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Far-ultraviolet optical rotatory dispersion and circular dichroism studies of bovine pancreatic ribonuclease A
BIOCHIMICAET BIOPHYSICAACTA
SHORT
583
COMMUNICATIONS
BBA 33O72
Far-ultraviolet optical rotatory dispersion and circular dichroism studies of bovine pancreatic ribonuclease A The recent reports of preliminary X-ray investigations on ribonuclease A* (ref. I) and its related derivative ribonuclease S** (ref. 2) prompts us to publish our results on the structure of ribonuclease A in solution. Although the rotatory properties of ribonuclease A in the ultraviolet region have been investigated extensively in recent years s-7, fewer studies have been carried out in the far-ultraviolet region. In this communication we present the results of optical rotatory dispersion (ORD) and circular dichroism (CD) measurements on ribonuclease A in the far ultraviolet. Shown in Fig. I is the ORD spectrum of ribonuclease A at pH 6.8. There is a trough at 228 m# ([rn']22s = --53oo ± 5oo) in good agreement with previous results 3 5, a crossover point at 215 m#, and a peak at about 2oo m#. A slight shoulder appears at 21o to 215 m/z which is suggestive of the a-helical conformation although the low values of the [m'] trough and the Ira'1 peak clearly indicate that only small portions of the protein could be in this conformation. Moreover the position of the trough at 228 m# excludes the possibility of a simple mixture of the a-helix and the unordered structure and suggests some contribution of the fl-structure 8. The CD spectrum of ribonuclease A is shown in Fig. 2. The curve is characterized by a minimum at 2o8 m# (typical of the a-helical peptide transition 9) with a broad shoulder up to about 22o m# and by a maximum at 193 to 195 m#. The broadening of the usual bimodal curve of the a-helix and the presence of other features which are also noted in the CD spectrum of lysozyme 1°, a protein which is known to contain both a-helix, fl-form and unordered form, agree with the suggestion of the presence of fl-form. Indeed it is known that the antiparallel fl-form shows a CD minimum at 217 m# and a maximum at 195 m# (see, e.g. ref. II). Further support for the presence of the/5-form of ribonuclease A in aqueous solution is obtained by the behaviour of the protein in sodium dodecyl sulfate solution. Previous observations in this medium3,~, TM have shown that certain optical parameters, such as b0 and the position of the trough in the ORD curve, are shifted towards values typical of the a-helical form. In Fig. i is shown the far-ultraviolet ORD curve of ribonuclease A in 0.05 M sodium dodecyl sulfate (pH 6.8). In addition to the previously observed3, 5 shift to 233 m# and the decreased intensity of the trough ([m'1233= --35 °0 ± 500) one can more easily recognize the shoulder at 21o to 225 m# (crossover point at 21o m/~). Similarly in the corresponding CD spectrum (Fig. 2), a minimum appears at 221 to 222 m/~ reminiscent of the n-~* transition of the a-helix 9. One must emphasize that both the CD and ORD curves in sodium dodecyl sulfate solution show an increase of the unordered form, as judged by the blue shift of the dichroic band present at 208 m# in aqueous solution to 205 m# and by the Abbreviations: ORD, optical rotatory dispersion; CD, circular dichroism. * The principal chromatographic component of bovine pancreatic ribonuclease. ~* Subtilisin-modified ribonuclease. Biochim. Biophys. Acta, 154 (1968) 583-585
