Proinsulin: Crystallization and Preliminary X-Ray Diffraction Studies* W

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Proceedings of the National Academy of Sciences Vol. 66, No. 4, pp. 1213-1219, August 1970 Proinsulin: Crystallization and Preliminary X-Ray Diffraction Studies* W. Wardle Fullerton, Reginald Pottert and Barbara W. Lowt

COLLEGE OF PHYSICIANS AND SURGEONS, COLUMBIA UNIVERSITY, NEW YORK, NEW YORK Communicatcd by Erwin Chargaff, Mlay 15, 1970 Abstract. Bovine proinsulin has been crystallized under a variety of conditions at both neutral and acid pH. Microtechniques were employed with sample weights of about 200 jig and volumes of 5-20 1AL The crystalline preparations all differ from each other morphologically. X-ray photographs of one form, tetragonal bipyramids grown at pH 3 with added ammonium sulphate solution, established the space group P41212 (or its enlantiomorph P43212). The cell dimensions are a = 50.8 i 0.2 A, c = 148.0 + 0.4 A. The asymmetric unit in this form is a dimer of proinsulin which is also the dominant species in solution at this pH.

Introduction. Following the studies of Steiner arid his co-w%-orkersl2 who established that insulin is synthesized via a single chain precursor protein, proinsulin has been isolated and characterized from bovine,3-6 porcine,' cod,8 anglerfish,9 and human10"1 tissues. The connecting peptide segment of proinsulin which links the carboxyl-terminal group of the insulin B chain to the amino-terminal group of the insulin A chain is cleaved in vivo by proteolysis. The lengths and sequences of the C-peptide vary markedly between species. In bovine proinsulin, where the connecting peptide is 30 amino acid residues long," the proinsulin molecule with a single 81 residue chain has a molecular weight of 868.5. Physicochemical studies of porcine proinsulin in solution'2"3 suggest that the aggregation of proinsulin molecules is comparable to that observed with insulin, both as a function of pH and in terms of interaction with zinc. The concentra- tion dependence of the monomer-dimer equilibrium in acid solution has been investigated. At neutral pH in the presence of zinc proinsulin forms hexamers, although in the absence of zinc, the solutions are heterogeneous with respect to particles in the molecular weight range 22,000-58,000. Jeffrey and Coates"4 reported that for insulin in the absence of zinc there is a mixture of even-ag- gregate states. Comparable solution studies of bovine proinsulin have not been made; the behavior of bovine proinsulin in solution, however, is probably analogous to that of porcine proinsulin. From studies of optical rotatory dispersion and circular dichroism spectra, Frank and Veros12 have concluded that the connecting peptide of porcine proinsulin probably exists in a random coil conformation. Their studies are con- sistent with the view that the conformation of the insulin segment in proinsulin is very similar, if not identical, at least so far as the regions of a-helix are con- cerned, to the conformation of insulin as an isolated molecule. Studies in this 1213 Downloaded by guest on October 1, 2021 1214 BIOCHEMISTRY: FULLERTO\N, POTTER, AND LOWV Ptoc. N. A. S.

