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Physical-chemical properties of ubiquitin
378
Biochimica et Biophysica Acta, 624 (1980) 378--385 © Elsevier/North-Holland Biomedical Press
BBA 38487
PHYSICAL-CHEMICAL PROPERTIES OF UBIQUITIN
J A M E S J E N S O N a, G I D E O N G O L D S T E I N a,, and E S T H E R B R E S L O W b , , ,
a Sloan-Kettering Institute, New York, N Y 10021 and h Department o f Biochemistry, Cornell University Medical College, New York, N Y 10021 (U.S.A.) (Received F e b r u a r y 6th, 1980)
Key words: Ubiquitin; Protein A24; Histone 2A; Tyrosine residue; (Circular dichroism, Fluorescence)
Summary The secondary structure of ubiquitin, the environment of its single tyrosine residue and its potential for interacting noncovalently with histone 2A or DNA, have been probed by circular dichroism (CD), ultraviolet absorbance, fluorescence and ancillary techniques. The results indicate that ubiquitin has a stable secondary structure containing only a low percentage of R-helix or ~-sheet. The ubiquitin tyrosine has an elevated pKa arising from the influence of a spatially proximate carboxylate which also causes a marked quenching of the tyrosine fluorescence at neutral pH; the influence of this carboxylate is lost when the protein is unfolded in 7 M guanidine. No evidence has been obtained for the presence of aUosteric noncovalent interactions between free ubiquitin and either histone 2A or purified unfractionated DNA. The results suggest that one function of ubiquitin (or of the ubiquitin segment of protein A24) may be to interact with a chromatin component other than histone 2A or DNA, and/or that ubiquitin functions within A24 as a steric blocking group of a region of the nucleosome.
Introduction Ubiquitin is a small protein (Mr, 8451) thought to be universally distributed in prokaryotes and eukaryotes [1,2]. The natural function of ubiquitin is not known. In mammals, it has been observed to stimulate lymphocyte differentiation and to activate adenyl cyclase, apparently by binding to catecholamine
* Present address: Ortho Pharmaceutical Corporation, Rarltan, NJ 08869, U.S.A. ** T o w h o m c o r r e s p o n d e n c e s h o u l d h e addressed.
379 receptors [1,2]. Ubiquitin has also been found in chromatin, both covalentlyconjugated to histone 2A within the chromosomal protein A24 [3] and in the free state in the nucleosome linker region [4]; there is some evidence that increases in free ubiquitin or decreases in A24 are associated with increased gene template activity [5,6]. The present studies were undertaken to characterize general aspects of ubiquitin conformation; little is known about this except that NMR studies have indicated that the conformation is stable over wide changes of pH and temperature [7]. It was of particular interest to probe the environment of the single ubiquitin tyrosine since a ubiquitin
Ubiquitin was purified as described previously [1]. The concentration of ubiquitin in solution was routinely determined by ultraviolet absorbance using the value 1750 for the molar extinction at 275 nm at neutral pH; the molar extinction was initially determined from a ubiquitin solution the concentration of which was determined by quantitative amino acid analysis. Ubiquitin pentapeptide was prepared as described elsewhere [9]. Histone 2A was isolated from Sigma calf thymus histone preparations using modifications of the 50 mM NaC1 P-60 chromatographic procedure of BShm et al. [10] to obtain histories 3 and 2A and the method of Sommer and Chalkley [11] to purify histone 2A. The product migrated identically to histone 2A prepared from fresh calf thymus when examined electrophoretically in 15% acrylamide slab gels (2.5 M urea/0.9 N acetic acid, pH 2.8) and in 0.1% SDS containing gels; its amino acid composition and the absence of a free aminoterminus were as expected for histone 2A [12]. The concentration of histone 2A in solution was determined by ultraviolet absorbance using the value 4620 for E27~ (molar extinction); this is an average of values reported elsewhere (cf. Refs. 13 and 14). Calf thymus DNA was purified by a minor modification of the method of Adler et al. [15] from Sigma Type V calf thymus DNA. Absorbance ratios, 230 : 260 and 280 : 260, and CD spectra indicated good purity [16]. DNA concentration in solution was estimated from known extinction coefficients (e.g., Ref. 16). CD studies were carried out using a Cary 60 spectropolarimeter with Model 6001 CD attachment. Fluorescence measurements were made on a PerkinElmer Model MPF 44A spectrofluorimeter; typical ubiquitin concentrations were 6 - - 1 2 . 1 0 - S M and, where appropriate, corrections for inner filter effects were made as previously described [17]. Ultraviolet absorption mea-
380
surements were carried out using Gilford Model 222A and Cary 15 spectrophotometers. Ultracentrifuge studies were carried out with Schlieren optics and a Beckman Model E ultracentrifuge. Results
CD studies of the unmodified protein Fig. 1 shows the far-ultraviolet CD spectrum of ubiquitin at pH 7; signal-tonoise ratios became increasingly poor below 205 nm, but the data indicated that 206 nm is the negative extremum of a band that returns to the baseline at 199 rim. The near-ultraviolet spectrum (not shown) indicates that the single tyrosine and two phenylalanine residues have only weak optical activity in this wavelength region. In Fig. 1, the far-ultraviolet spectrum is compared with a theoretical spectrum calculated for 6% a-helix, 10% ~-structure and 84% random structure using the parameters of Chen et al. [18]. The agreement above 210 nm indicates a low percentage in ubiquitin of organized repeating secondary structure. Below 210 nm, agreement between experimental and theoretical curves is not good. This situation is similar to that observed by Chen et al. [18] for chymotrypsin, the far-ultraviolet CD spectrum of which is strikingly similar to that we report for ubiquitin; the results suggest that the secondary structure content of ubiquitin and chymotrypsin may be similar. As indicated by previous NMR studies [7], the overall conformation of ubiquitin is stable to changes in pH. In accord with this, no significant changes in ultraviolet CD spectra were seen between pH 3 and 12. The low percentage of organized secondary structure in ubiquitin is also similar in some respects to that associated with histone conformation [13].