584
SHORT COMMUNICATIONS
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6000
400O I
2000
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-2000 I
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-6000 190 260 2JO 22r0 230 24'0 250 ~(mp)
190
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// 210
220 230 A(mp)
240
Fig. I. Far-ultraviolet ORD curves of ribonuclease A ill O.OI IV[ sodium phosphate buffer with 0.9% NaC1 (pH 6.8, Curve i) and in 0.05 M sodium dodecyl sulfate (pH 6.8, Curve 2). The concentrations used were 0.086% and 0.o93%, respectively, as determined spectrophotometrically. When necessary the solutions were filtered through sintered-glass filters. ORD measurements of sodium dodecyl sulfate solutions were made after 24 h. ORD was measured with a Cary 60 spectropolarimeter at 27 °. Cylindrical Optice]l quartz ceils were used with 0.05- and o.oi-cm optical path. The absence of optical artifacts was determined by recording the ORD of the same sample in cells of different optical path length. The accuracy of the readings was approx. ± 5-1o % above 225 m/*. At shorter wavelengths the noise increases rapidly with decreasing ~: the limits of the error are indicated in the figure. The symbol [m'] refers to the reduced mean residue rotation. The mean residue molecular weight of IiO was employed. Lyophilized ribonuclease A was obtained from Schwaxz BioResearch, Inc., Orangeburg, N.Y. Fig. 2. Far-ultraviolet CD curves of ribonuclease A in o.oi M sodium phosphate buffer with 0.9% NaCI (pH 6.8, Curve i) and in 0.05 M sodium dodecy] sulfate (pH 6.8, Curve 2). Solutions and experimental conditions were the same as in Fig. i. CD was measured with a Jouan Dichrograph model CD 185 at room temperature. The sensitivity was kept at i . io -s. The symbol [(9] represents the mean residue molecular ellipticity.
s h i f t o f t h e s h o u l d e r a t 210 t o 225 m # t o m o r e n e g a t i v e v a l u e s in t h e O R D s p e c t r u m . Recently there have been indications that sodium dodecyl sulfate eliminates the E - f o r m in s o m e proteinsS, a3 w h i l e in o t h e r s it c o n v e r t s t h e u n o r d e r e d f o r m t o a - h e l i x 12,14. W e t h i n k t h a t t h e d e t e r g e n t is a b l e in g e n e r a l t o i n d u c e t r a n s i t i o n s f r o m t h e / 5 - f o r m t o t h e u n o r d e r e d f o r m t o t h e a - h e l i x , a n d t h a t in r i b o n u c l e a s e A t h e t r a n s i t i o n is t e r m i n a t e d in t h e first s t e p . I n d e e d , a f t e r t r e a t m e n t w i t h s o d i u m d o d e c y l s u l f a t e , t h e p r e s e n c e of a - h e l i x is m o r e e a s i l y d e t e c t e d a l t h o u g h t h e t o t a l a m o u n t is n o t i n c r e a s e d , as i n d i c a t e d b o t h b y t h e l o w e r e d r o t a t i o n a n d e l l i p t i c i t y in t h e r e g i o n of t h e a m i d e
Biochim. Biophys. Acta, 154 (1968) 583-585
SHORT COMMUNICATIONS
585
