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Structural studies on polypeptide hormones
ARCHIVES
OF
BIOCHEMISTRY
AND
Structural
782-785 (1972)
BIO”HYSICS
160,
Studies
on Polypeptide
II. Parathyroid SALVATORE Clinical
Endocrinology
Branch,
ALOJ’
Hormones
Hormone AND
H. EDELHOCH
National Institute of Arthritis and Metabolic of Health, Bethesda, Maryland 2001.4
Received
February
28, 1972; accepted
March
Diseases, Xational
lastitutes
2, 1972
The molecular properties of bovine and porcine parathyroid hormone have been studied by circular dichroism and fluorescence. The effects of pH on the tryptophanyl fluorescence of both species and on the peptide optical activity of the bovine hormone have been evaluated. The data obtained show that there are constraints on the rotational freedom of the polypeptide backbone in both hormones. Therefore, parathyroid hormone cannot be characterized either as possessing the fully organized structure of a protein or the random coil structure of charged polylysine or polyglutamate.
There are several polypeptide hormones containing a sufficient number of residues to form ordered structures (glucagon-29, calcitonin-32, ACTH2-39) which, however, are essentially structureless in very dilute aqueous solutions (l-3). In solvents less polar than water, the chains fold into helices (1, 3) ; in fact, glucagon is largely helical when crystalline (4, 5). Preliminary data on bovine PTH indicated that it fell into the above class of polypeptides although it has more than twice as many residues, i.e., 84. The availability of small amounts of purified porcine PTH, which is missing the tyrosyl residue of bovine PTH, has permitted a comparison of its CD and fluorescence properties with that of bovine PTH (6, 7, 8). In addition, CD measurements have been extended on bovine PTH between pH 4 and 10. MATERIALS Highly purified porcine PTH were
AND
METHODS
preparations of bovine and a generous gift of Dr. G. D.
1 Visiting Associate, on leave from Istituto di Patologia Generale, Universita di Napoli, 1.80138, Naples, Italy. 2 The following abbreviations are used: ACTH, adrenocorticotropic hormone; PTH, parathyroid hormone: CD, circular dichroism. 782
Aurbach (NIH). The experimental procedures were essentially the same as reported previously (2). Quantum yields were obtained from the correctedspectraof the Turner 210 fluorometer. Acetyl tryptophanamide was used as a standard and its quantum yield was assigned a value of 0.13. RESULTS
The optical activity of the peptide chromophore affords information of the organization of the polypeptide backbone (9, 10). The fluorescent properties of the single tryptophanyl residue in both species of PTH can be used as an indicator of tertiary structure since this residue is the most “hydrophobic” amino acid (11) and is generally rigidly bound in t’he interior or on the surface of most proteins (12). These two methods have been selected since they require very small amounts of material and reflect different structural parameters. Fluorescence. The emission spectra of bovine and porcine PTH in 0.10 M KCl, 0.01 M lysine, at pH 3.8 are reproduced in Fig. 1. The emission maxima occur at identical wavelengths (343 nm) and are very similar except in the shorter wavelength region. Since porcine PTH does not contain a tyrosyl residue, the emission near 305 of bovine PTH should arise from it’s
STRUCTURAL
STUDIES
ON POLYPEPTIDE
HORMONES
783
2ols, , , , , , , ( , 300
320
340 WAVELENGTH
360
360
(nm)
FIG. 1. The emission spectra of bovine (------) and porcine (--) PTH in 0.1 M KC1-6.1 pH 3.8. Excitation was at 286 nm. Protein concentration is 0.036 mg/ml. T = 25” C.
FIG. 2. The pH dependence of tryptophanyl fluorescence of bovine (0) and porcine (0) PTH inO. M KC1-0.01 M lysine. Excitation and emission were at 286 nm and 345 nm, respectively. T = 25” C.