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Fig. 1. Far-ultraviolet CD s p e c t r u m o f u b i q u l t i n at p H 7 i n 0 . 1 6 M K C I at 2 5 ° C , r e p o r t e d as t h e m e a n zes/due e l l i p t i c i t y ( d e g r e e • c m 2 • d r o o l - I ) . T h e o b s e r v e d s p e c t r u m ( ) is c o m p a r e d w i t h a t h e o retical s p e c t r u m ( . . . . . . ) ' c a l c u l a t e d as d e s c r i b e d in t h e t e x t . Fig. 2. S p e c t r o p h o t o m e t r l e t i t r a t i o n o f t y r o s i n e in u b i q u i t i n and its dezlvative p e n t a p e p t i d e r e p o r t e d as t h e c h a n g e in m o l a r a b s o r p t i v i t y at 2 4 5 n m as a f u n c t i o n o f pH. C o n d i t i o n s : 0 . 1 6 M KCI. 1 • 1 0 - 2 M g]ycine, 25°C.
381 Histones undergo salt-dependent conformation changes [13]; we observed no change in ubiquitin conformation when the ionic strength was increased from 0 to 0.16 with NaC1.
Ultraeentrifugation studies The sedimentation velocity of ubiquitin (1.4 mg/ml) at pH 7 in 0.16 M KCI, 25°C was determined as 1.2 s, suggesting little self-association under these conditions; however, molecular weights were not directly investigated.
Studies of tyrosine environment Comparison of the near-ultraviolet absorption spectra of ubiquitin at pH 7 and 12 indicated two difference bands with maxima at 245 and 295 nm, characteristic of absorbance changes associated with tyrosine ionization. Spectrophotometric titration of the tyrosine, monitored at either 245 nm (Fig. 2) or at 295 rim, indicated an apparent pK of 11.1, a high value for tyrosine; titration curves were not associated with any time-dependent changes. By contrast, the ubiquitin~lerived lymphocyte-stimulating pentapeptide which contains the tyrosine, titrates with a normal tyrosine pK of 10.0 (Fig. 2). An abnormal tyrosine environment in ubiquitin is also evident from fluorescence data. Fig. 3 shows fluorescence emission spectra of ubiquitin excited at 276 rim. The emission maximum at 303 nm is characteristic of tyrosine; the weak-trailing long wavelength fluorescence is tentatively ascribed to an impurity. As estimated from the absorbance~orrected emission at 303 rim relative to a tyrosine amino acid standard at pH 6 [19], the ratio of the ubiquitin quantum yield at pH 7 to that of the free amino acid is less than 0.07; this is a low value for a protein tyrosine [19]. Decreasing the pH from 7 to 2 leads to a 400% increase in ubiquitin fluorescence. Additionally, at neutral pH
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Fig. 3. F l u o r e s c e n c e e n d s s l o n s p e e t z a o f u b i q u l t i n u n d e ¢ v a r i o u s c o n d i t i o n s . T e m p . , 2 6 ° C ; e x c i t i n g w a v e l e n g t h , 2 7 6 n m . F l u o r e s c e n c e i n t e n s i t i e s are =Iven o n an i n t e r n a l l y e o n s / s t e n t b u t arbitrary s c a l e . • . . . . . , p H 7 in 0 . 1 6 M NaCI; , p H 2 , i n 0 . 1 6 M NaCI; . . . . . . p H "/in 7 M s u a n i d l n i u m h y d r o c h l o r i d e . F i g . 4. E f f e c t o f p H o n u b i q u i t i n f l u o r e s c e n c e i n t e n s i t y . D a t a are r e p o s e d o n a n i n t e r n a l l y c o m d s t e n t b u t arbitrary s c a l e . T e m p e r a t u r e , 2 5 ° C . A r r o w indJeate~ a p p a r e n t t i t r a t i o n m i d p o i n t i n 0 . 1 6 M NaCI.
382 in 7 M guanidine, conditions under which ubiquitin is unfolded [7], fluorescence is increased b y 150% relative to that in the absence of guanidine. The pKa of the group responsible for the pH effect on tyrosine fluorescence in the absence of guanidine was determined as 3.9 b y monitoring fluorescence as a function of pH {Fig. 4). Also seen in Fig. 4 is that, fluorescence is largely independent of pH between pH 7 and 2 in 7 M guanidine. We have also studied the ubiquitin tyrosine environment b y solvent perturbation absorption [20] and b y iodide-quenching of fluorescence (e.g., Ref. 21). Solvent perturbation b y Me2SO at pH 4.9, suggested that the tyrosine was less than 50% exposed to solvent at this pH. Iodide-quenching studies, however, did not indicate a significant degree of tyrosine burial at either pH 7 or pH 2. We were also able to nitrate the ubiquitin tyrosine with tetmnitromethane under standard conditions [22]. The pKa of the nitrotyrosine was determined spectroscopically [22] as 6.85. This is a normal value for nitrotyrosine [22], as distinguished from the high tyrosine pK~ in the unmodified protein, and suggests that nitration disrupts the interactions which elevate the pKa of the unmodified protein.
