Nuclear magnetic resonance studies of the denaturation of ubiquitin

Bioch#nica et Biophysica Acta, 494 (1977) 126 [30 '.() Elsevier/North-Holland Biomedical Press

BBA 37740 N U C L E A R M A G N E T I C R E S O N A N C E STUDIES OF T H E D E N A T U R A T I O N OF UB1QUIT1N

ROBERT E. LENKINSKI b'c'e, DOUGLAS GIDEON GOLDSTEINa

M.

CHEN c, JERRY D. GLICKSON a'b'~'' and

Departments of aBiochemistry, ~Chemistry and ~The Comprehensive Cancer Center, The UniversiO, of Alabama in Birmh~gham, Birmingham, Ala. 35294; aThe Memorial SIoan-Kettering Cancer Center, New York, N.Y. 10021 ; and ¢The Department ~[ Physiology and Biophysics, UniversiO, ~[' Illinois" at the Medical Center, Chicago, Ill. 60680 (U.S.A.)

(Received April 6th, 1977)

SUMMARY The effects of pH, temperature and guanidine hydrochloride concentration on the structure of ubiquitin, a polypeptide which can activate adenylate cyclase and can mimic thymopoietin induced differentiation of prothymocytes, were monitored using nuclear magnetic resonance spectroscopy. This relatively small polypeptide (molecular weight of 8541) exhibits a remarkable stability towards pH and temperature changes. At 7 M guanidine hydrochloride concentration, the structure of ubiquitin is essentially a random coil.

INTRODUCTION Ubiquitin is a 74 amino acid polypeptide exhibiting a high degree of evolutionary conservation, manifested in similarities of immunological and functional properties when isolated from diverse sources [1]. This 8541-dalton polypeptide, which was first isolated from bovine thymus, was found to be present in the cells of bacteria, yeast, higher plants and mammals [1]. The amino acid sequence of the bovine ubiquitin has been determined [2] and the sequence of human ubiquitin was found to be identical [3]. A preliminary comparison of seven of the eight amino acid residues of the N-terminal sequence of bovine and celery ubiquitin (position 4 of the celery ubiquitin was not determined) showed six identical residues and substitution at position 1 [2]. The exact biological function of this molecule is undetermined. It has recently been shown by Hunt and Dayhoff that the amino terminal 37 residues of ubiquitin (both human and bovine) and of the nonhistone component of bovine nuclear protein A24 are identical [3, 4, 5]. These authors conclude that these two molecules perform a common basic cell function and have had a similar evolutionary origin. Ubiquitin has been shown to stimulate adenylate cyclase in sarcoma 180 cells (Bitensky, M., Wheeler, M. A. and Goldstein, G., manuscript in preparation), to mimic thymopoietin induced differentiation of prothymocytes and to activate B-cell differentiation [l, 6]. Because the adenylate cylase activation by ubiquitin is inhibited by

127 propranolol [l, 6], it is assumed to have a/3-adrenomimetic active site and thus act via a t~-adrenergic receptor. This in turn implies that the likely active site for adenylate cyclase stimulation is similar in conformation to the epinephrine molecule. Thus, the conformation of ubiquitin in solution is of interest in determining which residues are involved in its adenylate cyclase activity. We undertook a proton N M R investigation of this polypeptide in H20 and 2HzO. Ubiquitin was found to exhibit a well-defined three dimensional structure in solution. This was ascertained by a comparison of a random coil calculated spectrum for ubiquitin [7] with its observed N MR spectrum. We then examined the stability of this structure with respect to temperature, pH and chemical denaturants. The results of these denaturation experiments are reported here. EXPERIMENTAL SECTION Materials. Ubiquitin was isolated from bovine thymus as described by Goldstein et al. [1]. Guanidine hydrochloride was purchased from Sigma Chemical Co. and used without further purification. Methods. All N M R spectra were obtained on approximately 0.5 ml of solution containing approx. 10 mM ubiquitin in 5 m m sample tubes. The chemical denaturation studies were performed on the Carnegie-Mellon 250 MHz superconducting spectrometer operating in the correlation mode [8, 9]. As this spectrometer was not equipped for temperature variation, all spectra were obtained at ambient temperature (30 ~_ 1 °C). The acid and base denaturation studies were also performed on the above spectrometer system. All spectra obtained at this frequency are the sum of 20 scans. The variable temperature studies were performed on the Florida State Bruker H X-270 superconducting N MR spectrometer equipped with quadrature detection, operating in the FT mode. All spectra obtained at this frequency are the sum of 64 scans. All spectra were measured in 2H20 (99.7~o 2H). The pH meter reading is reported uncorrected for deuterium isotope effects. Tetramethylammonium chloride was used as an internal standard for the variable temperature studies.