M
lysine,
curve in alkali is more gradual in porcine than in bovine PTH. Circular dichroim. The far-ultraviolet CD spectrum of bovine PTH in 0.10 M KCl, 0.01 M lysine (DL), pH 3.8, revealed a symmetrical negative minimum centered at 200 nm with a weak shoulder near 220 nm (Fig. 3). Porcine PTH gave the same CD spectrum in the same solvent. At pH 7.1 the minimum in the spectrum of bovine PTH was shifted to 203.5 nm with only a slight decrease in negative ellipticity while the shoulder became more intense (Fig. 3). Both minima are shifted slightly toward the red at pH 10.0 and become more equal in intensity. In fact, the CD spectrum of bovine PTH now approaches that of the a-helix (9). DISCUSSION
tyrosyl group. The quantum yields of tryptophanyl emission have been determined from their spectra at pH 3.8 and found to be equal to 0.105 in both cases after correcting for the emission and absorption of the tyrosyl residue in bovine PTH. The pH dependence of tryptophanyl fluorescence of bovine and porcine PTH is shown in Fig. 2. The two curves are similar though there are some minor differences between them. The quenching
The folding of the polypeptide backbone could be analyzed for in terms of contributions from the a-helical, ,&structure, and random coil forms if the CD spectra of these structures were known accurately. Unfortunately, it is still not clear which are the best models to use to obtain this information. Saxena and Wetlaufer (13) have computed the spectra of the model structures from the relative amounts of the three forms in several proteins as determined by X-ray studies and from the CD spectra
784
ALOJ
AND
2c
: P n d
a 4 #I
+
I
I
I
200
1
I
,
220 W!a"ELENGTH I nm 1
/
I
240
FIG. 3. The pH dependence of the far ultraviolet CD spectra of bovine PTH in 0.1 M KCI-O.01 M lysine. T = 25” C. Protein concentration is 0.7
mg/d.
of the same proteins. It is unlikely that a single model for each type of structure can be applied universally to all proteins because environments in proteins differ and the a-helical and &structures that exist vary from the ideal. The approach recently suggested by Fasman et al. seems to be more reasonable in the current state of the art (14). The CD spectra of bovine and porcine PTH at pH 3.8 resemble those observed either with films of sodium polyglutamate and polylysine hydrochloride in unordered conformations or with certain proteins considered to be extensively unfolded, i.e., e,l-casein, oxidized ribonuclease, f-l histone, S-sulfonated p-lactoglobulin A, etc. (14). The structure of these molecules is considered by Fasman et al. as being without long-range order but where the rotational motions of parts of the polypeptide backbone and of some of the side chains are constrained by local interactions. Homologous polypeptides and proteins which can be considered as true random coils, such as aqueous solutions of t,he charged forms
EDELHOCH
of polyglutamate and polylysine (14), poly[N5-2(hvdroxyethyl-L-glutamine] (15), and phosvitin at pH 6.6 (16), show positive ellipticity in the 220-nm region and stronger minima in the 200-nm wavelength region. The unordered, but constrained, structure of bovine PTH does not appear to possess either the compactness or rigidity of the unordered parts (i.e., non-a-helical or p-structures) of globular proteins since the intensities of the two CD minima vary with pH between 3.8 and 10.0. The CD spectra of native globular proteins are generally independent of pH. Consequently, the extent of int’ramolecular interactions in bovine PTH should increase with increasing pH since the freedom of motion of the backbone peptide groups becomes more restricted at pH 10. This increase in organization presumably results