Behavior o f mixtures o f ubiquitin and histone 2A The effects of mixing ubiquitin and histone 2A were examined b y CD, fluorescence and gel chromatography. The far-ultraviolet CD spectrum of histone 2A that we observed and the effects of NaC1 on this spectrum were identical to results reported for histone 2A b y D'Anna and Isenberg [13]. At pH 7 in 0.16 M NaC1, the CD spectrum between 240 and 208 nm of a mixture of histone 2A (1.8 • 10 -4 M) and ubiquitin (2 • 10 -4 M) was the exact sum of the spectra of the isolated components, suggesting no effect of one protein on the conformation of the other; this result was obtained irrespective of whether the NaCl was added before or after the t w o proteins were mixed. Below 208 nm, signal-to-noise ratios were poor in these studies and data were unreliable. We did not study the CD spectra of mixtures of the t w o proteins in the absence of salt because of the possibility that ubiquitin would alter the conformation of the histone under these conditions b y nonspecific ionic effects. CD spectra of mixtures of the t w o proteins in NaC1 were observed not to change with time when followed for 1 week. A lack of interaction between ubiquitin and histone 2A was also suggested b y fluorescence and gel chromatography. The fluorescence emission spectra of mixtures of the t w o in 0.16 M NaC1 were within 2% of the sum of those of the individual components. These results strongly suggest that the environments of the single ubiquitin tyrosine and the three tyrosines of histone 2A are unaltered when the t w o proteins are mixed. We also observed no change in the accessibility of these tyrosines to I- (as measured b y quenching studies) on mixing the two proteins. On chromatography on Sephadex G-50 at pH 7 in 0.15 M NaCI, ubiquitin and histone 2A migrated as well-separated components when applied as a mixture containing 6 mg/ml ubiquitin and 13 mg/ml histone 2A. Behavior o f mixtures o f ubiquitin and DNA The CD spectra at pH 7 in 0.16 M NaC1 of mixture of ubiquitin and calf thymus DNA were identical to the sum of those of the isolated components
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X(nm) Fig. 5. CO s p e c t r a o f u b i q u l t i n , D N A a n d u b l q u i t L n / D N A m i x t u r e s . Cond/$1ons: T e m p e r a t u r e , 2 5 ° C ; l l g h t path, 1.0 c m ; p H = 7; 0 . 1 6 M NaCl. - - - , 0.38 m g / m l DNA" . . . . . . , 5.3 • 1 0 -6 M ( 0 . 0 4 5 m g / m l ) ubiq u i t i n ; • 1 : 1 m i x t u r e o f ubiquitln a n d D N A s o l u t i o n s ( e x p e r l m e n t a l v a l u e s ) ; . . . . . . , calculated spect r u m for 1 : 1 mixture of ubiqultin and D N A solutions assuming no interaction.
between 300 and 210 n m (Fig. 5). This wavelength region includes the strong near-ultraviolet D N A ellipticity bands as well as a good fraction of the farultraviolet bands of both components. Preliminary studies of the effect of ubiquitin on D N A melting transition temperature have also indicated no significant degree of interaction between ubiquitin and D N A . Discussion The notable features of ubiquitin conformation demonstrated here are that it contains very little CD-demonstrable repeating secondary structure and that its single tyrosine is in an abnormal environment. The data indicate that the abnormal tyrosine behavior reflects the proximity to the tyrosine of at least one side-chain carboxylate. Tyrosine fluorescence is known to be susceptible to quenching by deprotonated carboxyl groups [19]. Thus, the marked increase in tyrosine fluorescence accompanying protonation of a group with pK a = 3.9 can be interpreted most simply to reflect the loss of quenching associated with protonation of such a carboxyl group. The alternative possibility, that quenching occurs via another mechanism and is lost at low pH because of a carboxyl-controlled conformational change, is not supported by the virtual absence of CD changes between pH 7 and 3. Additionally, the high tyrosine pK in the native protein is clearly compatible with the influence of a proximal carboxylate. It is relevant that the residue preceding the tyrosine in the sequence is an aspartic acid [2]. While it is possible that this is the carboxyl group responsible for quenching, the absence of pH-dependent quenching in 7 M guanidine indicates that the interactions between the tyrosine and carboxylate are dependent on the integrity of the native conformation; therefore a carboxyl elsewhere in the sequence might be involved. The marked differences in tyrosine titration behavior between ubiquitin and its biologically-active derivative pentapeptide (which contains tyrosine at the amino terminus) indicate that the tyrosine environment is different in the two cases. The relevance of this lies in the observation that cleavage of tyrosine from the pentapeptide results in loss of its biological activity (unpublished
384
