Detergent CHAPS Research Articles

High Sensitivity Identification of Membrane Proteins by MALDI TOF-MASS Spectrometry Using Polystyrene Beads

Membrane proteins play a large variety of functions in life and represent 30% of all genomes sequenced. Due to their hydrophobic nature, they are tightly bound to their biological membrane, and detergents are always required to extract and isolate them before identification by mass spectrometry (MS). The latter, however remains difficult. Peptide mass fingerprinting methods using techniques such as MALDI-TOF MS, for example, have become an important analytical tool in the identification of proteins. However, PMF of membrane proteins is a real challenge for at least three reasons. First, membrane proteins are naturally present at low levels; second, most of the detergents strongly inhibit proteases and have deleterious effects on MALDI spectra; and third, despite the presence of detergent, membrane proteins are unstable and often aggregate. We took the mitochondrial uncoupling protein 1 (UCP1) as a model and showed that differential acetonitrile extraction of tryptic peptides combined with the use of polystirene Bio-Beads triggered high resolution of the MALDI-TOF identification of mitochondrial membrane proteins solubilized either with Triton-X100 or CHAPS detergents.

Two-dimensional gel electrophoresis of peripheral nerve proteins: Optimized sample preparation

For proteomic analysis, sample preparation plays a crucial role in two-dimensional gel electrophoresis (2DE), since, very often, each tissue or cell culture requires specific treatments. In the present paper, we report a sample preparation procedure suitable for 2DE that was done on peripheral nerve using bovine sciatic nerves and human sural nerve biopsies. We obtained an appreciable reduction of tissue heterogeneity using protein extracts obtained from nerve-fiber bundles instead of the entire nerve. In addition, we optimized 2DE protein separation using a combination of CHAPS, Triton X-100, and SB3-10 detergents in an isoelectric-focusing (IEF) buffer. The reported experimental procedures appear to be essential for 2DE separation of peripheral nerve proteins for the establishment of a reference map.

Purification and characterization of recombinant ligand-binding domains from the ecdysone receptors of four pest insects

Cloned EcR and USP cDNAs encoding the ecdysone receptors of four insect pests (Lucilia cuprina, Myzus persicae, Bemisia tabaci, Helicoverpa armigera) were manipulated to allow the co-expression of their ligand binding domains (LBDs) in insect cells using a baculovirus vector. Recombinant DE/F segment pairs (and additionally, for H. armigera, an E/F segment pair) from the EcR and USP proteins associated spontaneously with high affinity to form heterodimers that avidly bound an ecdysteroid ligand. This shows that neither ligand nor D-regions are essential for the formation of tightly associated and functional LBD heterodimers. Expression levels ranged up to 16.6mg of functional apo-LBD (i.e., unliganded LBD) heterodimer per liter of recombinant insect cell culture. Each recombinant heterodimer was affinity-purified via an oligo-histidine tag at the N-terminus of the EcR subunit, and could be purified further by ion exchange and/or gel filtration chromatography. The apo-LBD heterodimers appeared to be more easily inactivated than their ligand-containing counterparts: after purification, populations of the former were <40% active, whereas for the latter >70% could be obtained as the ligand–LBD heterodimer complex. Interestingly, we found that the amount of ligand bound by recombinant LBD heterodimer preparations could be enhanced by the non-denaturing detergent CHAPS (3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate). Purity, integrity, size and charge data are reported for the recombinant proteins under native and denaturing conditions. Certain intra- and intermolecular disulfide bonds were observed to form in the absence of reducing agents, and thiol-specific alkylation was shown to suppress this phenomenon but to introduce microheterogeneity.

Latex lipase of Euphorbia characias L.: An aspecific acylhydrolase with several isoforms

The objective of the present work was to contribute to the understanding of the physiological role of latex lipolytic activity in Euphorbia characias. To this end, the acid and basic lipolytic activity of E. characias latex, as well as the substrate specificity on various triacylglycerols, were measured during the plant's vegetative and reproductive stages. Both activities appeared to increase during the reproductive stage and to peak at the beginning of the vegetative stage, when new leaves and branches are formed. For the first time, the phospholipolytic and esterase activity of E. characias latex is also reported. An extraction method in aqueous medium with the zwitterionic detergent CHAPS was successfully used to extract lipolytic activity from latex. Extraction permitted the selective recovery of a single protein spot, with a molecular weight of 37 kDa, and presumably made of several acid isoforms which retained both lipolytic and phospholipolytic activity. The biochemical results suggest that lipolytic and phospholipolytic activity could depend on a single hydrolytic enzyme with several isoforms, equally expressed throughout the biological cycle of the plant. On the basis of the obtained results, we hypothesise that the E. characias latex lipase should be considered as an aspecific acylhydrolase with a combined lipase/phospholipase A activity.

