H. Groendijk

Structure of quinoprotein methylamine dehydrogenase at 2.25 A resolution.

The three‐dimensional structure of quinoprotein methylamine dehydrogenase from Thiobacillus versutus has been determined at 2.25 A resolution by a combination of multiple isomorphous replacement, phase extension by solvent flattening and partial structure phasing using molecular dynamics refinement. In the resulting map, the polypeptide chain for both subunits could be followed and an X‐ray sequence was established. The tetrameric enzyme, made up of two heavy (H) and two light (L) subunits, is a flat parallellepiped with overall dimensions of approximately 76 x 61 x 45 A. The H subunit, comprising 370 residues, is made up of two distinct segments: the first 31 residues form an extension which embraces one of the L subunits; the remaining residues are found in a disc‐shaped domain. This domain is formed by a circular arrangement of seven topologically identical four‐stranded antiparallel beta‐sheets, with approximately 7‐fold symmetry. In spite of distinct differences, this arrangement is reminiscent of the structure found in influenza virus neuraminidase. The L subunit consists of 121 residues, out of which 53 form a beta‐sheet scaffold of a central three‐stranded antiparallel sheet flanked by two shorter two‐stranded antiparallel sheets. The remaining residues are found in segments of irregular structure. This subunit is stabilized by six disulphide bridges, plus two covalent bridges involving the quinone co‐factor and residues 57 and 107 of this subunit. The active site is located in a channel at the interface region between the H and L subunits, and the electron density in this part of the molecule suggests that the co‐factor of this enzyme is not pyrrolo quinoline quinone (PQQ) itself, but might be instead a precursor of PQQ.

The transfer of protein crystals from their original mother liquor to a solution with a completely different precipitant

A procedure is described for the transfer of protein crystals from an ammonium sulfate-containing mother liquor to a solution with another precipitant, such as polyethylene glycol. The suitable concentration of the alternative precipitant is established via a novel protocol, using a hanging-drop equilibration method. This crystal transfer procedure is illustrated by experiments with crystals of trypanosomal triosephosphate isomerase and bacterial p-hydroxybenzoate hydroxylase, but it might have more general applicability.

Crystallization and preliminary X-ray investigation of lipoamide dehydrogenase from Azotobacter vinelandii.

The FAD-containing enzyme lipoamide dehydrogenase (EC 1.6.4.3. NADH: lipoamide oxidoreductase) of Azotobacter vinelandii has been crystallized from polyethylene glycol solutions. The space group is P2(1)2(1)2(1) with one dimer in the asymmetric unit. The cell dimensions are: a = 64.2, b = 83.8, c = 193 A. X-ray reflections extend to at least 2.2 A resolution.

Preliminary crystallographic studies of glycosomal glyceraldehyde phosphate dehydrogenase from Trypanosoma brucei brucei

Crystals of glyceraldehyde phosphate dehydrogenase from the glycosome of Trypanosoma brucei brucei have been grown, and a partial data set has been collected using synchrotron radiation. The crystals diffract initially to 2.3 A resolution. The space group is P2(1)2(1)2, with cell dimensions a = 135 A, b = 255 A, c = 115 A, so there are probably at least two tetramers in the asymmetric unit.

EXPERIMENTS IN MEMBRANE-PROTEIN CRYSTALLIZATION

Abstract Experiments have been carried out aimed at obtaining crystals of three integral membrane proteins: phospholipase A, cytochrome-c reductase and cytochrome-c oxidase. Crystals, in the form of platelets and bipyramids, as well as several micro-crystal forms have been obtained of phospholipase A from the outer membrane of Escherichia coli . The best crystals obtained so far were thin plates having spacegroup C222 1 with a = 72.0A, b = 107A, c = 75.8Aand one molecule per asymmetric unit. Reflections were observed out to 2.7Aresolution. Long red needles have been obtained of the respiratory chain complex-III, or cytochrome-c reductase, from beefheart.