n-~* transition. On the other hand, the unordered form increases and this points to a transition from the r-form to the unordered form. It must however be noted that these are only tentative qualitative interpretations, the confirmation of which needs more complete studies of the contribution of the non-peptide chromophores in the 19o-225 m/~ region. In conclusion, the general features of the structure of ribonuclease A in solution seem to be very similar to those found by X-ray techniques. Indeed in HARKER'S model of ribonuclease A (ref. I), about 17% of helix is present and three extended portions of the polypeptide chain run in roughly antiparallel sections, which, at least in part, could be in the r-conformation. Moreover the X-ray structure of ribonuclease S (ref. 2) closely resembles the structure of ribonuclease A and shows features including 15% of helix and appreciable antiparallel r-chain pairing. Thanks are due to Prof. E. SCOFFONE for helpful discussion, to Dr. P. SALVADORI for assistance with ORD and CD measurements, and to Dr. L. A. CARPINO for reading the manuscript. Centro Nazionale di Chimica delle Macromolecole del C.N.R., and Istituto di Chimica Organica dell' Universita' di Padova, Padova (Italy)
A. M. TAMBURRO A. SCATTURIN L. MORODER
I G. I'{ARTHA, J. BELLO AND D. HARKER, Nature, 2i 3 (1967) 862. 2 H. W. WYCKOFF, K. D. HARDMAN, N. M. ALLEWEL, T. INAGAMI, L. N. JOHNSON AND F . M . RICHARDS, J. Biol. Chem., 242 (1967) 3984. 3 1NT.A. GLAZER AND S. ]7~. SIMMONS, J. Am. Chem. Soc., 87 (1965) 3991. 4 R. E. CATHOU, G. G. HAMMES AND P. R. SCHIMMEL, Biochemistry, 4 (1965) 26875 R. T. SIMPSON AND ]~. L. VALLEE, Biochemistry, 5 (1966) 2531. 6 S. BEYCHOK, Proc. Natl. Acad. Sci. U.S., 53 (1965) 999. 7 S. BEYCHOK, Science, 154 (1966) 1288. 8 B. JIRGENSON, J. Biol. Chem., 241 (1966) 4855 . 9 G. HOLZWARTH AND P. DOTY, J. Am. Chem. Soc., 87 (1965) 218. IO S. lxT. TIMASHEFF, H. SusI, R. TOWNEND, L. STEVENS, M. J. GORBUNOFF AND T. F. KUMOSINSKI, in G. N. RAMACHANDRAN, Conformation of Biopolymers, Vol. i, Academic Press, London, 1967, p. 173. i i R. TOWNEND, T. F. KUMOSINSKI, S. •. TIMASHEFF AND G. D. FASMAN, Biochem. Biophys. Res. Commun., 23 (1966) 163. 12 B. JIRGENSON, J. Biol. Chem., 238 (1963) 2716. 13 G. V. TROITSKII, Biofizika, IO (1965) 895. 14 ]3. JIRGENSON AND L. S. HNILICA, jr. Am. Chem. Soc., 88 (1966) 2341.
Received November 3oth, 1967 Bioehim. Biophys. Acta, 154 (1968) 583-585
SHORT
583
COMMUNICATIONS
BBA 33O72
Far-ultraviolet optical rotatory dispersion and circular dichroism studies of bovine pancreatic ribonuclease A The recent reports of preliminary X-ray investigations on ribonuclease A* (ref. I) and its related derivative ribonuclease S** (ref. 2) prompts us to publish our results on the structure of ribonuclease A in solution. Although the rotatory properties of ribonuclease A in the ultraviolet region have been investigated extensively in recent years s-7, fewer studies have been carried out in the far-ultraviolet region. In this communication we present the results of optical rotatory dispersion (ORD) and circular dichroism (CD) measurements on ribonuclease A in the far ultraviolet. Shown in Fig. I is the ORD spectrum of ribonuclease A at pH 6.8. There is a trough at 228 m# ([rn']22s = --53oo ± 5oo) in good agreement with previous results 3 5, a crossover point at 215 m#, and a peak at about 2oo m#. A slight shoulder appears at 21o to 215 m/z which is suggestive of the a-helical conformation although the low values of the [m'] trough and the Ira'1 peak clearly indicate that only small portions of the protein could be in this conformation. Moreover the position of the trough at 228 m# excludes the possibility of a simple mixture of the a-helix and the unordered structure and suggests some contribution of the fl-structure 8. The CD spectrum of ribonuclease A is shown in Fig. 2. The curve is characterized by a minimum at 2o8 m# (typical of the a-helical peptide transition 9) with a broad