RESUMO
The measurement of protein digestibility is one of the key steps in determining the safety of a genetically modified crop that has been traditionally accomplished using antibodies. Membrane proteins are often extremely difficult to express with replicated authentic tertiary structure in heterologous systems. As a result raising antibodies for use in safety assessment may not be feasible. In this study, LC-MS based proteomics was used to characterise seven transmembrane enzymes from the docosahexaenoic acid biosynthetic pathway that had been introduced into canola. The application of a two-stage digestion strategy involving simulated gastric fluid followed by trypsin enabled the measurement of protein digestibility in vitro. Tryptic peptide markers that spanned the length of each desaturase protein were monitored and showed that these proteins were readily degraded (>95% within 5â¯min) and highlighted regions of the elongase enzymes that showed limited resistance to simulated gastric digestion. Traditional gel-based and Western blotting analysis of ω3-desaturase and Δ6-elongase revealed rapid hydrolysis of the intact proteins within seconds and no fragments (>3â¯kDa) remained after 60â¯min, complementing the novel approach described herein. The LC-MS approach was sensitive, selective and did not require the use of purified proteins.
Assuntos
Ácidos Docosa-Hexaenoicos/biossíntese , Enzimas/metabolismo , Proteômica/métodos , Sequência de Aminoácidos , Cromatografia Líquida/métodos , Espectrometria de Massas , Reprodutibilidade dos TestesRESUMO
Examining tissue-specific expression and the measurement of protein abundance are important steps when assessing the performance of genetically engineered crops. Liquid chromatography-mass spectrometry offers many advantages over traditional methods for protein quantitation, especially when dealing with transmembrane proteins that are often difficult to express or generate antibodies against. In this study, discovery proteomics was used to detect the seven transgenic membrane-bound enzymes from the docosahexaenoic acid (DHA) biosynthetic pathway that had been engineered into canola. Subsequently, a targeted LC-MS/MS method for absolute quantitation was developed and applied to the simultaneous measurement of the seven DHA biosynthetic pathway enzymes in genetically modified canola grown across three sites. The results of this study demonstrated that the enzymatic proteins that drive the production of DHA using seed-specific promoters were detected only in mature and developing seed of DHA canola. None of the DHA biosynthesis pathway proteins were detected in wild-type canola planted in the same site or in the non-seed tissues of the transgenic canola, irrespective of the sampling time or the tissues tested. This study describes a streamlined approach to simultaneously measure multiple membrane-bound proteins in planta.
Assuntos
Brassica rapa/enzimologia , Ácidos Docosa-Hexaenoicos/biossíntese , Espectrometria de Massas/métodos , Proteínas de Membrana/química , Proteínas de Plantas/química , Plantas Geneticamente Modificadas/enzimologia , Brassica rapa/química , Brassica rapa/genética , Brassica rapa/metabolismo , Engenharia Genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/química , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Sementes/química , Sementes/enzimologia , Sementes/genética , Sementes/metabolismoRESUMO
Transcription errors occur in all living cells; however, it is unknown how these errors affect cellular health. To answer this question, we monitor yeast cells that are genetically engineered to display error-prone transcription. We discover that these cells suffer from a profound loss in proteostasis, which sensitizes them to the expression of genes that are associated with protein-folding diseases in humans; thus, transcription errors represent a new molecular mechanism by which cells can acquire disease phenotypes. We further find that the error rate of transcription increases as cells age, suggesting that transcription errors affect proteostasis particularly in aging cells. Accordingly, transcription errors accelerate the aggregation of a peptide that is implicated in Alzheimer's disease, and shorten the lifespan of cells. These experiments reveal a previously unappreciated role for transcriptional fidelity in cellular health and aging.
Assuntos
Senescência Celular/genética , Chaperonas Moleculares/metabolismo , Agregação Patológica de Proteínas/metabolismo , Estresse Fisiológico , Transcrição Gênica , Linhagem Celular , Sobrevivência Celular/genética , Proteínas de Choque Térmico/metabolismo , Mutação , RNA Polimerase II/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
A family of 21 polyphenolic compounds consisting of those found naturally in danshen and their analogues were synthesized and subsequently screened for their anti-amyloidogenic activity against the amyloid beta peptide (Aß42) of Alzheimer's disease. After 24 h incubation with Aß42, five compounds reduced thioflavin T (ThT) fluorescence, indicative of their anti-amyloidogenic propensity (p < 0.001). TEM and immunoblotting analysis also showed that selected compounds were capable of hindering fibril formation even after prolonged incubations. These compounds were also capable of rescuing the yeast cells from toxic changes induced by the chemically synthesized Aß42. In a second assay, a Saccharomyces cerevisiae AHP1 deletant strain transformed with GFP fused to Aß42 was treated with these compounds and analyzed by flow cytometry. There was a significant reduction in the green fluorescence intensity associated with 14 compounds. We interpret this result to mean that the compounds had an anti-amyloid-aggregation propensity in the yeast and GFP-Aß42 was removed by proteolysis. The position and not the number of hydroxyl groups on the aromatic ring was found to be the most important determinant for the anti-amyloidogenic properties.
