Barbara W. Low

Molecular conformation of erabutoxin b; Atomic coordinates at 2.5 Å resolution

Abstract Atomic coordinates determined from a 2.5 A electron density map are given for erabutoxin b, a sea snake venom postsynaptic neurotoxin. The principal structural features, anti-parallel β pleated sheet, β bulge and β bends are described. The erabutoxin b structure is discussed as structural prototype of this class of homologous curare-mimetic neurotoxins from both land and sea snakes.

Precipitation and crystallization of insulin in the presence of lysozyme and salmine

Abstract 1. 1. During a search for new crystalline derivatives of insulin for X-ray study, a re-examination was carried out of the co-precipitation of insulin with salmine and lysozyme. A new lysozyme-containing insulin crystal was prepared and improved techniques of preparing the crystalline protamine-insulin described earlier by Krayenbuhl and Rosenberg were developed. 2. 2. We confirmed that insulin and salmine co-precipitate in a 5:1 molar ratio at pH 7.3, in accordance with earlier reports. The 8:1 molar ratio found for insulin-lysozyme precipitates at this pH was twice that reported previously. 3. 3. Crystalline derivatives were grown from phosphate buffer solutions of insulin containing NaCl, m -cresol, and relatively small amounts of either salmine or lysozyme (molar ratios: insulin/salmine = 28:1 and insulin/lysozyme = 47:1). Both preparations gave crystals large enough (about 250 μ) for X-ray measurements to be made. The salmine-containing crystals are tetragonal, and the lysozyme-containing crystals are orthorhombic. Amino acid analyses established the following molar ratios: insulin/ salmine = 12:1, insulin/lysozyme = 18:1. 4. 4. In both crystals, the amount of non-insulin peptide present is too small for the basic component to form part of the ordered crystal structure. Nonetheless, salmine or lysozyme is essential for crystal formation and growth, and determines the type of crystal produced.

Insulin-proinsulin, a new crystalline complex.

SINCE the first crystallisation of proinsulin in this laboratory1, we have tried to grow crystals appropriate for detailed X-ray crystal structure analysis. None of the pro-insulin crystal forms prepared proved ideal2, and we attempted to cocrystallise insulin and proinsulin. Such crystals, if both components were in ordered array, could provide a basis for the determination of the structure of proinsulin by the use of rigid-body vector search methods3,4 using the known stereochemistry of insulin5.

Proinsulin: Further crystallization and X-ray crystallographic studies of bovine and porcine prohormone

Abstract Further crystallization studies of bovine proinsulin have led to the preparation of large crystals of the neutral form (pH 7.25) grown from citrate buffer containing m-cresol and excess zinc. These are tetragonal, space group P41212 (or its enantiomorph P43212) with a = 157.8 ± 0.5 A and c = 132.0 ± 0.7 A . The protein component of the asymmetric unit is equivalent to three proinsulin hexamers. When citrate was replaced by zinc-free phosphate, crystals grew which are apparently isomorphous with these. Porcine proinsulin has also been crystallized at pH 7.25 in citrate with m-cresol and zinc present; the crystals are isomorphous with the bovine preparation described here; space group P41212 with a = 158.3 ± 0.5 A and c = 132.5 ± 0.7 A . Porcine proinsulin crystals have been grown at acid pH in the presence of ammonium sulfate. These are tetragonal bipyramids isomorphous with the previously reported bovine proinsulin crystals; space group P41212 with a = 52 ± 1 A and c = 151 ± 2 A . The marked differences in the connecting peptide region of these two prohormones have little if any effect on their crystalline intermolecular packing at both acid and neutral pH.

Insulin crystallization in the presence of basic proteins and peptides

Abstract The range of conditions under which tetragonal P41212 insulin-salmine crystals can be grown has been studied extensively and the required minimal salmine/insulin ratio established. Crystals grown in the presence of m-iodophenol show significant X-ray intensity differences from those observed when m-cresol is used. The results of tests for biological activity are reported for a preparation of insulin-salmine crystals of different composition from that of commercial Lilly NPH insulin. The phenomenon of isophane precipitation has been demonstrated between insulin and (a) ribonuclease, (b) a high-molecular-weight polyarginine (approx. 21 900) and (c) a low-molecular-weight polylysine (approx. 3000). With these materials crystals were grown above, at and below the appropriate isophane ratios. With one exception, all the crystals prepared were normal monoclinic insulin.

Crystalline insulin-ribonuclease: The presence of large molecular aggregates

A new insulin-ribonuclease crystalline form has been characterized and the conditions under which it grows from solutions of insulin and ribonuclease investigated. The weight of the protein component of the asymmetric unit in these monoclinic crystals is 288000 daltons, corresponding to a minimum of forty-eight insulin molecules in the aggregate. This is the largest known discreet aggregate of insulin molecules in a single particle. From analysis, it appears that the new crystals very probably correspond to true co-crystallization of ribonuclease and insulin, with an asymmetric unit of one ribonuclease and forty-eight insulin molecules. The crystals, space group P21, have unit cell dimensions a = 111 A, b = 224 A, c = 61 A; β = 105°; the cell volume, υ = 1.48·106A3. This crystal form has been prepared only at approx. 3°. The crystals are soluble in their mother liquor at 23° although they are relatively stable at approx. 20–21° at which temperature the X-ray photographs were taken. They may be grown from solutions of insulin and ribonuclease, which on standing at room temperature would yield either rhombohedral (in the absence of a phenol) or monoclinic (in the presence of a phenol) insulin crystals. Both normal, monoclinic insulin and the new insulin-ribonuclease crystals have been grown from solutions in the same vial at approx. 3° and remained stable for several months at that temperature. A clathrate-type structure may be an appropriate model for this new crystal form. The significance both in vino and in vitro of aggregate initiation of protein molecules of the kind absorbed is discussed.