observations) a fact originally suggesting that the tyrosine ring was involved in this activity. The very different environments of tyrosine in the t w o cases suggests that this is not the case, and is in accord with a very recent observation that the tyrosine side-chain per se can be deleted without activity effects. We see no evidence of intermolecular noncovalent interactions between free ubiquitin and histone 2A, particularly any which result in conformational change. While these results do not strictly preclude an effect of ubiquitin on histone 2A properties when the t w o proteins are linked covalently within A24, t h e y suggest that noncovalent allosteric interactions between the t w o within A24 are likely to be weak. This is in accord with recent demonstrations that A24 can substitute for histone 2A as a c o m p o n e n t of the nucleosome histone core and retains the ability to interact with histone 2B [23], and that free histone 2A and the histone 2A components o f A24 are similarly acetylated and phosphorylated in chromatin [24]. We have also obtained no evidence, thus far, indicating interaction between ubiquitin and DNA. A lack of interaction between ubiquitin (as an A24 component) and DNA has also been reported elsewhere [24,25]. Thus, the function of ubiquitin in chromatin remains puzzling. While it remains possible that ubiquitin, either free or within A24, is capable of interaction with a very small subpopulation o f the chromatin DNA (cf. Ref. 4), our results suggest that ubiquitin might interact with another, as yet unidentified, chromatin component, and/or that the covalent attachment of ubiquitin to histone 2A in A24 serves simply to sterically block a region of the nucleosome. In accord with this, Matsui et al. [26] have recently obtained evidence suggesting that, during the interphase stage of the mitosis cycle, ubiquitin attachment to histone 2A physically blocks chromatin condensation. Acknowledgment This research was supported b y Grant GM-17528 to E.B. from the National Institutes of Health. References 1 Goldstein, G., Sheid, M.S., Hammcrling, V., Boysc, E.A., Schlesinger, D.H. and Nial], H.D. (1975) PToc. Natl. Acad. ScL U.S.A. 72, 11--15 2 Schles~ge~, D.H., Goldsteln° G. and Niall, H~D. (1975) Biochemistry 14, 2214--2218 3 Hunt, L.T. and Dayhoff, M.O. (1977) B i o c h e m . B i o p h y s . Res. Commun. 74, 650---655 4 Watson° D.C., Levy° W.B. and Dixon, G.H. (1978) Nature 276, 196--198 5 Ballsl, N.R,, Kang, Y.-J., Olson, M . O J . and Busch, H. (1975) J. Biol. Chem. 250, 5921--5925 6 Goldknoff, I.L., French, M.F., Daskal, Y. and Busch, H. (1978) Biochem. Biophys. Res. Commun. 84, 786--793 7 Lenkinski, R.E., Chen, D.M.° Glickson, J.D. and Goldstetn, G. (1977) Biochim. Biophys. Acta 494° 126--130 8 Schlestnger, D.H., Goldstein, G., Seheid, M.P. and Bitensky, M. (1977) Expetentia 34, 703--704 9 Goldstein , G. and Schlesinger, D.H, (1978) Patent No. 864122, Belgium Patent O f f i c e 10 BShm, E.L., Strickland, W.M., Strickland, N., Thwaits, B.H., van der Westhuizen, D.R. and Von Holt, C. (1973) FEBS Lett. 34, 217--221 11 Sommer, K.R. and Chalkley, R. (1974) Biochemistry 13, 1022--1032 12 Johns, E.W. (1977) Methods Cell Biol. 16° 183--203 13 D'Anna, J.A. and Isenberg, I, (1974) Biochemistry 13° 2093--2098 14 Oh, Y.H. (1970) J. Biol. Chem. 245, 6404--6416
385 15 16 17 18 19 20 21 22 23 24 25 26
Adler, A.J., Schaffhausen, B., Langan, T.A. and F u m a n , G.D. (1971) Biochemistry 10, 909--913 Gross-Bellard, M., Oudet, P. and Chambon, P. (1973) Eur. J. Biochem. 36, 32---38 Stir, S.S., Rabbani, L., Libman, L. and Breslow, E. (1979) Biochernls~y 18, 1026--1036 Chen, Y.-H., Yang, J.T. and Martlnez, H.M. (1972) Biochemistry 11, 4120---4131 Cowg~Ul, R.W. (1976) in Biochemical Fluorescence (Chen, R.F. and Edelhoch, H,, eds.), Vol. II, pp. 441---486, Marcel Dekker, New York Hesskovits, T.T. and Laskowski, M J r . (1962) J. Biol. Chem. 237, 3418--3422 Ugurbil, K. and Bersohn, R. (1977) Biochemistry 16,895--901 Riordan, J.F.0 Sokolovsky, V. and Vallee, B.L. (1967) Biochemistry 6, 358--361 M~tlnson, H.G., True. R.0 Butch, J.B.E. and Kunkel, G. (1979) Proc. Natl. Acad. Sol. U.S.A. 76, 1030--1034 Goldknopf, I.L., Rosenbaum, F., Sterner, R.0 Vidali, G., Allfrey, V.G. and Busch, H. (197"9) Biochem. Biophys. Res. Commun. 90, 269--277 Goldknopf, I.L., French, M.F.0 BAII~I, N.R.0 Daskal, I. and Busch, H. (1977) Proc. Am. Assoc. Cancer Res. 18, 83 Matsui, S.-I., Seon, B.K. and Sandberg, A.A. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6386--6390
Biochimica et Biophysica Acta, 624 (1980) 378--385 © Elsevier/North-Holland Biomedical Press
BBA 38487
PHYSICAL-CHEMICAL PROPERTIES OF UBIQUITIN
J A M E S J E N S O N a, G I D E O N G O L D S T E I N a,, and E S T H E R B R E S L O W b , , ,
a Sloan-Kettering Institute, New York, N Y 10021 and h Department o f Biochemistry, Cornell University Medical College, New York, N Y 10021 (U.S.A.) (Received F e b r u a r y 6th, 1980)
Key words: Ubiquitin; Protein A24; Histone 2A; Tyrosine residue; (Circular dichroism, Fluorescence)
