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Preliminary X-ray investigation of bovine seminal ribonuclease
J. Mol.
Biol.
Preliminary
(1972) 64, 311-312
X-ray Investigation
of Bovine Seminal Ribonuclease
Single crystals of bovine seminal ribonucleaae suitable for high-resolution X-ray diffraction studies were obtained. The space group is P212,21 8nd unit cell dimensions 8re a = 3tM A, b = 66.6 A, c = 107.4 A. The asymmetric unit contains one molecule, of molecular weight about 29.000, which consists of two polypeptide chains related by 8 non-cryst8llogrephio e-fold or pseudo-e-fold axis. D’Alessio & Leone, (1963) have reported the purification of a ribonuclesse isolated from bovine seminal fluid (RN&se-BSl). It appears very similar to the well-known pancreatic RN&se A, with which it shares most of the characteristio catalytic properties (bond specificity snd kinetic
parameters) ; it is, however, also active towards double-stranded RNA (Libonati & Floridi, 1969). RNase B&l has a molecular weight of 29,000, and differs from RNase A in amino-acid composition, especially for a higher content of basic residues.Recent experiments, mainly basedon quantitation of N and C termini and on peptide mapping, as well as studies on the molecular size of the denatured and reduced protein, would suggestthat the native protein is made up of two similar, possibly identical, polypeptide chains (D’Alessio & Leone, personal communication). As the structure of the bovine pancreatic RNase is now very well established (Kartha, Bello & Harker, 1967; Wyckoff et d., 1970), it will be extremely interesting to solve the structure of RNase-BSl in order to compare, on structural grounds, the function of the two enzymes. Chemical work on seminal RNase is also in progress to establish functionel properties and primary sequenceof the protein (Leone, personal communication). RNase-BSl w&s first crystallized by Floridi (1968) from ammonium sulphate solutions containing phosphoric acid. Numerous attempts to prepare crystals suitable for X-ray examination by use of alcoholic solutions in the acid pH range following the method of crystallizing bovine pancreatic RN&se (King, Magdoff, Adehnan & Harker, 1956)were unsuccessful. Satisfactory results were obtained from ammonium sulphate solution after a number of experiments varying and controlling the conditions of concentration, temperature and pH. An aqueous solution of the enzyme (20 mg/ml.) was adjusted to pH 4.2 with phosphorio aoid and solid ammonium sulphate was added to a ooncentration of 3-l M. After rt small precipitate formed in this way had been discarded by centrifugation, small amounts of solid ammonium sulphete were added to 0.2~ml. samplesof this solution. Large crystals were obtained after standing this solution at 20°C for two weeks. The crystals can be left for several months solution (3.3 M) without deterioration. The crystals,
at 20°C in ammonium up to 1 mm in length,
sulphate are pris-
matic with well developed (001) and (010) faces. X-ray diffraction photographs were recorded on a Buerger preoessioncamera using CuKor radiation. The unit oell is orthorhombic with dimensions: a = 36.6 A, b = 66.6 A, c = 107.4A and V = 2.62 x lo6 A3. 311
312
8. CAPASSO
ET
AL.