in part from the decrease in the positive charge of bovine PTH between pH 3.8 and 10 since there are more basic (9 lys, 5 arg, 4 his) than acidic (6 glu, 6 asp) amino acids (8). The reduction in electrostatic interactions would allow the formation of additional intramolecular contacts by decreasing the unfavorable electrostatic free energy bet’ween segments of the polypeptide chain. The variations in fluorescence intensity are another reflection of pH-dependent structural alterations in PTH. The quenching of tryptophanyl fluorescence cannot be explained by the ionization of side-chain amino groups since the dissociation of the amino group in model tryptophanyl peptides increases its quantum yield nor by base quenching since this occurs at higher pH values (17). It was suggested previously that the ionization of the phenolic side chain of bovine PTH quenched its tryptophanyl fluorescence by radiationless energy transfer (2). This cannot be the mechanism of the alkaline quenching of PTH fluorescence since one of the seven substitutions in porcine PTH is a histidine residue for the tyrosine in bovine PTH. The quenching is, therefore, due to structural modifications in PTH. The pH dependence of fluorescence of the tryptophanyl residue in PTH
STRUCTURAL
STUDIES
ON POLYPEPTIDE
is slightly affected by the substitution of seven amino acids. Since the closest substitutions occur at residues 18 and 42! (6-8) positions which are not close to the tryptophanyl residue at 23, the influence of the new residues suggest that the interactions of the tryptophanyl residue are altered and modify its fluorescence. The much weaker quenching in acid may be due to the protonation of vicinal side chain groups, i.e., imidazole or carboxyl since tryptophanyl fluorescence is partially quenched in model peptides containing these groups (18, 19). REFERENCES W. B., BEAVEN, G. H., RATTLE, 1. GRATZER, H. E. W., AND BR.4DBURY, E. M. (1968) Eur. J. Biochem. 3, 276. 2. EDELHOCII, H., AND LIPPOLDT, R. E. (1969) J. Biol. Chem. 244.3876. ~.~BREWER, B. H., .~ND EDELHOCH, H. (1970) J. Biol. Chem. 246, 2402. 4. KING, V. M. (1965) J. Mol. Biol. 11,549. 5. HAUGEN, W. P., .~ND LIPSCOMB, W. N. (1969) Acta Crystallogr. Sect. A 26 (Suppl.), S185 (XV-25). 6. WOODHEaD, J. S., O’RIORDAN, J. L. H., KEUTMANN, H. T., STALTZ, M. L., LAWSON, B. F., NIALL, H. D., ROBINSON, C. J., AND POTTS, J. T., JR. (1971) Biochemistry 10, 2787.
HORMONES
785
7. POTTS, J. T., JR., TREGEON, G. W., KEUTM~NN, H. T., NIALL, H. D., SAUER, R., DEFTOS, L. J., DAWSON, B. F., HOGAN, M. L., AND G. D., (1971) Proc. Nat. Acad. AURBACH, Sci. U. S. A. 68,63. 8. BREWER, B. H., AND RONAN, R. (1970) Proc. IVat. Acad. Sci. U. S. A. 67, 1862. G., AND DOTY, P. (1965) J. Amer. 9. HOLZWARTH, Chem. Sot. 87, 218. 10. SARHAR, P., AND DOTY, P. (1966) Proc. Nat. Acad. Sci. U. S. A. 66, 981. Y., AND TANFORD, C. (1971) J. Biol. 11. NOZAKI, Chem. 246,221l. 12. TIMASHEFF, S. N., .~ND GORBUNOFF, M. J. (1967) Ann. Rev. Biochem. 36, 13. D. B. (1971) 13. SAXENA, V. P., .~ND WETLAUFER, Proc. Nat. Acad. Sci. U. S. A. 68, 969. 14. FASMAN, G. D., HOVING, H., AND TIMASHEFF, S. N. (1970) Biochemistry 9, 3316. 15. ADLER, A. J., HOVING, P., POTTER, J. WELLS, M., AND FASMAN, G. D. (1968) J. Amer. Chem. Sot. 90, 4736. S. N., TOWNEND, R., AND PERL16. TIM.~SHEFF, MANN, G. (1967). J. Biol. Chem. 242,229O. H., BR.4ND, L., SND WILCHECK, 17. EDELHOCH, M. (1968) Biochemistry 7, 547. M., AND KATCHALSKY, E. (1968) 18. SCHINITZKY, in Molecular Associat,ion in Biology (Pullman, B., ed.), p. 361. Academic Press, New York. H., AND STEINER, 19. CHEN, R. F., EDELHOCH, R. F. (1969) in Physical Principles and Techniques of Protein Chemistry (Leach, S. J., ed.), p. 191. Academic Press, New York.
