Biochemical and X-Ray Diffraction Analysis of Concanavalin B Crystals from Jack Bean

Abstract

Parallel biochemical and crystallographic studies have been carried out on the protein concanavalin B from Jack Bean (Canavalisensiformis). The studies show the protein to be a monomer of 33,000 daltons with unexceptional amino acid composition and no covalently bound carbohydrate. The molecule contains a single, firmly bound zinc ion and we present evidence that the protein specifically binds nucleotide coenzymes including at least NADPH and flavin mononucleotide. An electron density map of the concanavalin B crystals (space group P61, a = b = 80.9 A, c = 102.2 A) has been calculated from phases based on six isomorphous heavy atom derivatives. A description of the structure of the protein based on the 5.0 A resolution x-ray diffraction study is provided. The structure appears to contain a substantial amount of a-helix as well as an extensive,a-sheet and these are organized into two domains of unequal size. The position of the bound zinc is indicated by anomalous difference Fouriers and the nucleotide binding site by conventional difference Fourier maps.Concanavalin B is a protein present at substantial levels in aqueous extracts of the seeds of Canavalis ensiformis. It is one of four proteins from the Jack Bean crystallized by J. B. Summer early in this century, the others being concanavalin A (17), urease (18), and canavalin (19). The only distinctive property of concanavalin B that brought it to the attention of biochemists was its propensity to crystallize from crude solutions as long, hexagonal needles like those shown in Figure 1. Thus, it represented one of the few 'proven pure' proteins available for study. Its function in neither the seed nor the plant is known. It is not a lectin like its companion concanavalin A nor does it share other of its characteristics. Its physical and chemical properties do not place it in either the classical vicillin or legumin classes of storage proteins (2). Virtually no biochemical characterization has been attempted on concanavalin B so that its precise properties were, until this study, obscure. This is not to say that they are clear even now.Encouraged by its ease of crystallization, we initiated preliminary experiments and single crystal analyses ( 12) with the objective of elucidating its three dimensional structure. An example of its x-ray diffraction pattern is seen in Figure 2. It was determined that the space group ofthe hexagonal prisms was P61 with a = b = 80.9 A and c = 102.2 A. Electron micrographs indicated the crystals to be relatively well hydrated implying that the unit cell contained asymmetric units composed of single molecules. The mol wt of these molecules was estimated, using the ultracentrifuge, by Sumner et al. (20) Amino Acid Analysis. Three mg of protein was hydrolyzed under vacuum at 11 5C for 24 h with 6 N HCI. At the end of hydrolysis, 1.0 ml of 3.5 N NaOH was added to the hydrolysate and this was then diluted to 5.0 ml with water. One-ml aliquots containing 0.1 mg protein were applied to a Beckman 121 automated amino acid analyzer. ...

Solvent-freeze-out (SFO) technology: A controlled crystallization process—Case study of jack bean urease

“…2, octahedral crystals showed up after 24h and were always accompanied by needle shaped crystals. It was found that the needle shaped 11 crystals are concanavalin B (Morrison et al, 1984), indicating that the one time crystallization is not suitable for the here used meal. Fig.2 Comparison between crystallization and recrystallization.…”

Structural and functional evolution of chitinase‐like proteins from plants

“…Morrison et al. showed presence of a firmly bound zinc ion, NADPH, and flavin mononucleotide . ConB has an extended loop between the β2 and α2‐helix .…”

“…Some of the CLPs whose crystal structures are available include ConB from Canavalia ensiformis, narbonin from Vicia narbonensis, xylanase inhibitor protein I (XIP-I) from Triticum aestivum (var. Soisson), xylanase and ␣-amylase inhibitor protein (XAIP) from Scadoxus multiflorus, Parkia platycephala lectin 2 (PPL2), and tamarind CTL lectin (TCLL) from Tamarindus indica [12][13][14][15][16][17] (Fig. 1).…”

“…ConB has an extra disulfide bond Cys41-Cys93 in comparison to narbonin, this bond is engaged in stabilization of L␤2␣2 and L␤3␣3. Morrison et al showed presence of a firmly bound zinc ion, NADPH, and flavin mononucleotide [15]. ConB has an extended loop between the ␤2 and ␣2-helix [16].…”

Crystallization of seed globulins from legumes

“…The addition of divalent metal ions (10mM barium acetate) together with an increase of the ethanol concentration resulted in rather stable rhombohedral crystals (Table 1). This Morrison et al (1984) crystallization condition is very reproducible and was applied in the COSIMA protein crystal growth facility on unmanned satellites (Hennig et al, 1994). By slow cooling of a very concentrated (80 mg m1-1) protein solution from 333 to 277 K in 24 h during dialysis against distilled water we obtained cubic crystals (Fig.…”