Summary The secondary structure of ubiquitin, the environment of its single tyrosine residue and its potential for interacting noncovalently with histone 2A or DNA, have been probed by circular dichroism (CD), ultraviolet absorbance, fluorescence and ancillary techniques. The results indicate that ubiquitin has a stable secondary structure containing only a low percentage of R-helix or ~-sheet. The ubiquitin tyrosine has an elevated pKa arising from the influence of a spatially proximate carboxylate which also causes a marked quenching of the tyrosine fluorescence at neutral pH; the influence of this carboxylate is lost when the protein is unfolded in 7 M guanidine. No evidence has been obtained for the presence of aUosteric noncovalent interactions between free ubiquitin and either histone 2A or purified unfractionated DNA. The results suggest that one function of ubiquitin (or of the ubiquitin segment of protein A24) may be to interact with a chromatin component other than histone 2A or DNA, and/or that ubiquitin functions within A24 as a steric blocking group of a region of the nucleosome.
Introduction Ubiquitin is a small protein (Mr, 8451) thought to be universally distributed in prokaryotes and eukaryotes [1,2]. The natural function of ubiquitin is not known. In mammals, it has been observed to stimulate lymphocyte differentiation and to activate adenyl cyclase, apparently by binding to catecholamine
* Present address: Ortho Pharmaceutical Corporation, Rarltan, NJ 08869, U.S.A. ** T o w h o m c o r r e s p o n d e n c e s h o u l d h e addressed.
379 receptors [1,2]. Ubiquitin has also been found in chromatin, both covalentlyconjugated to histone 2A within the chromosomal protein A24 [3] and in the free state in the nucleosome linker region [4]; there is some evidence that increases in free ubiquitin or decreases in A24 are associated with increased gene template activity [5,6]. The present studies were undertaken to characterize general aspects of ubiquitin conformation; little is known about this except that NMR studies have indicated that the conformation is stable over wide changes of pH and temperature [7]. It was of particular interest to probe the environment of the single ubiquitin tyrosine since a ubiquitin
Ubiquitin was purified as described previously [1]. The concentration of ubiquitin in solution was routinely determined by ultraviolet absorbance using the value 1750 for the molar extinction at 275 nm at neutral pH; the molar extinction was initially determined from a ubiquitin solution the concentration of which was determined by quantitative amino acid analysis. Ubiquitin pentapeptide was prepared as described elsewhere [9]. Histone 2A was isolated from Sigma calf thymus histone preparations using modifications of the 50 mM NaC1 P-60 chromatographic procedure of BShm et al. [10] to obtain histories 3 and 2A and the method of Sommer and Chalkley [11] to purify histone 2A. The product migrated identically to histone 2A prepared from fresh calf thymus when examined electrophoretically in 15% acrylamide slab gels (2.5 M urea/0.9 N acetic acid, pH 2.8) and in 0.1% SDS containing gels; its amino acid composition and the absence of a free aminoterminus were as expected for histone 2A [12]. The concentration of histone 2A in solution was determined by ultraviolet absorbance using the value 4620 for E27~ (molar extinction); this is an average of values reported elsewhere (cf. Refs. 13 and 14). Calf thymus DNA was purified by a minor modification of the method of Adler et al. [15] from Sigma Type V calf thymus DNA. Absorbance ratios, 230 : 260 and 280 : 260, and CD spectra indicated good purity [16]. DNA concentration in solution was estimated from known extinction coefficients (e.g., Ref. 16). CD studies were carried out using a Cary 60 spectropolarimeter with Model 6001 CD attachment. Fluorescence measurements were made on a PerkinElmer Model MPF 44A spectrofluorimeter; typical ubiquitin concentrations were 6 - - 1 2 . 1 0 - S M and, where appropriate, corrections for inner filter effects were made as previously described [17]. Ultraviolet absorption mea-
380
surements were carried out using Gilford Model 222A and Cary 15 spectrophotometers. Ultracentrifuge studies were carried out with Schlieren optics and a Beckman Model E ultracentrifuge. Results
CD studies of the unmodified protein Fig. 1 shows the far-ultraviolet CD spectrum of ubiquitin at pH 7; signal-tonoise ratios became increasingly poor below 205 nm, but the data indicated that 206 nm is the negative extremum of a band that returns to the baseline at 199 rim. The near-ultraviolet spectrum (not shown) indicates that the single tyrosine and two phenylalanine residues have only weak optical activity in this wavelength region. In Fig. 1, the far-ultraviolet spectrum is compared with a theoretical spectrum calculated for 6% a-helix, 10% ~-structure and 84% random structure using the parameters of Chen et al. [18]. The agreement above 210 nm indicates a low percentage in ubiquitin of organized repeating secondary structure. Below 210 nm, agreement between experimental and theoretical curves is not good. This situation is similar to that observed by Chen et al. [18] for chymotrypsin, the far-ultraviolet CD spectrum of which is strikingly similar to that we report for ubiquitin; the results suggest that the secondary structure content of ubiquitin and chymotrypsin may be similar. As indicated by previous NMR studies [7], the overall conformation of ubiquitin is stable to changes in pH. In accord with this, no significant changes in ultraviolet CD spectra were seen between pH 3 and 12. The low percentage of organized secondary structure in ubiquitin is also similar in some respects to that associated with histone conformation [13].