OF
BIOCHEMISTRY
AND
Structural
782-785 (1972)
BIO”HYSICS
160,
Studies
on Polypeptide
II. Parathyroid SALVATORE Clinical
Endocrinology
Branch,
ALOJ’
Hormones
Hormone AND
H. EDELHOCH
National Institute of Arthritis and Metabolic of Health, Bethesda, Maryland 2001.4
Received
February
28, 1972; accepted
March
Diseases, Xational
lastitutes
2, 1972
The molecular properties of bovine and porcine parathyroid hormone have been studied by circular dichroism and fluorescence. The effects of pH on the tryptophanyl fluorescence of both species and on the peptide optical activity of the bovine hormone have been evaluated. The data obtained show that there are constraints on the rotational freedom of the polypeptide backbone in both hormones. Therefore, parathyroid hormone cannot be characterized either as possessing the fully organized structure of a protein or the random coil structure of charged polylysine or polyglutamate.
There are several polypeptide hormones containing a sufficient number of residues to form ordered structures (glucagon-29, calcitonin-32, ACTH2-39) which, however, are essentially structureless in very dilute aqueous solutions (l-3). In solvents less polar than water, the chains fold into helices (1, 3) ; in fact, glucagon is largely helical when crystalline (4, 5). Preliminary data on bovine PTH indicated that it fell into the above class of polypeptides although it has more than twice as many residues, i.e., 84. The availability of small amounts of purified porcine PTH, which is missing the tyrosyl residue of bovine PTH, has permitted a comparison of its CD and fluorescence properties with that of bovine PTH (6, 7, 8). In addition, CD measurements have been extended on bovine PTH between pH 4 and 10. MATERIALS Highly purified porcine PTH were
AND
METHODS
preparations of bovine and a generous gift of Dr. G. D.
1 Visiting Associate, on leave from Istituto di Patologia Generale, Universita di Napoli, 1.80138, Naples, Italy. 2 The following abbreviations are used: ACTH, adrenocorticotropic hormone; PTH, parathyroid hormone: CD, circular dichroism. 782
Aurbach (NIH). The experimental procedures were essentially the same as reported previously (2). Quantum yields were obtained from the correctedspectraof the Turner 210 fluorometer. Acetyl tryptophanamide was used as a standard and its quantum yield was assigned a value of 0.13. RESULTS
The optical activity of the peptide chromophore affords information of the organization of the polypeptide backbone (9, 10). The fluorescent properties of the single tryptophanyl residue in both species of PTH can be used as an indicator of tertiary structure since this residue is the most “hydrophobic” amino acid (11) and is generally rigidly bound in t’he interior or on the surface of most proteins (12). These two methods have been selected since they require very small amounts of material and reflect different structural parameters. Fluorescence. The emission spectra of bovine and porcine PTH in 0.10 M KCl, 0.01 M lysine, at pH 3.8 are reproduced in Fig. 1. The emission maxima occur at identical wavelengths (343 nm) and are very similar except in the shorter wavelength region. Since porcine PTH does not contain a tyrosyl residue, the emission near 305 of bovine PTH should arise from it’s
STRUCTURAL
STUDIES
ON POLYPEPTIDE
HORMONES
783
2ols, , , , , , , ( , 300
320
340 WAVELENGTH
360
360
(nm)
FIG. 1. The emission spectra of bovine (------) and porcine (--) PTH in 0.1 M KC1-6.1 pH 3.8. Excitation was at 286 nm. Protein concentration is 0.036 mg/ml. T = 25” C.
FIG. 2. The pH dependence of tryptophanyl fluorescence of bovine (0) and porcine (0) PTH inO. M KC1-0.01 M lysine. Excitation and emission were at 286 nm and 345 nm, respectively. T = 25” C.