Assuntos
Amiloide/efeitos dos fármacos , Extratos Vegetais/farmacologia , Polifenóis/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Peptídeos beta-Amiloides/toxicidade , Fragmentos de Peptídeos/toxicidade , Peroxirredoxinas/genética , Extratos Vegetais/química , Polifenóis/química , Saccharomyces cerevisiae/metabolismo , Salvia miltiorrhiza/química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
The tendency of amyloid ß (Aß42) peptide to misfold and aggregate into insoluble amyloid fibrils in Alzheimer's disease (AD) has been well documented. Accumulation of Aß42 fibrils has been correlated with abnormal apoptosis and unscheduled cell division which can also trigger the death of neuronal cells, while oligomers can also exhibit similar activities. While investigations using chemically-synthesized Aß42 peptide have become common practice, there appear to be differences in outcomes from different preparations. In order to resolve this inconsistency, we report 2 separate methods of preparing chemically-synthesized Aß42 and we examined their effects in yeast. Hexafluoroisopropanol pretreatment caused toxicity while, ammonium hydroxide treated Aß42 induced cell proliferation in both C. glabrata and S. cerevisiae. The hexafluoroisopropanol prepared Aß42 had greater tendency to form amyloid on yeast cells as determined by thioflavin T staining followed by flow cytometry and microscopy. Both quiescent and non-quiescent cells were analyzed by these methods of peptide preparation. Non-quiescent cells were susceptible to the toxicity of Aß42 compared with quiescent cells (p < 0.005). These data explain the discrepancy in the previous publications about the effects of chemically-synthesized Aß42 on yeast cells. The effect of Aß42 on yeast cells was independent of the size of the peptide aggregates. However, the Aß42 pretreatment determined whether the molecular conformation of peptide resulted in proliferation or toxicity in yeast based assays.
Assuntos
Peptídeos beta-Amiloides/química , Candida glabrata/citologia , Fragmentos de Peptídeos/química , Saccharomyces cerevisiae/citologia , Doença de Alzheimer/metabolismo , Apoptose , Benzotiazóis , Citometria de Fluxo , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Neurônios/metabolismo , Peptídeos/química , Propanóis/química , Ligação Proteica , Conformação Proteica , Tiazóis/químicaRESUMO
Self association of the amyloid-ß (Aß42) peptide into oligomers, high molecular weight forms, fibrils and ultimately neuritic plaques, has been correlated with progressive cognitive decline in Alzheimer's disease. Thus, insights into the drivers of the aggregation pathway have the capacity to significantly contribute to our understanding of disease mechanism. Functional assays and a three-dimensional crystal structure of the P3 amyloidogenic region 18-41 of Aß were used to identify residues important in self-association and to design novel non-aggregating variants of the peptide. Biophysical studies (gel filtration, SDS-PAGE, dynamic light scattering, thioflavin T assay, and electron microscopy) demonstrate that in contrast to wild type Aß these targeted mutations lose the ability to self-associate. Loss of aggregation also correlates with reduced neuronal toxicity. Our results highlight residues and regions of the Aß peptide important for future targeting agents aimed at the amelioration of Alzheimer's disease.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Peptídeos/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/genética , Animais , Benzotiazóis , Microscopia Eletrônica de Varredura , Mutação , Células PC12 , Peptídeos/química , Ratos , Tiazóis/metabolismoRESUMO
Alzheimer's disease is the most common neurodegenerative disease and is one of the main causes of death in developed countries. Consumption of foods rich in polyphenolics is strongly correlated with reduced incidence of Alzheimer's disease. Our study has investigated the biological activity of previously untested polyphenolic compounds in preventing amyloid ß aggregation. The anti-aggregatory potential of these compounds was assessed using the Thioflavin-T assay, transmission electron microscopy, dynamic light scattering and size exclusion chromatography. Two structurally related compounds, luteolin and transilitin were identified as potent inhibitors of Aß fibril formation. Computational docking studies with an X-ray derived oligomeric structure offer a rationale for the inhibitory activity observed and may facilitate development of improved inhibitors of Aß aggregation and toxicity.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Fragmentos de Peptídeos/metabolismo , Polifenóis/farmacologia , Agregados Proteicos/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Humanos , Modelos Moleculares , Estrutura Molecular , Polifenóis/química , Polifenóis/isolamento & purificação , Agregação Patológica de Proteínas/prevenção & controle , Relação Estrutura-AtividadeRESUMO