shoulder up to about 22o m# and by a maximum at 193 to 195 m#. The broadening of the usual bimodal curve of the a-helix and the presence of other features which are also noted in the CD spectrum of lysozyme 1°, a protein which is known to contain both a-helix, fl-form and unordered form, agree with the suggestion of the presence of fl-form. Indeed it is known that the antiparallel fl-form shows a CD minimum at 217 m# and a maximum at 195 m# (see, e.g. ref. II). Further support for the presence of the/5-form of ribonuclease A in aqueous solution is obtained by the behaviour of the protein in sodium dodecyl sulfate solution. Previous observations in this medium3,~, TM have shown that certain optical parameters, such as b0 and the position of the trough in the ORD curve, are shifted towards values typical of the a-helical form. In Fig. i is shown the far-ultraviolet ORD curve of ribonuclease A in 0.05 M sodium dodecyl sulfate (pH 6.8). In addition to the previously observed3, 5 shift to 233 m# and the decreased intensity of the trough ([m'1233= --35 °0 ± 500) one can more easily recognize the shoulder at 21o to 225 m# (crossover point at 21o m/~). Similarly in the corresponding CD spectrum (Fig. 2), a minimum appears at 221 to 222 m/~ reminiscent of the n-~* transition of the a-helix 9. One must emphasize that both the CD and ORD curves in sodium dodecyl sulfate solution show an increase of the unordered form, as judged by the blue shift of the dichroic band present at 208 m# in aqueous solution to 205 m# and by the Abbreviations: ORD, optical rotatory dispersion; CD, circular dichroism. * The principal chromatographic component of bovine pancreatic ribonuclease. ~* Subtilisin-modified ribonuclease. Biochim. Biophys. Acta, 154 (1968) 583-585
584
SHORT COMMUNICATIONS
12000
DO00
8000
6000
400O I
2000
",\ ',\
-2000 I
\ 2
'/ { I I t
// i I
/ I
l
i
//
I
I
I
-4000I
\
-6000 190 260 2JO 22r0 230 24'0 250 ~(mp)
190
200
// 210
220 230 A(mp)
240
Fig. I. Far-ultraviolet ORD curves of ribonuclease A ill O.OI IV[ sodium phosphate buffer with 0.9% NaC1 (pH 6.8, Curve i) and in 0.05 M sodium dodecyl sulfate (pH 6.8, Curve 2). The concentrations used were 0.086% and 0.o93%, respectively, as determined spectrophotometrically. When necessary the solutions were filtered through sintered-glass filters. ORD measurements of sodium dodecyl sulfate solutions were made after 24 h. ORD was measured with a Cary 60 spectropolarimeter at 27 °. Cylindrical Optice]l quartz ceils were used with 0.05- and o.oi-cm optical path. The absence of optical artifacts was determined by recording the ORD of the same sample in cells of different optical path length. The accuracy of the readings was approx. ± 5-1o % above 225 m/*. At shorter wavelengths the noise increases rapidly with decreasing ~: the limits of the error are indicated in the figure. The symbol [m'] refers to the reduced mean residue rotation. The mean residue molecular weight of IiO was employed. Lyophilized ribonuclease A was obtained from Schwaxz BioResearch, Inc., Orangeburg, N.Y. Fig. 2. Far-ultraviolet CD curves of ribonuclease A in o.oi M sodium phosphate buffer with 0.9% NaCI (pH 6.8, Curve i) and in 0.05 M sodium dodecy] sulfate (pH 6.8, Curve 2). Solutions and experimental conditions were the same as in Fig. i. CD was measured with a Jouan Dichrograph model CD 185 at room temperature. The sensitivity was kept at i . io -s. The symbol [(9] represents the mean residue molecular ellipticity.