Myelin basic protein-dependent plasma membrane reorganization in the formation of myelin

During vertebrate development, oligodendrocytes wrap their plasma membrane around axons to produce myelin, a specialized membrane highly enriched in galactosylceramide (GalC) and cholesterol. Here, we studied the formation of myelin membrane sheets in a neuron-glia co-culture system. We applied different microscopy techniques to visualize lipid packing and dynamics in the oligodendroglial plasma membrane. We used the fluorescent dye Laurdan to examine the lipid order with two-photon microscopy and observed that neurons induce a dramatic lipid condensation of the oligodendroglial membrane. On a nanoscale resolution, using stimulated emission depletion and fluorescence resonance energy transfer microscopy, we demonstrated a neuronal-dependent clustering of GalC in oligodendrocytes. Most importantly these changes in lipid organization of the oligodendroglial plasma membrane were not observed in shiverer mice that do not express the myelin basic protein. Our data demonstrate that neurons induce the condensation of the myelin-forming bilayer in oligodendrocytes and that MBP is involved in this process of plasma membrane rearrangement. We propose that this mechanism is essential for myelin to perform its insulating function during nerve conduction.

Physical and Functional Association of Human Protein O-Mannosyltransferases 1 and 2

A defect of protein O-mannosylation causes congenital muscular dystrophy with brain malformation and structural eye abnormalities, so-called Walker-Warburg syndrome. Protein O-mannosylation is catalyzed by protein O-mannosyltransferase 1 (POMT1) and its homologue, POMT2. Coexpression of POMT1 and POMT2 is required to show O-mannosylation activity. Here we have shown that POMT1 forms a complex with POMT2 and the complex possesses protein O-mannosyltransferase activity. Results indicate that POMT1 and POMT2 associate physically and functionally in vivo. Recently, three mutations were reported in the POMT1 gene of patients who showed milder phenotypes than typical Walker-Warburg syndrome. We coexpressed these mutant POMT1s with POMT2 and found that none of them had any activity. However, all POMT1 mutants, including previously identified POMT1 mutants, coprecipitated with POMT2. These results indicate that the mutant POMT1s could form heterocomplexes with POMT2 but that such complexes are insufficient for enzymatic activity.

Identification and Active Expression of the Mycobacterium tuberculosis Gene Encoding 5-Phospho-α-D-ribose-1-diphosphate: Decaprenyl-phosphate 5-Phosphoribosyltransferase, the First Enzyme Committed to Decaprenylphosphoryl-D-arabinose Synthesis

Decaprenylphosphoryl-d-arabinose, the lipid donor of mycobacterial d-arabinofuranosyl residues, is synthesized from phosphoribose diphosphate rather than from a sugar nucleotide. The first committed step in the process is the transfer of a 5-phosphoribosyl residue from phosphoribose diphosphate to decaprenyl phosphate to form decaprenylphosphoryl-5-phosphoribose via a 5-phospho-alpha-d-ribose-1-diphosphate:decaprenyl-phosphate 5-phospho-ribosyltransferase. A candidate for the gene encoding this enzyme (Rv3806c) was identified in Mycobacterium tuberculosis, primarily via its homology to one of four genes responsible for d-arabinosylation of nodulation factor in Azorhizobium caulinodans. The resulting protein was predicted to contain eight or nine transmembrane domains. The gene was expressed in Escherichia coli, and membranes from the expression strain of E. coli but not from a control strain of E. coli were shown to convert phosphoribose diphosphate and decaprenyl phosphate into decaprenylphosphoryl-5-phosphoribose. Neither UDP-galactose nor GDP-mannose was active as a sugar donor. The enzyme favored polyprenyl phosphate with 50-60 carbon atoms, was unable to use C-20 polyprenyl phosphate, and used C-75 polyprenyl phosphate less efficiently than C-50 or C-60. It requires CHAPS detergent and Mg(2+) for activity. The Rv3806c gene encoding 5-phospho-alpha-d-ribose-1-diphosphate:decaprenyl-phosphate 5-phosphoribosyltransferase is known to be essential for the growth of M. tuberculosis, and the tuberculosis drug ethambutol inhibits other steps in arabinan biosynthesis. Thus the Rv3806c-encoded enzyme appears to be a good target for the development of new tuberculosis drugs.