Extended X-ray absorption fine structure spectroscopy, mass spectrometry, dynamic light scattering and density functional theory are combined to derive structural models for the interaction of neurotoxicity-ablating platinum-based compounds with the amyloid-ß peptide.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Complexos de Coordenação/química , Platina/química , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Complexos de Coordenação/síntese química , Complexos de Coordenação/metabolismo , Humanos , Luz , Fenantrolinas/química , Ligação Proteica , Espalhamento de Radiação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Alzheimer's disease is the leading cause of dementia in the elderly. Pathologically it is characterized by the presence of amyloid plaques and neuronal loss within the brain tissue of affected individuals. It is now widely hypothesised that fibrillar structures represent an inert structure. Biophysical and toxicity assays attempting to characterize the formation of both the fibrillar and the intermediate oligomeric structures of Aß typically involves preparing samples which are largely monomeric; the most common method by which this is achieved is to use the fluorinated organic solvent 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP). Recent evidence has suggested that this method is not 100% effective in producing an aggregate free solution. We show, using dynamic light scattering, size exclusion chromatography and small angle X-ray scattering that this is indeed the case, with HFIP pretreated Aß peptide solutions displaying an increased proportion of oligomeric and aggregated material and an increased propensity to aggregate. Furthermore we show that an alternative technique, involving treatment with strong alkali results in a much more homogenous solution that is largely monomeric. These techniques for solubilising and controlling the oligomeric state of Aß are valuable starting points for future biophysical and toxicity assays.
RESUMO
Alzheimer's disease is the most common form of dementia in humans and is related to the accumulation of the amyloid-ß (Aß) peptide and its interaction with metals (Cu, Fe, and Zn) in the brain. Crystallographic structural information about Aß peptide deposits and the details of the metal-binding site is limited owing to the heterogeneous nature of aggregation states formed by the peptide. Here, we present a crystal structure of Aß residues 1-16 fused to the N-terminus of the Escherichia coli immunity protein Im7, and stabilized with the fragment antigen binding fragment of the anti-Aß N-terminal antibody WO2. The structure demonstrates that Aß residues 10-16, which are not in complex with the antibody, adopt a mixture of local polyproline II-helix and turn type conformations, enhancing cooperativity between the two adjacent histidine residues His13 and His14. Furthermore, this relatively rigid region of Aß (residues, 10-16) appear as an almost independent unit available for trapping metal ions and provides a rationale for the His13-metal-His14 coordination in the Aß1-16 fragment implicated in Aß metal binding. This novel structure, therefore, has the potential to provide a foundation for investigating the effect of metal ion binding to Aß and illustrates a potential target for the development of future Alzheimer's disease therapeutics aimed at stabilizing the N-terminal monomer structure, in particular residues His13 and His14, and preventing Aß metal-binding-induced neurotoxicity.
Assuntos
Peptídeos beta-Amiloides/química , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/metabolismo , Escherichia coli , Humanos , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Latrepirdine (Dimebon), an anti-histamine, has shown some benefits in trials of neurodegenerative diseases characterized by accumulation of aggregated or misfolded protein such as Alzheimer's disease (AD) and has been shown to promote the removal of α-synuclein protein aggregates in vivo. An important pathway for removal of aggregated or misfolded proteins is the autophagy-lysosomal pathway, which has been implicated in AD pathogenesis, and enhancing this pathway has been shown to have therapeutic potential in AD and other proteinopathies. Here we use a yeast model, Saccharomyces cerevisiae, to investigate whether latrepirdine can enhance autophagy and reduce levels of amyloid-ß (Aß)42 aggregates. Latrepirdine was shown to upregulate yeast vacuolar (lysosomal) activity and promote transport of the autophagic marker (Atg8) to the vacuole. Using an in vitro green fluorescent protein (GFP) tagged Aß yeast expression system, we investigated whether latrepirdine-enhanced autophagy was associated with a reduction in levels of intracellular GFP-Aß42. GFP-Aß42 was localized into punctate patterns compared to the diffuse cytosolic pattern of GFP and the GFP-Aß42 (19:34), which does not aggregate. In the autophagy deficient mutant (Atg8Δ), GFP-Aß42 showed a more diffuse cytosolic localization, reflecting the inability of this mutant to sequester GFP-Aß42. Similar to rapamycin, we observed that latrepirdine significantly reduced GFP-Aß42 in wild-type compared to the Atg8Δ mutant. Further, latrepirdine treatment attenuated Aß42-induced toxicity in wild-type cells but not in the Atg8Δ mutant. Together, our findings provide evidence for a novel mechanism of action for latrepirdine in inducing autophagy and reducing intracellular levels of GFP-Aß42.