RESULTS AND DISCUSSION The 270 M H z spectrum of ubiquitin is shown in (a) of Fig. 1. From the amino acid sequence [2], it is known that ubiquitin contains only one histidine and one tyrosine residue, histidine-68 and tyrosine-59, respectively. Ubiquitin also has two phenylalanines at sequence positions 4 and 45, respectively. Consequently, the aromatic region of the spectrum can be assigned easily. The two singlets at around 6.8 p p m and 7.5 ppm are the resonances of the histidine C-2 and C-4 hydrogens, respectively. The AB doublets at around 6.9 and 7.2 correspond to the expected pattern for tyrosine. The remaining resonances at around 7.0, 7.2 and 7.6 ppm can be assigned to the aromatic hydrogens of the two phenylalanines. As can be expected, the two histidine imidazole resonances shift with changes in pH with a characteristic pK~, of around 6.7. In the upfield portion of the ubiquitin spectrum, there are a number of high field shifted methyl resonances originating from methyl groups situated near the faces of aromatic side chains. Such peaks are characteristic of a well defined folded conformation.

128

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The spectrum of ubiquitin was measured over a range of temperatures between 23 °C to 80 °C. Surprisingly, over this entire temperature range, no apparent denaturation takes place in the ubiquitin molecule. The two spectra obtained at the extreme temperatures are shown in Fig. 1. With the exception of the sharpening of the resonances observed in the spectrum obtained at 80 °C both spectra are identical.

pH ~ 848

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512

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Fig. 2. The effects of pH on the upfield portion of the spectrum of ubiquitin.

129 The sharpening of the resonances with increases in temperature stems from a decrease in the rotational correlation time of the ubiquitin at higher temperatures. The variation with pH of the upfield portion of the spectrum of ubiquitin is shown in Fig. 2. Comparison of these spectra show little or no changes in the methyl region. Variations are observed in the region between 1.0 and 3.0 ppm. These are probably caused by the titration of the aspartic acid and glutamic acid residues of which there are six and twelve respectively in ubiquitin. Again, this polypeptide shows a high degree of stability with respect to acid or base denaturation.

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Fig. 3. The effects of various concentrations of guanidine hydrochloride (indicated on the right ~and side of the spectra) on the upheld portion of the spectrum of ubiquitin. Spectra of ubiquitin at various concentrations of guanidine hydrochloride appear in Fig. 3. Addition of the denaturant results in a decrease in the intensity of resonances associated with the native protein and a concomitant increase in the spectral intensity of resonances of the denatured protein. This is most readily observed for the high field shifted methyl peaks. A similar transition has been observed in globular proteins such as.lysozyme [10]. The spectrum of ubiquitin in 7 M guanidine corresponds essentially to the random coil conformation. This conclusion is borne out by the two spectra shown in Fig. 4. The upper spectrum is the random coil calculation. The lower spectrum is the ubiquitin spectrum in the presence of 7 M guanidine. In conclusion, we note a remarkable stability of the structure of this relatively small polypeptide. Further investigations into the characterization of ubiquitin and its interactions are under investigation in our laboratories.

130

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A C K N O W L E D G E M ENTS T h i s w o r k w a s s u p p o r t e d b y U.S. P u b l i c H e a l t h Service G r a n t s C A - 1 3 1 4 8 (JDG), CA-08747 (GG), and Contract Grant CB-53868 (GG). REFEREN(7ES 1 Goldstein, G., Scheid, M. S., Hammerling, V., Boyse, E. A., Schlesinger, D. H. and Niall, H. D. (1975) Proc. Natl. Acad. Sci. U.S. 72(1), 11 15 2 Schlesinger, D. H., Goldstein, G. and Niall, H. D. (1975) Biochemistry 14, 2214-.2218 3 Schlesinger, D. H. and Goldstein, G. (1975) Nature 225, 423-424 4 Hunt, L. T. and Dayhoff, M. O. (1977) Biochem. Biophys. Res. Commun. 74, 650 655 5 0 l s o n , M. O. J., Goldknopf, I. L., Guetzow, K. A., James, G. T., Hawkins, T. C., Mays-Rothberg, (7. J. and Busch, H. (1976) J. Biol. Chem. 251, 5901-5903 6 Scheid, M. S., Goldstein, G., Hammerling, V. and Boyse, E. A. (1975) Ann. N.Y. Acad. Sci. 249, 531 535 7 McDonald, (7. C. and Phillips, W. D. (1969) J. Am. (Them. Soc. 91, 1513 1521 8 Dadok, J. and Sprecher, R. F. (1974} J. Magn. Res. 13, 243-248 9 Gupta, R. K., Ferretti, J. A. and Becker, E. D. (1974) J. Magn. Res. 13, 275 290 10 McDonald, C. C., Phillips, W. D. and GlJckson, J. D. (1971) J. Am. Chem. Soc. 93,235 246