Structure of the 7S globulin (vicilin) from pea (Pisumsativum) K. J. l'Anson, M. J. Miles, J. R. Bacon, H. J. Carr, N. Lambert, V. J. Morris and D. J. Wright AFRC Institute of Food Research, Norwich Laboratory, Colney Lane, Norwich NR4 7UA, UK (Received 3 March 1988) The Daresbury synchrotron has been used to obtain X-ray scattering data on solutions of the pea 7S globulin (vicilin) to an angular resolution correspondino to the modulus of the scattering vector Ik[ < 14.5 nm- 1. The angular ranoe 0.45 nm- 1 < [kI <~ 6.5 nm- 1 reveals scattering details characteristic of the subunit size, shape and arrangement. The protein can be modelled in terms of three touching non-spherical subunits each containing two spherical scattering domains of unequal radii. Circular dichroism studies indicate that the ordered protein secondary structure contains mainly fl-sheet and little it-helix. Structure-prediction calculations on the primary sequence of the subunit suggest that the domains may correspond to regions of fl-sheet. Keywords: Synchrotron X-ray scattering; circular dichroism; Pisum sativum; vicilin; seed globulins Introduction Vicilin is the trivial name for the 7S storage protein obtained from peas (Pisum sativum). Biochemical studies have revealed the presence of three chemically distinct subunits of similar molecular weight and the complete amino acid sequence of the subunits is known 1'2. However, little is known about the three-dimensional structure of the protein. Vicilin has not as yet been crystallized, and isolation of individual subunits leads to their irreversible denaturation. The radius of gyration from small angle X-ray scattering studies 3 is 4.0__+ 0.3 nm. Such studies using conventional X-ray sources are limited to an angular range, defined by the modulus of the scattering vector (Ikl), of 0.01 am -t <lkl<~ 2.0nm-'. After smoothing and desmearing corrections, such data provide information on molar mass and the overall size and shape of the protein but little information on the internal structure. The near-ideal geometry of the synchrotron X-ray source eliminates the need for desmearing corrections and the high intensity permits measurements at wider angles4'5. This article describes the use of the synchrotron to obtain X-ray scattering data on vicilin solutions to an angular range of Ikl z 14.5 nm- 1 and the use of such data to model the protein structure to a resolution of ~0.90nm (Ikl~x=6.Snm-t). Our approach to the modelling of this protein has been to construct the simplest model which is consistent with all the experimental data, including secondary-structure predictions from sequence data using computer-based algorithms, circular dichroism (c.d.) measurements and published transmission electron micrographs. Experimental Vicilin was isolated from defatted pea flour (Pisum sativum cv Birte) using the methods described by Lambert et al. ~. Earlier studies 3 have shown that the X-ray scattering varies linearly with protein concentrations for protein concentrations < 99 mg ml- 1. In the present studies the protein concentration was ~ 50mg ml-1 in 50 mm Tris buffer, 200 mm NaC1 adjusted to pH 8.2 with HCI. X-ray scattering data were collected at station 7.2 on the Daresbury synchrotron, Daresbury Laboratory, Warrington, Cheshire, UK. The detailed methodology of the measurement and the subsequent preliminary data analysis have been discussed in a previous publication 5. The experimental scattering curves presented in this paper are an average of several independent measurements. Scattering data were collected to [k(< 14.5 nm-1. The present modelling has been restricted to the angular range Ikl<6.5nm -1. This angular range corresponds to features greater than 0.9nm. In order to facilitate comparison with model curves, the contribution to the scattered intensity over this range from features of smaller sizes than this can be subtracted from the data using the method described by Miiller 7: the scattered intensity due to the high-resolution features of the scattering curve was treated as producing a constant background intensity Ih which was evaluated using Porod's law. lim (Ikl'I(k)} = Alk l` ÷ I~ Ik]-~ oo where A is a constant. This procedure was checked using the Indirect Transformation Programme s,9 (ITP), which also provides a reliable extrapolation value for the zero- angle scattered intensity, Io. This value was then used to normalize the experimental data to the Io of the calculated scattering curves for the models, i.e. Io set at l0 s . This facilitates the comparison of the model and experimental curves and contains the implicit ass_umption that each of the models would produce the same Mwvalue. This will be considered in more detail later. The scale of the models was also adjusted to obtain a match between the measured value of radius of gyration, P~, and that 0141-8130/88/050311-07503.00 © 1988 Butterworth & Co. (Publishers) Ltd. Int. J. Biol. Macromol., 1988, Vol. 10, October 311