laboratory,"5 on the other hand, of published sequences for both porcine and bovine proinsulin employing criteria developed here'6 have suggested that there are probably two short regions of a-helix in both of the connecting peptides. We began crystallization studies with a bovine proinsulin preparation4 given to us by Prof. Steiner. The material is salt free; if it contained zinc, this would imply a strong atypical (unlike insulin) zinc-binding capacity. We were aware of some similarities in solution properties between insulin and proinsulin and sought to exploit them. In particular, salt (sodium chloride) was employed to enhance ionic strength; a phenol' was also used in some studies. Only 8 mg of proinsulin was initially available. M\licrotechniques were required not, only for the crystallization studies but also for the preliminary solution studies made to determine proinsulin solubility over a broad range of pH and salt concentra- tion. In order to enhance the possibility that the material could be crystallized we chose deliberately to explore several different sets of would-be optimal conditions. In all cases, crystals were obtained. Experimental Procedures. Preliminary solution studies suggested that proinsulin is more soluble than insulin and we were able to prepare solutions at neutral pH containing as much as 20-30 mg/ml and at pH 2-3 over 50 mg/ml. Proinsulin is less soluble at 30C than at room temperature and crystallization proceeds more rapidly in the cold. Salt concentrations appropriate to the preparation of proinsulin crystals were determined by preliminary diffusion experiments (see below). Survey studies were all performed at room temperature and the crystals which appeared, usually after about 4 weeks, were very small and ill-formed. In consequence, although some solutions were made up and mixed at room temperature, all subsequent crystallization studies were made in a cold room at approximately 30C. Mlost of the crystallization studies were conducted in thin- walled (10 Am) glass or quartz capillaries, 1 mm in diameter. These are normally used to mount protein crystals for x-ray study. The glass capillaries must be washed in acid and subsequently rinsed in distilled water to remove surface alkali. Some of the capillaries were coated with a bovine serum albumin solution (10 mg/ml in 50% acetic acid) to prevent interaction of proinsulin at the glass wall. The capillaries are approximately 60 mm long with a further thicker-walled flared end. The proinsulin preparations were usually made up at room temperature and allowed to stand in the cold (about 30C). Proinsulin was brought out of aqueous solution by one or more of several effects: temperature-dependent solubility, salting out, pH change, or slow evaporation of the solvent. Two variations in crystallization procedure were initially employed: (a) The capillary was heat-sealed at the thin-walled end. Solutions were added with a syringe, mixed using a micro-glass stirring rod, and finally sealed with a plug of embedding wax. Here, as elsewhere, the capillaries were maintained vertical at all times. (b) The narrow thin- walled end of the capillary was sealed with acrylamide gel, using the procedure of Zep- pezauer et al.1' The proinsulin solution was introduced into the capillary as before, the narrow end of the capillary was then immersed in a glass container of precipitating solution which diffuses slowly through the gel. Once some crystalline material had formed, the capillary was removed from the container to halt diffusion and sealed at both ends with embedding wax. Glass apparatus (including glass syringe needles) was employed throughout except that steel syringe needles were used when a Swinny filter was employed. Sometimes material to be employed in one crystallization (of the order of 200/Ag to be dissolved in about 5-20 Al) was weighed out separately in the capillary tube. Stock solutions prepared with 1 mg of proinsulin in 25-100 Mil were also employed. These solutions were usually filtered through a Jena sintered-glass crucible (IG4-porosity 5-10 Mm) or sometimes filtered through a Swinny filter. Crystallization of proinsulin was studied under the following conditions: (1) In phosphate buffer at pH 7.25: Proinsulin (10-40 mg/ml) was dissolved in 0.0133 M phosphate Downloaded by guest on October 1, 2021 VOL. 66, 1970 BIOCHEMISTRY: FULLERTON, POTTER, AND LOW1212154

buffer, which contained 22 mg/ml sodium chloride and also, when a phenol was employed, 3 mg/ml of m-cresol. The capillary tubes were filled at room temperature and then removed to the cold room. (2) In citrate buffer at pH 7.25: Proinsulin (10-20 mg/ml) was dissolved in 0.05 M citrate buffer containing 22 mg/ml sodium chloride with 0.04% zinc as zinc chloride and, when a phenol was used, 3 mg/ml m-cresol. The capillary tubes were filled at room temperature and then removed to the cold room. (3) In citrate buffer at approximately pH 3: With the exceptions noted, all manipula- tions were conducted in the cold. Proinsulin (30-40 mg/ml) was dissolved in 0.1 -M citric acid solution containing 1.75% potassium chloride. Cold 0.08 M di-ammonium citrate solution or cold-saturated ammonium sulfate solution was either added to slight turbidity or permitted to dialyze through acrylamide gel until a few crystals appeared. The tubes were then sealed at both ends with embedding wax. A few experiments were performed by the initial addition of the ammonium sulfate solution at room temperature followed by removal of the preparation to the cold room. The first x-ray studies of proinsulin crystals were made without removing them from the thin-walled glass capillaries in which they were grown. In order to cut down excess background scattering, most of the mother liquor about the crystals to be photographed was removed with a microsyringe and only a meniscus of liquid left. Copper radiation from a conventional normal-focus GE x-ray source was employed. At a later stage of x-ray study, crystals were removed from the vessels in which they were grown and mounted in the usual way. X-ray precession photographs were taken with copper radiation from a Jarrell Ash microfocus unit. A pinhole collimator was used to limit the beam to a diameter of about 150 Am. The densities of two crystals which had been used for x-ray study were measured at 230C in a water-saturated bromobenzene-xylene gradient column made up to cover the calculated range of possible densities. Results. In the studies with phosphate buffer those crystals which appeared after 1 week in the presence of m-cresol were essentially sphenoids, with some curved high-order faces still evident; some crystals were truncated (see Fig. IA and B). The crystals did not grow larger on further standing; the largest specimens observed were about 50 X 30 X 10 ,m. In the absence of m-cresol, crystals appeared after standing for 3 weeks. These were well-formed nonequant three-sided plates 30 Mm in average diameter and 10 Mm thick and showed oblique extinction. Crystals grown from citrate buffer in the presence of m-cresol appeared after 1 week. They were thin blades of wedge-shaped cross section, about 10-20 Mm long. (Fig. iC). In the absence of m-cresol intergrowths of ill-formed plates, of approximately square (20 Am) cross section. appeared after 3 weeks (Fig. ID). In the studies at acid pH in citrate buffer, the use of ammonium citrate led to the separation after about 3 weeks of small thin rectangular plates 10-20 Mm in length, which show parallel extinction. The crystals did not grow on further standing. Those crystals obtained from ammonium sulphate solutions appeared in 2 days and reached maximum size after 1 week. They were well- formed, bipyramids, about 90 X 40 X 20 Am, and extinguished parallel to the long axis (Fig. 1E). X-ray photographs of this form taken with a normal focus source were poor and inadequate to establish unit cell dimensions or space group. In all the preparations described, the crystals immersed in their mother liquor were stable in the cold for a period of more than 10 months and at room temperature for at least several weeks. A second-run crystallization was Downloaded by guest on October 1, 2021 1216 BIOCHEMISTRY: FULLERTON, POTTER, AND LOW Pitoc. N. A. S.