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Fig. 1. Far-ultraviolet CD s p e c t r u m o f u b i q u l t i n at p H 7 i n 0 . 1 6 M K C I at 2 5 ° C , r e p o r t e d as t h e m e a n zes/due e l l i p t i c i t y ( d e g r e e • c m 2 • d r o o l - I ) . T h e o b s e r v e d s p e c t r u m ( ) is c o m p a r e d w i t h a t h e o retical s p e c t r u m ( . . . . . . ) ' c a l c u l a t e d as d e s c r i b e d in t h e t e x t . Fig. 2. S p e c t r o p h o t o m e t r l e t i t r a t i o n o f t y r o s i n e in u b i q u i t i n and its dezlvative p e n t a p e p t i d e r e p o r t e d as t h e c h a n g e in m o l a r a b s o r p t i v i t y at 2 4 5 n m as a f u n c t i o n o f pH. C o n d i t i o n s : 0 . 1 6 M KCI. 1 • 1 0 - 2 M g]ycine, 25°C.
381 Histones undergo salt-dependent conformation changes [13]; we observed no change in ubiquitin conformation when the ionic strength was increased from 0 to 0.16 with NaC1.
Ultraeentrifugation studies The sedimentation velocity of ubiquitin (1.4 mg/ml) at pH 7 in 0.16 M KCI, 25°C was determined as 1.2 s, suggesting little self-association under these conditions; however, molecular weights were not directly investigated.
Studies of tyrosine environment Comparison of the near-ultraviolet absorption spectra of ubiquitin at pH 7 and 12 indicated two difference bands with maxima at 245 and 295 nm, characteristic of absorbance changes associated with tyrosine ionization. Spectrophotometric titration of the tyrosine, monitored at either 245 nm (Fig. 2) or at 295 rim, indicated an apparent pK of 11.1, a high value for tyrosine; titration curves were not associated with any time-dependent changes. By contrast, the ubiquitin~lerived lymphocyte-stimulating pentapeptide which contains the tyrosine, titrates with a normal tyrosine pK of 10.0 (Fig. 2). An abnormal tyrosine environment in ubiquitin is also evident from fluorescence data. Fig. 3 shows fluorescence emission spectra of ubiquitin excited at 276 rim. The emission maximum at 303 nm is characteristic of tyrosine; the weak-trailing long wavelength fluorescence is tentatively ascribed to an impurity. As estimated from the absorbance~orrected emission at 303 rim relative to a tyrosine amino acid standard at pH 6 [19], the ratio of the ubiquitin quantum yield at pH 7 to that of the free amino acid is less than 0.07; this is a low value for a protein tyrosine [19]. Decreasing the pH from 7 to 2 leads to a 400% increase in ubiquitin fluorescence. Additionally, at neutral pH
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Fig. 3. F l u o r e s c e n c e e n d s s l o n s p e e t z a o f u b i q u l t i n u n d e ¢ v a r i o u s c o n d i t i o n s . T e m p . , 2 6 ° C ; e x c i t i n g w a v e l e n g t h , 2 7 6 n m . F l u o r e s c e n c e i n t e n s i t i e s are =Iven o n an i n t e r n a l l y e o n s / s t e n t b u t arbitrary s c a l e . • . . . . . , p H 7 in 0 . 1 6 M NaCI; , p H 2 , i n 0 . 1 6 M NaCI; . . . . . . p H "/in 7 M s u a n i d l n i u m h y d r o c h l o r i d e . F i g . 4. E f f e c t o f p H o n u b i q u i t i n f l u o r e s c e n c e i n t e n s i t y . D a t a are r e p o s e d o n a n i n t e r n a l l y c o m d s t e n t b u t arbitrary s c a l e . T e m p e r a t u r e , 2 5 ° C . A r r o w indJeate~ a p p a r e n t t i t r a t i o n m i d p o i n t i n 0 . 1 6 M NaCI.
382 in 7 M guanidine, conditions under which ubiquitin is unfolded [7], fluorescence is increased b y 150% relative to that in the absence of guanidine. The pKa of the group responsible for the pH effect on tyrosine fluorescence in the absence of guanidine was determined as 3.9 b y monitoring fluorescence as a function of pH {Fig. 4). Also seen in Fig. 4 is that, fluorescence is largely independent of pH between pH 7 and 2 in 7 M guanidine. We have also studied the ubiquitin tyrosine environment b y solvent perturbation absorption [20] and b y iodide-quenching of fluorescence (e.g., Ref. 21). Solvent perturbation b y Me2SO at pH 4.9, suggested that the tyrosine was less than 50% exposed to solvent at this pH. Iodide-quenching studies, however, did not indicate a significant degree of tyrosine burial at either pH 7 or pH 2. We were also able to nitrate the ubiquitin tyrosine with tetmnitromethane under standard conditions [22]. The pKa of the nitrotyrosine was determined spectroscopically [22] as 6.85. This is a normal value for nitrotyrosine [22], as distinguished from the high tyrosine pK~ in the unmodified protein, and suggests that nitration disrupts the interactions which elevate the pKa of the unmodified protein.