s h i f t o f t h e s h o u l d e r a t 210 t o 225 m # t o m o r e n e g a t i v e v a l u e s in t h e O R D s p e c t r u m . Recently there have been indications that sodium dodecyl sulfate eliminates the E - f o r m in s o m e proteinsS, a3 w h i l e in o t h e r s it c o n v e r t s t h e u n o r d e r e d f o r m t o a - h e l i x 12,14. W e t h i n k t h a t t h e d e t e r g e n t is a b l e in g e n e r a l t o i n d u c e t r a n s i t i o n s f r o m t h e / 5 - f o r m t o t h e u n o r d e r e d f o r m t o t h e a - h e l i x , a n d t h a t in r i b o n u c l e a s e A t h e t r a n s i t i o n is t e r m i n a t e d in t h e first s t e p . I n d e e d , a f t e r t r e a t m e n t w i t h s o d i u m d o d e c y l s u l f a t e , t h e p r e s e n c e of a - h e l i x is m o r e e a s i l y d e t e c t e d a l t h o u g h t h e t o t a l a m o u n t is n o t i n c r e a s e d , as i n d i c a t e d b o t h b y t h e l o w e r e d r o t a t i o n a n d e l l i p t i c i t y in t h e r e g i o n of t h e a m i d e
Biochim. Biophys. Acta, 154 (1968) 583-585
SHORT COMMUNICATIONS
585
n-~* transition. On the other hand, the unordered form increases and this points to a transition from the r-form to the unordered form. It must however be noted that these are only tentative qualitative interpretations, the confirmation of which needs more complete studies of the contribution of the non-peptide chromophores in the 19o-225 m/~ region. In conclusion, the general features of the structure of ribonuclease A in solution seem to be very similar to those found by X-ray techniques. Indeed in HARKER'S model of ribonuclease A (ref. I), about 17% of helix is present and three extended portions of the polypeptide chain run in roughly antiparallel sections, which, at least in part, could be in the r-conformation. Moreover the X-ray structure of ribonuclease S (ref. 2) closely resembles the structure of ribonuclease A and shows features including 15% of helix and appreciable antiparallel r-chain pairing. Thanks are due to Prof. E. SCOFFONE for helpful discussion, to Dr. P. SALVADORI for assistance with ORD and CD measurements, and to Dr. L. A. CARPINO for reading the manuscript. Centro Nazionale di Chimica delle Macromolecole del C.N.R., and Istituto di Chimica Organica dell' Universita' di Padova, Padova (Italy)
A. M. TAMBURRO A. SCATTURIN L. MORODER
I G. I'{ARTHA, J. BELLO AND D. HARKER, Nature, 2i 3 (1967) 862. 2 H. W. WYCKOFF, K. D. HARDMAN, N. M. ALLEWEL, T. INAGAMI, L. N. JOHNSON AND F . M . RICHARDS, J. Biol. Chem., 242 (1967) 3984. 3 1NT.A. GLAZER AND S. ]7~. SIMMONS, J. Am. Chem. Soc., 87 (1965) 3991. 4 R. E. CATHOU, G. G. HAMMES AND P. R. SCHIMMEL, Biochemistry, 4 (1965) 26875 R. T. SIMPSON AND ]~. L. VALLEE, Biochemistry, 5 (1966) 2531. 6 S. BEYCHOK, Proc. Natl. Acad. Sci. U.S., 53 (1965) 999. 7 S. BEYCHOK, Science, 154 (1966) 1288. 8 B. JIRGENSON, J. Biol. Chem., 241 (1966) 4855 . 9 G. HOLZWARTH AND P. DOTY, J. Am. Chem. Soc., 87 (1965) 218. IO S. lxT. TIMASHEFF, H. SusI, R. TOWNEND, L. STEVENS, M. J. GORBUNOFF AND T. F. KUMOSINSKI, in G. N. RAMACHANDRAN, Conformation of Biopolymers, Vol. i, Academic Press, London, 1967, p. 173. i i R. TOWNEND, T. F. KUMOSINSKI, S. •. TIMASHEFF AND G. D. FASMAN, Biochem. Biophys. Res. Commun., 23 (1966) 163. 12 B. JIRGENSON, J. Biol. Chem., 238 (1963) 2716. 13 G. V. TROITSKII, Biofizika, IO (1965) 895. 14 ]3. JIRGENSON AND L. S. HNILICA, jr. Am. Chem. Soc., 88 (1966) 2341.
Received November 3oth, 1967 Bioehim. Biophys. Acta, 154 (1968) 583-585