Structure at 1.6 A resolution of the protein from gene locus At3g22680 from Arabidopsis thaliana.

The gene product of At3g22680 from Arabidopsis thaliana codes for a protein of unknown function. The crystal structure of the At3g22680 gene product was determined by multiple-wavelength anomalous diffraction and refined to an R factor of 16.0% (Rfree = 18.4%) at 1.60 A resolution. The refined structure shows one monomer in the asymmetric unit, with one molecule of the non-denaturing detergent CHAPS {3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate} tightly bound. Protein At3g22680 shows no structural homology to any other known proteins and represents a new fold in protein conformation space.

Enhanced detergent extraction for analysis of membrane proteomes by two-dimensional gel electrophoresis

BackgroundThe analysis of hydrophobic membrane proteins by two-dimensional gel electrophoresis has long been hampered by the concept of inherent difficulty due to solubility issues. We have optimized extraction protocols by varying the detergent composition of the solubilization buffer with a variety of commercially available non-ionic and zwitterionic detergents and detergent-like phospholipids.ResultsAfter initial analyses by one-dimensional SDS-PAGE, quantitative two-dimensional analyses of human erythrocyte membranes, mouse liver membranes, and mouse brain membranes, extracted with buffers that included the zwitterionic detergent MEGA 10 (decanoyl-N-methylglucamide) and the zwitterionic lipid LPC (1-lauroyl lysophosphatidylcholine), showed selective improvement over extraction with the common 2-DE detergent CHAPS (3 [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate). Mixtures of the three detergents showed additive improvements in spot number, density, and resolution. Substantial improvements in the analysis of a brain membrane proteome were observed.ConclusionThis study demonstrates that an optimized detergent mix, coupled with rigorous sample handling and electrophoretic protocols, enables simple and effective analysis of membrane proteomes using two-dimensional electrophoresis.

Glycogen- and PP1c-targeting Subunit GM Is Phosphorylated at Ser48 by Sarcoplasmic Reticulum-bound Ca2+-Calmodulin Protein Kinase in Rabbit Fast Twitch Skeletal Muscle

Multifunctional Ca(2+)-calmodulin-dependent protein kinase (CaMKII) is a Ser/Thr protein kinase uniformly distributed within the sarcoplasmic reticulum (SR) of skeletal muscle. In fast twitch muscle, no specific substrates of CaMKII have yet been identified in nonjunctional SR. Previous electron microscopy data showed that glycogen particles containing glycogen synthase (GS) associate with SR at the I band level. Furthermore, recent evidence implicates CaMKII in regulation of glucose and glycogen metabolism. Here, we demonstrate that the glycogen- and protein phosphatase 1-targeting subunit, also known as G(M), selectively localizes to the SR membranes of rabbit skeletal muscle and that G(M) and GS co-localize at the level of the I band. We further show that G(M), GS, and PP1c assemble in a structural complex that selectively localizes to nonjunctional SR and that G(M) is phosphorylated by SR-bound CaMKII and dephosphorylated by PP1c. On the other hand, no evidence for a structural interaction between G(M) and CaMKII was obtained. Using His-tagged G(M) recombinant fragments and site-directed mutagenesis, we demonstrate that the target of CaMKII is Ser(48). Taken together, these data suggest that SR-bound CaMKII participates in the regulation of GS activity through changes in the phosphorylation state of G(M). Based on these findings, we propose that SR-bound CaMKII participates in the regulation of glycogen metabolism, under physiological conditions involving repetitive raises elevations of [Ca(2+)](i).