Assuntos
Peptídeos beta-Amiloides/antagonistas & inibidores , Autofagia/fisiologia , Proteínas de Fluorescência Verde/metabolismo , Indóis/farmacologia , Líquido Intracelular/metabolismo , Fragmentos de Peptídeos/antagonistas & inibidores , Saccharomyces cerevisiae/metabolismo , Peptídeos beta-Amiloides/metabolismo , Autofagia/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Proteínas de Fluorescência Verde/antagonistas & inibidores , Humanos , Líquido Intracelular/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Sortase-mediated protein ligation is a biological covalent conjugation system developed from the enzymatic cell wall display mechanism found in Staphylococcus aureus. This three-component system requires: (i) purified Sortase A (SrtA) enzyme; (ii) a substrate containing the LPXTG peptide recognition sequence; and (iii) an oligo-glycine acceptor molecule. We describe cloning of the single-chain antibody sc528, which binds to the extracellular domain of the epidermal growth factor receptor (EGFR), from the parental monoclonal antibody and incorporation of a LPETGG tag sequence. Utilizing recombinant SrtA, we demonstrate successful incorporation of biotin from GGG-biotin onto sc528. EGFR is an important cancer target and is over-expressed in human tumor tissues and cancer lines, such as the A431 epithelial carcinoma cells. SrtA-biotinylated sc528 specifically bound EGFR expressed on A431 cells, but not negative control lines. Similarly, when sc528 was labeled with fluorescein we observed antigen-specific labeling. The ability to introduce functionality into recombinant antibodies in a controlled, site-specific manner has applications in experimental, diagnostic, and potentially clinical settings. For example, we demonstrate addition of all three reaction components in situ within a biosensor flow cell, resulting in oriented covalent capture and presentation of sc528, and determination of precise affinities for the antibody-receptor interaction.
Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Bactérias/metabolismo , Cisteína Endopeptidases/metabolismo , Receptores ErbB/antagonistas & inibidores , Anticorpos de Cadeia Única/metabolismo , Coloração e Rotulagem/métodos , Aminoaciltransferases/genética , Anticorpos Bloqueadores/genética , Anticorpos Bloqueadores/imunologia , Anticorpos Bloqueadores/metabolismo , Proteínas de Bactérias/genética , Biotina/metabolismo , Linhagem Celular Tumoral , Cisteína Endopeptidases/genética , Receptores ErbB/imunologia , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologiaRESUMO
This study has found that the Maltose binding protein Aß42 fusion protein (MBP-Aß42) forms soluble oligomers while the shorter MBP-Aß16 fusion and control MBP did not. MBP-Aß42, but neither MBP-Aß16 nor control MBP, was toxic in a dose-dependent manner in both yeast and primary cortical neuronal cells. This study demonstrates the potential utility of MBP-Aß42 as a reagent for drug screening assays in yeast and neuronal cell cultures and as a candidate for further Aß42 characterization.
Assuntos
Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/toxicidade , Neurônios/efeitos dos fármacos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/toxicidade , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/toxicidade , Peptídeos beta-Amiloides/genética , Animais , Apoptose , Córtex Cerebral/citologia , Proteínas Ligantes de Maltose/química , Proteínas Ligantes de Maltose/genética , Proteínas Ligantes de Maltose/toxicidade , Camundongos , Fragmentos de Peptídeos/genética , Multimerização Proteica , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/efeitos dos fármacos , SolubilidadeRESUMO
6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the Mg(2+)-dependent transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP), forming 6-hydroxymethyl-7,8-dihydropterin pyrophosphate, which is a critical step in the de novo folic acid-biosynthesis pathway. Diffraction-quality crystals of HPPK from the medically relevant species Staphylococcus aureus were grown in the presence of ammonium sulfate or sodium malonate and diffracted to better than 1.65 A resolution. The crystals belonged to space group P2(1), with unit-cell parameters a = 36.8, b = 76.6, c = 51.5 A, alpha = gamma = 90.0, beta = 100.2 degrees . The crystals contained two molecules per asymmetric unit, with a volume per protein weight (V(M)) of 2.04 A(3) Da(-1) and an estimated solvent content of 39.6%.