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FIG. 1.-Crystals of bovine proinsulin: (A) and (B) From phosphate buffer at pH 7.25 containing m-cresol. (C) From citrate buffer at pH 7.25 containing m-cresol and zinc. (D) From citrate buffer at pH 7.25 containing zinc. (E) and (F) From citrate buffer at pH 3 containing ammonium sulphate.

attempted on the same scale and under similar conditions with a further 5 mg of material in order to establish that the crystallization procedures were repro- ducible. There were no differences in time of appearance, growth rate, or final size of the crystals obtained in this second study. Larger scale crystallization was evidently necessary for the preparation of Downloaded by guest on October 1, 2021 VOL. 66, 1970 BIOCHEMISTRY: FULLERTON, POTTER, AND LOW* 1217

bigger crystals and the m-cresol-free preparations were combined and chromato- graphed on Bio-Gel P-2 with 0.1 'M acetic acid at room temperature according to the procedure of Steiner.'9 The proinsulin, the first material to come off the column and identified by its absorbance at 275 nm with a Beckman-DB spectro- photometer, was freeze-dried. Because acid crystallization procedures with ammonium sulphate had yielded the largest, well-formed crystals, the whole of the reworked material was initially employed for recrystallization of this acid form on a larger scale. The study further provided an opportunity to investigate solid-state aggregation of proinsulin at acid pH. These crystals may, in spite of their size and development, be expected to be of poorer quality than those grown at neutral pH if the analogy between proinsulin and insulin is maintained. The large scale crystallization studies were made in culture tubes (6 X 30 mm) or vials (cut to 9 X 5 mm), each containing about 1 mg of proinsulin. The crystallization procedures employed in the culture tubes were completely analogous to those in the thin-walled glass capillaries. The vials, however, were not stoppered but left open in a larger closed vessel to permit slow evaporation. Crystals up to 200 X 100 X 75 ,um were obtained with both procedures within one week (see lF'ig. IF). X-ray precession photographs were taken on the microfocus unit. Photo- graphs of hOl, OH, and Iki zones showed systematic absences indicating the space group P4,212 or its enantimorph P43212. The unit cell dimensions are a = 50.S + 0.2 A, c = 148.0 + 0.4 A, with a cell volume of 3.82 X 1&5 A3. The minimum spacing observed for relatively short exposures was 2 A. The crystals appear unusually resistant to radiation damage and withstood x-ray exposure for more than 100 hr. The equation

1/P, = Vc = A1VV + (1 - fp)VL (1) defines the relationship between weight fraction and other variables, where 1V, is the specific volume of the crystal, f, is the weight fraction of the protein, and UL is the partial specific volume of the liquid of crystallization. If it is assumed that the partial specific volume of the protein in the crystal is that determined in dilute solution, and that the liquid of crystallization is identical with the buffer solution used to prepare the crystals, then the equation can be solved for f,. The crystals employed for density determination were small (100 and 120 ,um long, respectively). This introduces error-wiping the crystal free of mother liquor rapidly enough to prevent air drying is very difficult. The measured density p = 1.20 + 0.01 g/cm3 (estimated error) therefore must be considered approximate. The density of the protein-free buffer is 1.028 + 0.002 g/cm3. The partial specific volume of proinsulin has not yet been determined experi- mentallv. In employing Eq. (1) to solve for fp, we have used two limiting values for the partial specific volume of insulin up = 0.71 or 0.72.20 This procedure gives values for the weight fraction of protein in the crystal of 0.53 and 0.55. respectively Downloaded by guest on October 1, 2021 1218 BIOCHEMISTRY: FULLERTON, POTTER, AND LOW Pitoc. N. A. S.

