THE JOURNAL OF BIOLOGICAL CHEMISTRY Printed in U.S.A. Vol. 257, No. 15, Issue of August IO, pp. 88968897, 1982

Crystallization and Preliminary X-ray Data for a Glycosylated Form of Bovine Pancreatic Ribonuclease*

(Received for publication, February 24, 1982)

Byron Rubin, Vasili Carperos, and Edwin Kezar From the Department of Chemistry, Emory University, Atlanta, Georgia 30322

A glycosylated form of bovine pancreatic ribonucle- ase, ribonuclease B, has been crystallized in a form suitable for three-dimensional x-ray structure deter- mination. Crystals grown from polyethylene glycol so- lutions display high resolution diffraction patterns in- dicating the orthorhombic space group P212121 with a = 69.2 (kO.l), b = 56.0 (+0.1), and c = 81.0 (k0.2) a, V = 2.7 X 10' A3. With 8 molecules/unit cell V,,, = 2.28 A3/ dalton. The systematically weak intensity of all reflec- tions with their 1 indices odd suggests the likely align- ment of a noncrystallographic 2-fold axis near the crys- tal c axis.

Of the large number of known glycoproteins relatively few occur naturally in both their glycosylated and carbohydrate- free forms. Moreover relative to their natural abundance, few glycoproteins have been crystallized (1-3). Bovine pancreatic ribonuclease is secreted in several forms of which 15-2076 bears covalently bound carbohydrate (4, 5). Plummer et al. (6) have identified a single site, Asn-34, at which the carbo- hydrate is bound in bovine pancreatic ribonuclease B (RNase B). The chemical structure of the single covalently bound sugar chain has been determined as shown below (7).

Manal \6.

(Manal-2)o.s 3rMana1 ' \

Manal / 6,

.3> ManPl+ 4GlcNAcP1+ 4GlcNAc

Manal /

In the other glycosylated forms of bovine pancreatic ribonu- clease, designated C and D, the carbohydrate chains have modified chemical structures but are also attached at Asn-34 (5, 6).

Although the crystal structures of several forms of ribonu- clease have been reported previously (8-12), there is no prec- edent for the crystallization and x-ray characterization of any of the glycosylated forms. Having the structure of both the glycosylated and carbohydrate free forms, however, would provide an opportunity to observe the structural consequences of carbohydrate attachment and the structure of the carbo- hydrate. We report here the crystallization and initial char- acterization of ribonuclease B.

26905 and GM 27907 from the National Institutes of Health and *This research was supported in part by research Grants GM

Grant PCM 05749 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduer- tisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

EXPERIMENTAL PROCEDURES

Protein Purification-Purified ribonuclease B was obtained com- mercially from Sigma (Type XII-B) and was purified further by affinity chromatography on concanavalin A-sepharose 4B (Pharma- cia) at 4 "C following the procedure of Baynes and Wold (13). After the column step, the purity of the RNase B was confirmed by its appearance as a single band on sodium dodecyl sulfate-gel electro- phoresis. Protein recovery was best when the elution with 10% methyl a - ~ galactopyranoside was begun after washing the bound RNase B with only 2-4 column volumes of buffer. The combined ribonuclease fractions were then dialyzed against 0.1 M Tris-HC1 at pH 7.3, and concentrated by ultrafiltration on an Amicon YMlO membrane to a protein concentration of approximately 10-12 mg/ml. This solution was then stored at 4 "C and used in the crystallization.

Crystallization-Crystals were grown using a batch technique. Into a flat-bottomed, quarter-dram vial the following solutions were added 40 pl of 40% (w/v) polyethylene glycol 6000 (Fisher), 30 p1 0.2 M sodium phosphate, pH 6.2, and 30 pl of the above RNase B solution. Several seed crystals from a previous crystallization were transferred to the vial and allowed to stand until the seed crystals began to dissolve (15-30 min) after which 10 p1 of the 40% polyethylene glycol solution was added. If no apparent growth had taken place by the second day following seeding, an additional 5 pl of the polyethylene glycol solution was added. The crystals normally grow over the course of 2-3 weeks and appear as plates having dimensions often as large as 1 x 1 X 0.2 mm.

X-ray Difiuction Measurements-For each diffraction experi- ment, a crystal is transferred to a thin walled quartz capillary which is selected to allow the crystal to float midway down the capillary before becoming wedged. This method of mounting prevented crack- ing the relatively fragde plates as the mother liquor was being removed. Otherwise, when they were deformed on the concave surface of the capillary wall, they often cracked. Precession photographs were taken at room temperature using a Rigaku RU3V rotating anode, nickel-fdtered Cu source operating at 40 kV and 60 to 70 mA. Unit cell dimensions measured from the T i s were in agreement with those obtained by diffractometer measurement. For the diffractome- ter unit cell measurements, several crystals were examined. In each case, 15 reflections with 20 from 12 to 18" were machine-centered on a Nicolet R3 goniostat. These reflections were then used in an unconstrained least squares refinement of the unit cell parameters.

RESULTS AND DISCUSSION

Precession photographs of the hl1 and hhl are displayed in Figs. 1 and 2. In both photographs it is apparent that reflec- tions with 1 odd are particularly weak relative to those with 1 even. Along each axis reflections with odd indices are absent; suggesting the space group P21212, with a = 59.2(1), b = 56.0(1), c = 81.0(2) A. The unit cell volume is 2.3 X lo5 k'. With 8 molecules/unit cell, 2 molecules in the asymmetric unit, the ratio of the cell volume to the cell contents, molecular mass, V,, is 2.28 A3/dalton, well within the range of observed values for other proteins (14). The systematically weak inten- sity of reflections with their 1 indices odd suggests the likely alignment of a noncrystallographic 2-fold axis near the crystal c axis.

The appearance of 2 molecules in the asymmetric unit is not unique to this form of ribonuclease. In the solution of

8896

X-ra.y Data for Ribonuclease B 8897

FIG. 1. h l l precession photograph of ribonuclease B. The c* axis is vertical. The relative intensities of the reflections with 1 odd are systematically low. Those along (011) appear with some promi- nence. The photograph was taken with a precession angle ji = 12'. crystal-to-film distance of 6.0 cm. Exposure time was 30 hr using a Ni-filtered CuKa source a t 2.8 kilowatts.

monoclinic form of ribonuclease S, Mitsui and Wyckoff ob- served 2 molecules in the asymmetric unit (12). Crestfield, Stein and Moore (15) observed aggregation of ribonuclease A and Capasso et al. (16) have observed a dimeric form of ribonuclease from bovine semen which has a homologous sequence. It is possible that the covalently bound sugar is involved in the aggregation, since both chemical and x-ray characterization of the protein-protein contacts involve the same region of protein as that to which the polysaccharide is attached. High resolution data collection and screening for heavy atom derivatives is now under way.

Achnoudedgments-We wish to thank Susan Bovles for her tech- nical assistance and Dr. David Goldsmith for his help in reproducing the diffraction patterns.

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FIG. 2. hhl precession photographs of ribonuclease B. The c' axis is vertical. The photograph was taken with a precession angle ,ii = 15O, crystal to film distance of 6.0 cm. Exposure time was 14 h using a Ni-filtered CuKa source at 2.4 kilowatts.

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