Assuntos
Difosfotransferases/química , Staphylococcus aureus/enzimologia , Cristalização , Cristalografia por Raios XRESUMO
Inhibitors of phenylethanolamine N-methyltransferase [PNMT, the enzyme that catalyzes the final step in the biosynthesis of epinephrine (Epi)] may be of use in determining the role of Epi in the central nervous system. Here we describe the synthesis and characterization of 7-SCN tetrahydroisoquinoline as an affinity label for human PNMT.
Assuntos
Feniletanolamina N-Metiltransferase/metabolismo , Tetra-Hidroisoquinolinas/síntese química , Tetra-Hidroisoquinolinas/farmacologia , Animais , Epinefrina/metabolismo , Epinefrina/fisiologia , Humanos , Feniletanolamina N-Metiltransferase/antagonistas & inibidores , Ratos , Tetra-Hidroisoquinolinas/química , Fatores de TempoRESUMO
The crystal structures of human phenylethanolamine N-methyltransferase in complex with S-adenosyl-l-homocysteine (7, AdoHcy) and either 7-iodo-1,2,3,4-tetrahydroisoquinoline (2) or 8,9-dichloro-2,3,4,5-tetrahydro-1H-2-benzazepine (3, LY134046) were determined and compared with the structure of the enzyme complex with 7 and 7-aminosulfonyl-1,2,3,4-tetrahydroisoquinoline (1, SK&F 29661). The enzyme is able to accommodate a variety of chemically disparate functional groups on the aromatic ring of the inhibitors through adaptation of the binding pocket for this substituent and by subtle adjustments of the orientation of the inhibitors within the relatively planar binding site. In addition, the interactions formed by the amine nitrogen of all three inhibitors reinforce the hypothesis that this functional group mimics the beta-hydroxyl of norepinephrine rather than the amine. These studies provide further clues for the development of improved inhibitors for use as pharmacological probes.
Assuntos
Epinefrina/biossíntese , Feniletanolamina N-Metiltransferase/antagonistas & inibidores , Benzazepinas/química , Sítios de Ligação , Cristalografia por Raios X , Humanos , Isoquinolinas/química , Modelos Moleculares , Feniletanolamina N-Metiltransferase/química , Feniletanolamina N-Metiltransferase/metabolismo , Ligação Proteica , Quinolinas/química , S-Adenosil-Homocisteína/química , Especificidade por SubstratoRESUMO
Single-chain variable fragments (scFvs) of anti-Lewis(y) hu3S193 humanized antibody were constructed by joining the V(H) and V(L) domains with either +2 residues, +1 residue, or by directly linking the domains. In addition two constructs were synthesized in which one or two C-terminal residues of the V(H) domain were removed (-1 residue, -2 residue) and then joined directly to the V(L) domain. An scFv construct in the reverse orientation with the V(L) joined directly to the V(H) domain was also synthesized. Upon transformation into Escherichia coli all scFv constructs expressed active protein. Binding activity, multimeric status, and multivalent properties were assessed by flow cytometry, size exclusion chromatography, and biosensor analysis. The results for hu3S193 scFvs are consistent with the paradigm that scFvs with a linker of +3 residues or more associate to form a non-covalent dimer, and those with a shorter linker or directly linked associate predominantly to form a non-covalent trimer and tetramer that are in equilibrium. While the association of V domains to form either a dimer or trimer/tetramer is governed by the length of the linker, the stability of the trimer/tetramer in the equilibrium mixture is dependent on the affinity of the interaction of the individual V domains to associate to form the larger Fv module.
Assuntos
Fragmentos de Imunoglobulinas/imunologia , Antígenos do Grupo Sanguíneo de Lewis/imunologia , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Fluorescência , Humanos , Fragmentos de Imunoglobulinas/genéticaRESUMO
Phenylethanolamine N-methyltransferase, PNMT, utilizes the methylating cofactor S-adenosyl-L-methionine to catalyse the synthesis of adrenaline. Human PNMT has been crystallized in complex with an inhibitor and the cofactor product S-adenosyl-L-homocysteine using the hanging-drop technique with PEG 6000 and lithium chloride as precipitant. A critical requirement for crystallization was a high enzyme concentration (>90 mg ml(-1)) and cryocrystallography was used for high-quality data measurement. Diffraction data measured from a cryocooled crystal extend to a resolution of 2.3 A. Cryocooled crystals belong to space group P4(3)2(1)2 and have unit-cell parameters a = b = 94.3, c = 187.7 A.