Precessionphotographs (17”) of the hO1and Ok1zones, covering spacing down to 2.7 A, showed the systematic absences: hO0, h odd; OkO,k odd; 001,l odd. The spacegroup is therefore probably P2,212,. A precessionphotograph of the hO1zone is shown in Plate I. The density of the wet crystals was measuredon a bromobenzene-benzene densitygradient column calibrated with small drops of sucrosesolutions of known density. A value of 1.31 g/ml. was obtained. Using the partial specific volume of 0.718 ml./g (D’Alessio t Leone personal communication) the solvent content by volume is calculated to be 46%, and the molecular weight is 29,600 on the basisof four molecules per unit cell, in closeagreement with the experimental value. Inspection of the three-dimensional reciprocal lattice showsthat the hkl reflections are almost absent at very low resolution when h + k+ I# 2n. This limiting condition would imply a body-centered lattice. If one assumesthat the molecular centres are located at a 4-fold set of positions of 2-fold symmetry of the space group 12,2,2,, only small departures from a-fold symmetry or from the positional requirements of the body-centered lattice leads to the proper spacegroup P212,2,. Thus it can be concluded that the asymmetric unit of structure is one molecule of the enzyme, which is composedof two polypepticle chains related by a non-crystallographic 2-f&l or pseudo-2-fold axis. The determination and exploitation of the non-crystallographic symmetry is now in progressusing rotation and translation functions. However it should be noted that the structure determination to atomic resolution is needed to distinguish between a true molecular ayd displaced from a crystallographic axis ancl an approximate molecular ayfbd. The extension of the diffraction pattern to a resolution of 1.8 A and the quality of the crystals make them suitable for a three-dimensional X-ray analysis. For this purpose isomorphous heavy-atom derivatives are now being prepared. about
We thank Professor E. Leone and Professor G. D’Alessio for the gift of the RNase-BSI used in this study and for stimulating discussions. This investigation was partly supported by the Consiglio Nazionale delle Ricerche, Roma, Italy. Istituto Chimico dell’Universita, Via Mezzocannone, 4 80134, Napoli, Italy.
8. F. L. A.
&PASS0 C~IORD~O -LLA RIPAMONTI
Received 2 June 1971 REFERENCES D’Alessio, G. & Leone, E. (1963). Biochem. J. 89, 7P. Floridi, A. (1968). Bhchen. Biophya. Rea. Cm. 99, 179. Kartha, G., Bello, J. L Harker, D. (1967). Natwe, 213, 862. King, M. V., Magdoff, B. S., Adehnan, M. B. BE Harker, D. (1966). A&a. Cry&. 9, 460. Libonati, M. & Floridi, A. (1969). Europ. J. Bgochem. 8, 81. Wyckoff, H. W., Tsernoglou, D. Hsnson, A. W., Knox, J. R., Lee, B. & Richards, F. M. (1970). J. Bid. C&m. 24~5, 306.
PLATE
I.
A
17”
precession
photograph
of h0Z
zone
of bovine
seminal
ribonucleasn.
Biol.
Preliminary
(1972) 64, 311-312
X-ray Investigation
of Bovine Seminal Ribonuclease
Single crystals of bovine seminal ribonucleaae suitable for high-resolution X-ray diffraction studies were obtained. The space group is P212,21 8nd unit cell dimensions 8re a = 3tM A, b = 66.6 A, c = 107.4 A. The asymmetric unit contains one molecule, of molecular weight about 29.000, which consists of two polypeptide chains related by 8 non-cryst8llogrephio e-fold or pseudo-e-fold axis. D’Alessio & Leone, (1963) have reported the purification of a ribonuclesse isolated from bovine seminal fluid (RN&se-BSl). It appears very similar to the well-known pancreatic RN&se A, with which it shares most of the characteristio catalytic properties (bond specificity snd kinetic
parameters) ; it is, however, also active towards double-stranded RNA (Libonati & Floridi, 1969). RNase B&l has a molecular weight of 29,000, and differs from RNase A in amino-acid composition, especially for a higher content of basic residues.Recent experiments, mainly basedon quantitation of N and C termini and on peptide mapping, as well as studies on the molecular size of the denatured and reduced protein, would suggestthat the native protein is made up of two similar, possibly identical, polypeptide chains (D’Alessio & Leone, personal communication). As the structure of the bovine pancreatic RNase is now very well established (Kartha, Bello & Harker, 1967; Wyckoff et d., 1970), it will be extremely interesting to solve the structure of RNase-BSl in order to compare, on structural grounds, the function of the two enzymes. Chemical work on seminal RNase is also in progress to establish functionel properties and primary sequenceof the protein (Leone, personal communication). RNase-BSl w&s first crystallized by Floridi (1968) from ammonium sulphate solutions containing phosphoric acid. Numerous attempts to prepare crystals suitable for X-ray examination by use of alcoholic solutions in the acid pH range following the method of crystallizing bovine pancreatic RN&se (King, Magdoff, Adehnan & Harker, 1956)were unsuccessful. Satisfactory results were obtained from ammonium sulphate solution after a number of experiments varying and controlling the conditions of concentration, temperature and pH. An aqueous solution of the enzyme (20 mg/ml.) was adjusted to pH 4.2 with phosphorio aoid and solid ammonium sulphate was added to a ooncentration of 3-l M. After rt small precipitate formed in this way had been discarded by centrifugation, small amounts of solid ammonium sulphete were added to 0.2~ml. samplesof this solution. Large crystals were obtained after standing this solution at 20°C for two weeks. The crystals can be left for several months solution (3.3 M) without deterioration. The crystals,
at 20°C in ammonium up to 1 mm in length,
sulphate are pris-
matic with well developed (001) and (010) faces. X-ray diffraction photographs were recorded on a Buerger preoessioncamera using CuKor radiation. The unit oell is orthorhombic with dimensions: a = 36.6 A, b = 66.6 A, c = 107.4A and V = 2.62 x lo6 A3. 311
312
8. CAPASSO
ET
AL.