Behavior o f mixtures o f ubiquitin and histone 2A The effects of mixing ubiquitin and histone 2A were examined b y CD, fluorescence and gel chromatography. The far-ultraviolet CD spectrum of histone 2A that we observed and the effects of NaC1 on this spectrum were identical to results reported for histone 2A b y D'Anna and Isenberg [13]. At pH 7 in 0.16 M NaC1, the CD spectrum between 240 and 208 nm of a mixture of histone 2A (1.8 • 10 -4 M) and ubiquitin (2 • 10 -4 M) was the exact sum of the spectra of the isolated components, suggesting no effect of one protein on the conformation of the other; this result was obtained irrespective of whether the NaCl was added before or after the t w o proteins were mixed. Below 208 nm, signal-to-noise ratios were poor in these studies and data were unreliable. We did not study the CD spectra of mixtures of the t w o proteins in the absence of salt because of the possibility that ubiquitin would alter the conformation of the histone under these conditions b y nonspecific ionic effects. CD spectra of mixtures of the t w o proteins in NaC1 were observed not to change with time when followed for 1 week. A lack of interaction between ubiquitin and histone 2A was also suggested b y fluorescence and gel chromatography. The fluorescence emission spectra of mixtures of the t w o in 0.16 M NaC1 were within 2% of the sum of those of the individual components. These results strongly suggest that the environments of the single ubiquitin tyrosine and the three tyrosines of histone 2A are unaltered when the t w o proteins are mixed. We also observed no change in the accessibility of these tyrosines to I- (as measured b y quenching studies) on mixing the two proteins. On chromatography on Sephadex G-50 at pH 7 in 0.15 M NaCI, ubiquitin and histone 2A migrated as well-separated components when applied as a mixture containing 6 mg/ml ubiquitin and 13 mg/ml histone 2A. Behavior o f mixtures o f ubiquitin and DNA The CD spectra at pH 7 in 0.16 M NaC1 of mixture of ubiquitin and calf thymus DNA were identical to the sum of those of the isolated components
383
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-151 ;' 200
210
220
230
240
250
260
270
280
290
300
X(nm) Fig. 5. CO s p e c t r a o f u b i q u l t i n , D N A a n d u b l q u i t L n / D N A m i x t u r e s . Cond/$1ons: T e m p e r a t u r e , 2 5 ° C ; l l g h t path, 1.0 c m ; p H = 7; 0 . 1 6 M NaCl. - - - , 0.38 m g / m l DNA" . . . . . . , 5.3 • 1 0 -6 M ( 0 . 0 4 5 m g / m l ) ubiq u i t i n ; • 1 : 1 m i x t u r e o f ubiquitln a n d D N A s o l u t i o n s ( e x p e r l m e n t a l v a l u e s ) ; . . . . . . , calculated spect r u m for 1 : 1 mixture of ubiqultin and D N A solutions assuming no interaction.
between 300 and 210 n m (Fig. 5). This wavelength region includes the strong near-ultraviolet D N A ellipticity bands as well as a good fraction of the farultraviolet bands of both components. Preliminary studies of the effect of ubiquitin on D N A melting transition temperature have also indicated no significant degree of interaction between ubiquitin and D N A . Discussion The notable features of ubiquitin conformation demonstrated here are that it contains very little CD-demonstrable repeating secondary structure and that its single tyrosine is in an abnormal environment. The data indicate that the abnormal tyrosine behavior reflects the proximity to the tyrosine of at least one side-chain carboxylate. Tyrosine fluorescence is known to be susceptible to quenching by deprotonated carboxyl groups [19]. Thus, the marked increase in tyrosine fluorescence accompanying protonation of a group with pK a = 3.9 can be interpreted most simply to reflect the loss of quenching associated with protonation of such a carboxyl group. The alternative possibility, that quenching occurs via another mechanism and is lost at low pH because of a carboxyl-controlled conformational change, is not supported by the virtual absence of CD changes between pH 7 and 3. Additionally, the high tyrosine pK in the native protein is clearly compatible with the influence of a proximal carboxylate. It is relevant that the residue preceding the tyrosine in the sequence is an aspartic acid [2]. While it is possible that this is the carboxyl group responsible for quenching, the absence of pH-dependent quenching in 7 M guanidine indicates that the interactions between the tyrosine and carboxylate are dependent on the integrity of the native conformation; therefore a carboxyl elsewhere in the sequence might be involved. The marked differences in tyrosine titration behavior between ubiquitin and its biologically-active derivative pentapeptide (which contains tyrosine at the amino terminus) indicate that the tyrosine environment is different in the two cases. The relevance of this lies in the observation that cleavage of tyrosine from the pentapeptide results in loss of its biological activity (unpublished