M
lysine,
curve in alkali is more gradual in porcine than in bovine PTH. Circular dichroim. The far-ultraviolet CD spectrum of bovine PTH in 0.10 M KCl, 0.01 M lysine (DL), pH 3.8, revealed a symmetrical negative minimum centered at 200 nm with a weak shoulder near 220 nm (Fig. 3). Porcine PTH gave the same CD spectrum in the same solvent. At pH 7.1 the minimum in the spectrum of bovine PTH was shifted to 203.5 nm with only a slight decrease in negative ellipticity while the shoulder became more intense (Fig. 3). Both minima are shifted slightly toward the red at pH 10.0 and become more equal in intensity. In fact, the CD spectrum of bovine PTH now approaches that of the a-helix (9). DISCUSSION
tyrosyl group. The quantum yields of tryptophanyl emission have been determined from their spectra at pH 3.8 and found to be equal to 0.105 in both cases after correcting for the emission and absorption of the tyrosyl residue in bovine PTH. The pH dependence of tryptophanyl fluorescence of bovine and porcine PTH is shown in Fig. 2. The two curves are similar though there are some minor differences between them. The quenching
The folding of the polypeptide backbone could be analyzed for in terms of contributions from the a-helical, ,&structure, and random coil forms if the CD spectra of these structures were known accurately. Unfortunately, it is still not clear which are the best models to use to obtain this information. Saxena and Wetlaufer (13) have computed the spectra of the model structures from the relative amounts of the three forms in several proteins as determined by X-ray studies and from the CD spectra
784
ALOJ
AND
2c
: P n d
a 4 #I
+
I
I
I
200
1
I
,
220 W!a"ELENGTH I nm 1
/
I
240
FIG. 3. The pH dependence of the far ultraviolet CD spectra of bovine PTH in 0.1 M KCI-O.01 M lysine. T = 25” C. Protein concentration is 0.7
mg/d.
of the same proteins. It is unlikely that a single model for each type of structure can be applied universally to all proteins because environments in proteins differ and the a-helical and &structures that exist vary from the ideal. The approach recently suggested by Fasman et al. seems to be more reasonable in the current state of the art (14). The CD spectra of bovine and porcine PTH at pH 3.8 resemble those observed either with films of sodium polyglutamate and polylysine hydrochloride in unordered conformations or with certain proteins considered to be extensively unfolded, i.e., e,l-casein, oxidized ribonuclease, f-l histone, S-sulfonated p-lactoglobulin A, etc. (14). The structure of these molecules is considered by Fasman et al. as being without long-range order but where the rotational motions of parts of the polypeptide backbone and of some of the side chains are constrained by local interactions. Homologous polypeptides and proteins which can be considered as true random coils, such as aqueous solutions of t,he charged forms
EDELHOCH
of polyglutamate and polylysine (14), poly[N5-2(hvdroxyethyl-L-glutamine] (15), and phosvitin at pH 6.6 (16), show positive ellipticity in the 220-nm region and stronger minima in the 200-nm wavelength region. The unordered, but constrained, structure of bovine PTH does not appear to possess either the compactness or rigidity of the unordered parts (i.e., non-a-helical or p-structures) of globular proteins since the intensities of the two CD minima vary with pH between 3.8 and 10.0. The CD spectra of native globular proteins are generally independent of pH. Consequently, the extent of int’ramolecular interactions in bovine PTH should increase with increasing pH since the freedom of motion of the backbone peptide groups becomes more restricted at pH 10. This increase in organization presumably results