Precessionphotographs (17”) of the hO1and Ok1zones, covering spacing down to 2.7 A, showed the systematic absences: hO0, h odd; OkO,k odd; 001,l odd. The spacegroup is therefore probably P2,212,. A precessionphotograph of the hO1zone is shown in Plate I. The density of the wet crystals was measuredon a bromobenzene-benzene densitygradient column calibrated with small drops of sucrosesolutions of known density. A value of 1.31 g/ml. was obtained. Using the partial specific volume of 0.718 ml./g (D’Alessio t Leone personal communication) the solvent content by volume is calculated to be 46%, and the molecular weight is 29,600 on the basisof four molecules per unit cell, in closeagreement with the experimental value. Inspection of the three-dimensional reciprocal lattice showsthat the hkl reflections are almost absent at very low resolution when h + k+ I# 2n. This limiting condition would imply a body-centered lattice. If one assumesthat the molecular centres are located at a 4-fold set of positions of 2-fold symmetry of the space group 12,2,2,, only small departures from a-fold symmetry or from the positional requirements of the body-centered lattice leads to the proper spacegroup P212,2,. Thus it can be concluded that the asymmetric unit of structure is one molecule of the enzyme, which is composedof two polypepticle chains related by a non-crystallographic 2-f&l or pseudo-2-fold axis. The determination and exploitation of the non-crystallographic symmetry is now in progressusing rotation and translation functions. However it should be noted that the structure determination to atomic resolution is needed to distinguish between a true molecular ayd displaced from a crystallographic axis ancl an approximate molecular ayfbd. The extension of the diffraction pattern to a resolution of 1.8 A and the quality of the crystals make them suitable for a three-dimensional X-ray analysis. For this purpose isomorphous heavy-atom derivatives are now being prepared. about
We thank Professor E. Leone and Professor G. D’Alessio for the gift of the RNase-BSI used in this study and for stimulating discussions. This investigation was partly supported by the Consiglio Nazionale delle Ricerche, Roma, Italy. Istituto Chimico dell’Universita, Via Mezzocannone, 4 80134, Napoli, Italy.
8. F. L. A.
&PASS0 C~IORD~O -LLA RIPAMONTI
Received 2 June 1971 REFERENCES D’Alessio, G. & Leone, E. (1963). Biochem. J. 89, 7P. Floridi, A. (1968). Bhchen. Biophya. Rea. Cm. 99, 179. Kartha, G., Bello, J. L Harker, D. (1967). Natwe, 213, 862. King, M. V., Magdoff, B. S., Adehnan, M. B. BE Harker, D. (1966). A&a. Cry&. 9, 460. Libonati, M. & Floridi, A. (1969). Europ. J. Bgochem. 8, 81. Wyckoff, H. W., Tsernoglou, D. Hsnson, A. W., Knox, J. R., Lee, B. & Richards, F. M. (1970). J. Bid. C&m. 24~5, 306.
PLATE
I.
A
17”
precession
photograph
of h0Z
zone
of bovine
seminal
ribonucleasn.