemistry3,480-482. 18. SUGETA, H., Go, A., AND MIYAZAWA, T. (1972) Chem. Lett. 83-86. 19. SUGETA, H., Go, A., AND MIYAZAWA, T. (1973) Bull. Chem. Sot. Japan 46,3407-3411. 20. VAN WART, H. E., AND SCHERAGA, H. A. (1976a) J. Phys. Chem. 80,1812-1822. 21. VAN WART, H. E., AND SCHERAGA, H. A. (1976b) J Phys. Chem. 80,1823-1832. 22. OZAKI, Y., MIZUNO, A., ITOH, K., AND IRIYAMA, K. (1987) J. Biol. Chem. 262,15545-15551. 23. Yu, N.-T., DENAGEL, D. C., JUI-YUAN Ho, D., AND K~JCK, J. F. R. (1987) in Biological Applications of Raman Spectroscopy (Spiro, T., Ed.), Vol. 1, pp. 47-81, Wiley, New York. 24. BARE, G. H., ALBEN, J. O., AND BROMBERG, P. A. (1975) Biochemistry 14,1578-1583. 25. MORGAN, R. S., TATSCH, C. E., GUSHARD, R. H., MCADON, J. M., AND WARME, P. K. (1978) Int. J. Pept. Protein Res. 11,209-217. 26. MORGAN, R. S., AND MCADON, J. M. (1980) Znt. J. Pept. Protein Res. 15,177-180. 27. MOH, P. P., FIAMINGO, F. G., AND ALBEN, J. 0. (1987) Biochemistry 26,6243-6249. 28. FRUSHOUR, B. G., AND KOENIG, J. L. (1975) in Advances in Infrared and Raman Spectroscopy (Clark, R. J. H., and Hester, R. E., Eds.), Vol. 1, pp. 35-97, Heydon & Son, London. 29. SIAMWIZA, M. N., LORD, R. C., CHEN, M. C., TAKAMATSU, T., HARADA, I., MATSUURA, H., AND SHIMANOUCHI, T. (1975) Biochemistry 14,48704876. 30. CRAIG, W. S., AND GABER, B. P. (1977) J. Amer. Chem. Sot. 99,4130-4134. 31. YU, N.-T. (1974) J. Amer. Chem. Sot. 96,4664-4668. 32. KITAGAWA, T., AZUMA, T., AND HAMAGUCHI, K. (1979) Biopolymers l&451-465. 33. MANDAL, K., CHAKRABARTI, B., THOMSON, J., AND SIEZEN, R. J. (1987) J. Biol. Chem. 262, 80968102.