in part from the decrease in the positive charge of bovine PTH between pH 3.8 and 10 since there are more basic (9 lys, 5 arg, 4 his) than acidic (6 glu, 6 asp) amino acids (8). The reduction in electrostatic interactions would allow the formation of additional intramolecular contacts by decreasing the unfavorable electrostatic free energy bet’ween segments of the polypeptide chain. The variations in fluorescence intensity are another reflection of pH-dependent structural alterations in PTH. The quenching of tryptophanyl fluorescence cannot be explained by the ionization of side-chain amino groups since the dissociation of the amino group in model tryptophanyl peptides increases its quantum yield nor by base quenching since this occurs at higher pH values (17). It was suggested previously that the ionization of the phenolic side chain of bovine PTH quenched its tryptophanyl fluorescence by radiationless energy transfer (2). This cannot be the mechanism of the alkaline quenching of PTH fluorescence since one of the seven substitutions in porcine PTH is a histidine residue for the tyrosine in bovine PTH. The quenching is, therefore, due to structural modifications in PTH. The pH dependence of fluorescence of the tryptophanyl residue in PTH
STRUCTURAL
STUDIES
ON POLYPEPTIDE
is slightly affected by the substitution of seven amino acids. Since the closest substitutions occur at residues 18 and 42! (6-8) positions which are not close to the tryptophanyl residue at 23, the influence of the new residues suggest that the interactions of the tryptophanyl residue are altered and modify its fluorescence. The much weaker quenching in acid may be due to the protonation of vicinal side chain groups, i.e., imidazole or carboxyl since tryptophanyl fluorescence is partially quenched in model peptides containing these groups (18, 19). REFERENCES W. B., BEAVEN, G. H., RATTLE, 1. GRATZER, H. E. W., AND BR.4DBURY, E. M. (1968) Eur. J. Biochem. 3, 276. 2. EDELHOCII, H., AND LIPPOLDT, R. E. (1969) J. Biol. Chem. 244.3876. ~.~BREWER, B. H., .~ND EDELHOCH, H. (1970) J. Biol. Chem. 246, 2402. 4. KING, V. M. (1965) J. Mol. Biol. 11,549. 5. HAUGEN, W. P., .~ND LIPSCOMB, W. N. (1969) Acta Crystallogr. Sect. A 26 (Suppl.), S185 (XV-25). 6. WOODHEaD, J. S., O’RIORDAN, J. L. H., KEUTMANN, H. T., STALTZ, M. L., LAWSON, B. F., NIALL, H. D., ROBINSON, C. J., AND POTTS, J. T., JR. (1971) Biochemistry 10, 2787.
HORMONES
785
7. POTTS, J. T., JR., TREGEON, G. W., KEUTM~NN, H. T., NIALL, H. D., SAUER, R., DEFTOS, L. J., DAWSON, B. F., HOGAN, M. L., AND G. D., (1971) Proc. Nat. Acad. AURBACH, Sci. U. S. A. 68,63. 8. BREWER, B. H., AND RONAN, R. (1970) Proc. IVat. Acad. Sci. U. S. A. 67, 1862. G., AND DOTY, P. (1965) J. Amer. 9. HOLZWARTH, Chem. Sot. 87, 218. 10. SARHAR, P., AND DOTY, P. (1966) Proc. Nat. Acad. Sci. U. S. A. 66, 981. Y., AND TANFORD, C. (1971) J. Biol. 11. NOZAKI, Chem. 246,221l. 12. TIMASHEFF, S. N., .~ND GORBUNOFF, M. J. (1967) Ann. Rev. Biochem. 36, 13. D. B. (1971) 13. SAXENA, V. P., .~ND WETLAUFER, Proc. Nat. Acad. Sci. U. S. A. 68, 969. 14. FASMAN, G. D., HOVING, H., AND TIMASHEFF, S. N. (1970) Biochemistry 9, 3316. 15. ADLER, A. J., HOVING, P., POTTER, J. WELLS, M., AND FASMAN, G. D. (1968) J. Amer. Chem. Sot. 90, 4736. S. N., TOWNEND, R., AND PERL16. TIM.~SHEFF, MANN, G. (1967). J. Biol. Chem. 242,229O. H., BR.4ND, L., SND WILCHECK, 17. EDELHOCH, M. (1968) Biochemistry 7, 547. M., AND KATCHALSKY, E. (1968) 18. SCHINITZKY, in Molecular Associat,ion in Biology (Pullman, B., ed.), p. 361. Academic Press, New York. H., AND STEINER, 19. CHEN, R. F., EDELHOCH, R. F. (1969) in Physical Principles and Techniques of Protein Chemistry (Leach, S. J., ed.), p. 191. Academic Press, New York.