384
observations) a fact originally suggesting that the tyrosine ring was involved in this activity. The very different environments of tyrosine in the t w o cases suggests that this is not the case, and is in accord with a very recent observation that the tyrosine side-chain per se can be deleted without activity effects. We see no evidence of intermolecular noncovalent interactions between free ubiquitin and histone 2A, particularly any which result in conformational change. While these results do not strictly preclude an effect of ubiquitin on histone 2A properties when the t w o proteins are linked covalently within A24, t h e y suggest that noncovalent allosteric interactions between the t w o within A24 are likely to be weak. This is in accord with recent demonstrations that A24 can substitute for histone 2A as a c o m p o n e n t of the nucleosome histone core and retains the ability to interact with histone 2B [23], and that free histone 2A and the histone 2A components o f A24 are similarly acetylated and phosphorylated in chromatin [24]. We have also obtained no evidence, thus far, indicating interaction between ubiquitin and DNA. A lack of interaction between ubiquitin (as an A24 component) and DNA has also been reported elsewhere [24,25]. Thus, the function of ubiquitin in chromatin remains puzzling. While it remains possible that ubiquitin, either free or within A24, is capable of interaction with a very small subpopulation o f the chromatin DNA (cf. Ref. 4), our results suggest that ubiquitin might interact with another, as yet unidentified, chromatin component, and/or that the covalent attachment of ubiquitin to histone 2A in A24 serves simply to sterically block a region of the nucleosome. In accord with this, Matsui et al. [26] have recently obtained evidence suggesting that, during the interphase stage of the mitosis cycle, ubiquitin attachment to histone 2A physically blocks chromatin condensation. Acknowledgment This research was supported b y Grant GM-17528 to E.B. from the National Institutes of Health. References 1 Goldstein, G., Sheid, M.S., Hammcrling, V., Boysc, E.A., Schlesinger, D.H. and Nial], H.D. (1975) PToc. Natl. Acad. ScL U.S.A. 72, 11--15 2 Schles~ge~, D.H., Goldsteln° G. and Niall, H~D. (1975) Biochemistry 14, 2214--2218 3 Hunt, L.T. and Dayhoff, M.O. (1977) B i o c h e m . B i o p h y s . Res. Commun. 74, 650---655 4 Watson° D.C., Levy° W.B. and Dixon, G.H. (1978) Nature 276, 196--198 5 Ballsl, N.R,, Kang, Y.-J., Olson, M . O J . and Busch, H. (1975) J. Biol. Chem. 250, 5921--5925 6 Goldknoff, I.L., French, M.F., Daskal, Y. and Busch, H. (1978) Biochem. Biophys. Res. Commun. 84, 786--793 7 Lenkinski, R.E., Chen, D.M.° Glickson, J.D. and Goldstetn, G. (1977) Biochim. Biophys. Acta 494° 126--130 8 Schlestnger, D.H., Goldstein, G., Seheid, M.P. and Bitensky, M. (1977) Expetentia 34, 703--704 9 Goldstein , G. and Schlesinger, D.H, (1978) Patent No. 864122, Belgium Patent O f f i c e 10 BShm, E.L., Strickland, W.M., Strickland, N., Thwaits, B.H., van der Westhuizen, D.R. and Von Holt, C. (1973) FEBS Lett. 34, 217--221 11 Sommer, K.R. and Chalkley, R. (1974) Biochemistry 13, 1022--1032 12 Johns, E.W. (1977) Methods Cell Biol. 16° 183--203 13 D'Anna, J.A. and Isenberg, I, (1974) Biochemistry 13° 2093--2098 14 Oh, Y.H. (1970) J. Biol. Chem. 245, 6404--6416
385 15 16 17 18 19 20 21 22 23 24 25 26
Adler, A.J., Schaffhausen, B., Langan, T.A. and F u m a n , G.D. (1971) Biochemistry 10, 909--913 Gross-Bellard, M., Oudet, P. and Chambon, P. (1973) Eur. J. Biochem. 36, 32---38 Stir, S.S., Rabbani, L., Libman, L. and Breslow, E. (1979) Biochernls~y 18, 1026--1036 Chen, Y.-H., Yang, J.T. and Martlnez, H.M. (1972) Biochemistry 11, 4120---4131 Cowg~Ul, R.W. (1976) in Biochemical Fluorescence (Chen, R.F. and Edelhoch, H,, eds.), Vol. II, pp. 441---486, Marcel Dekker, New York Hesskovits, T.T. and Laskowski, M J r . (1962) J. Biol. Chem. 237, 3418--3422 Ugurbil, K. and Bersohn, R. (1977) Biochemistry 16,895--901 Riordan, J.F.0 Sokolovsky, V. and Vallee, B.L. (1967) Biochemistry 6, 358--361 M~tlnson, H.G., True. R.0 Butch, J.B.E. and Kunkel, G. (1979) Proc. Natl. Acad. Sol. U.S.A. 76, 1030--1034 Goldknopf, I.L., Rosenbaum, F., Sterner, R.0 Vidali, G., Allfrey, V.G. and Busch, H. (197"9) Biochem. Biophys. Res. Commun. 90, 269--277 Goldknopf, I.L., French, M.F.0 BAII~I, N.R.0 Daskal, I. and Busch, H. (1977) Proc. Am. Assoc. Cancer Res. 18, 83 Matsui, S.-I., Seon, B.K. and Sandberg, A.A. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 6386--6390