corresponding to weights for the protein component of the asymmetric unit of 18,000 and 19,000 daltons. The molecular weight of bovine proinsulin is 8685. If the approximate value for the protein component of the asymmetric unit corresponds to the two molecules of proinsulin, the appropriate crystal density calculated from Eq. (1) is 1.19, or 1.185 corresponding to iU, = 0.71 and ip = 0.72 respectively. Discussion. The close agreement found between the observed and calculated density is certainly adequate to establish that a bovine proinsulin dimer is the asymmetric unit in the crystal form studied. The dimer association found in solution at acid pH therefore exists also in the solid state. Proinsulin crystals grown at acid pH show greater than ideal mosaicity and the extent of the diffraction pattern observed (2 A) is limiting for structure studies. Before the crystalline phase most appropriate for detailed x-ray study is chosen, other crystal forms should be investigated. A larger scale preparation of the other forms described is now under way. We note that the crystals grown in the presence of added zinc from citrate buffer appear poorer than those grown in the absence of added zinc from phosphate buffer. The relative roles of zinc and buffer are now under investigation. Zinc is required for the preparation of both rhombohedral21 and monoclinicl insulin crystals at neutral pH. The proinsulin provided, as we observed earlier, may already have contained tightly bound zinc. As porcine proinsulin differs so markedly from bovine proinsulin in the connecting peptide region, crystals of this species may not be formally isomorphous with those from bovine proinsulin-in contrast to the situation which exists for insulins from the two species. The crystallization of porcine proinsulin will be investigated. We are happy to acknowledge generous gifts of bovine proinsulin from Prof. D. F. Steiner and thank him for his interest. * This work was supported by grants from the National Institutes of Health (AMI 01320) and the National Science Foundation (GB 7272). t On leave from the Department of Physics, University College, Cardiff, Wales. $ Requests for reprints may be addressed to Dr. B. W. Low, Department of Biochemistry, College of Physicians and Surgeons, Columbia University, 630 W. 168th Street, New York, N.Y. 10032. 1 Steiner, D. F., and P. Oyer, these PROCEEDINGS, 57, 473 (1967). 2 Steiner, D. F., D. D. Cunningham, L. Spigelman, and B. Aten, Science, 157, 697 (1967). 3 Yip, C. C., and B. J. Lin, Biochem. Biophys. Res. Commun., 29, 382 (1967). 4Steiner, D. F., 0. Hallund, A. Rubenstein, S. Cho, and C. Bayliss, Diabetes, 17, 725 (1968). 5 Schmidt, D. D., and A. Arens, Hoppe-Seyler's Z. Physiol. Chem., 349, 1157 (1968). 6 Nolan, C., and E. Margoliash, cited in Recent Prog. Horm. Res., 25, 250 (1969), by D. F. Steiner, J. L. Clark, C. Nolan, H. A. Rubenstein, E. Margoliash, B. Aten and P. E. Oyer. 7Chance, R. E., R. M. Ellis, and W. W. Bromer, Science, 161, 165 (1968). 8 Grant, P. T., and K. B. M. Reid, Biochem. J., 110, 281 (1968). 9 Trakatellis, A. C., and G. P. Schwartz, Nature, 225, 548 (1970). 10 Clark, J. L., S. Cho, A. H. Rubenstein, and D. F. Steiner, Biochem. Biophys. Res. Commun., 35, 456 (1969). Oyer, P. E., S. Cho, and D. F. Steiner, Fed. Proc., 29, 533 (1970). 12 Frank, B. H., and A. J. Veros, Biochem. Biophys. Res. Commun., 32, 155 (1968). 13 Frank, B. H., and A. J. Veros, Biochem. Biophys. Res. Commun., 38, 284 (1970). 14 Jeffrey, P. D., and J. H. Coates, Biochemistry, 5, 489 (1966). Downloaded by guest on October 1, 2021 VOL. 66, 1970 BIOCHEMISTRY: FULLERTON, POTTER, AND LOW 1219

15 Rudko, A. D., and B. W. Low, unpublished studies. 16 Low, B. W., F. M. Lovell, and A. D. Rudko, these PROCEEDINGS, 60, 1519 (1968). 17 Schlichtkrull, J., in Insulin Crystals (Copenhagen: Munksgaard, 1958), p. 52. 18 Zeppezauer, M., H. Eklund, and E. S. Zeppezauer, Arch. Biochem. Biophys., 126, 564 (1968). 19 Steiner, D. F., personal communication. 20 Oncley, J. L., E. Ellenbogen, D. Gitlin, and F. R. N. Gurd, J. Phys. Chem., 56, 85 (1952). 21 Scott, D. A., Biochein. J., 28, 1.592 (1934). Downloaded by guest on October 1, 2021