Localization of a novel 252-kDa plasma membrane protein that binds Cry1A toxins in the midgut epithelia of Bombyx mori

We previously demonstrated that a novel 252-kDa protein (P252) isolated from brush border membranes (BBM) of Bombyx mori, hybrid Shurei×Shogetsu, specifically bound Cry1Aa, Cry1Ab and Cry1Ac toxins with a Kd of 29, 179 and 20 nM, respectively. P252 was found in a Triton X-100–soluble fraction of BBM from first, third and fifth instar larvae, suggesting that it may be an important element of midgut epithelial cell membranes. P252 was not partitioned into a Triton X-100–insoluble BBM fraction and nearly identical partitioning of P252 was observed using detergents CHAPS and Igepal. These results suggested that P252 localized in non-raft regions of BBM. Immunofluorescence analysis using anti-P252 antiserum demonstrated the existence of P252 in BBMV. Cy3-labeled Cry1Aa, Cry1Ab and Cry1Ac were shown to bind to BBMV, and the addition of anti-P252 antiserum reduced the number of BBMV showing Cy3 fluorescence by 30%. This clearly suggested an important role for P252 in Cry1A binding to BBMV. CD spectra of a mixture of purified P252 with either Cry1Aa, Cry1Ab or Cry1Ac were compared with those of respective free Cry1A toxins and only one of the mixtures (Cry1Aa/P252) was shown to be significantly changed compared to that of native Cry1Aa.

Developmental partitioning of myelin basic protein into membrane microdomains

Specific membrane microdomains (including lipid rafts) exist in myelin but have not been fully characterized. Myelin basic protein (MBP) maintains the compactness of the myelin sheath and is highly posttranslationally modified. Thus, it has been suggested that MBP might also have other functions, e.g., in signal transduction. Here, the distribution of MBP and its modified forms was studied, spatially and temporally, by detailed characterization of membrane microdomains from developing and mature bovine myelin. Myelin membranes were extracted with three different detergents (Brij 96V, CHAPS, or Triton X-100) at 4 degrees C. The detergent-resistant membranes (DRMs), representing coalesced lipid rafts, were isolated as low-buoyant-density fractions on a sucrose density gradient. These myelin rafts were disrupted when cholesterol was depleted with methyl-beta-cyclodextrin. The use of CHAPS detergent led to enrichment of several myelin proteins, including phospho-Thr97-MBP, in the DRMs from mature myelin. Citrullinated and methylated MBP remained in "nonraft" microdomains. In contrast, the DRMs from early myelin were enriched in Golli-MBP, Fyn, Lyn, and CNP. The localization of various proteins in DRMs was further supported by the colocalization of these lipid raft components in cultured mouse oligodendrocytes. Thus, there is a developmental regulation of posttranslationally modified forms of MBP into specific membrane microdomains.

Role of cholesterol in ligand binding and G-protein coupling of serotonin 1A receptors solubilized from bovine hippocampus

The serotonin 1A (5-HT 1A) receptor is an important member of the superfamily of seven transmembrane domain G-protein-coupled receptors. We report here that solubilization of the hippocampal 5-HT 1A receptor by the zwitterionic detergent CHAPS is accompanied by loss of membrane cholesterol which results in a reduction in specific agonist binding activity and extent of G-protein coupling. Importantly, replenishment of cholesterol to solubilized membranes using MβCD–cholesterol complex restores the cholesterol content of the membrane and significantly enhances the specific agonist binding activity and G-protein coupling. These novel results provide useful information on the role of cholesterol in solubilization of G-protein-coupled receptors, an important step for molecular characterization of these receptors.

Expression of Bacillus anthracis protective antigen in transgenic chloroplasts of tobacco, a non-food/feed crop

The Centers for Disease Control (CDC) lists Bacillus anthracis as a category A agent and estimates the cost of an anthrax attack to exceed US$ 26 billion per 100,000 exposed individuals. Concerns regarding anthrax vaccine purity, a requirement for multiple injections, and a limited supply of the protective antigen (PA), underscore the urgent need for an improved vaccine. Therefore, the 83 kDa immunogenic Bacillus anthracis protective antigen was expressed in transgenic tobacco chloroplasts. The PA gene ( pag) was cloned into a chloroplast vector along with the psbA regulatory signals to enhance translation. Chloroplast integration of the transgenes was confirmed by PCR and Southern blot analyses. Crude plant extracts contained up to 2.5 mg full length PA/g of fresh leaf tissue and this showed exceptional stability for several months in stored leaves or crude extracts. Maximum levels of expression were observed in mature leaves under continuous illumination. Co-expression of the ORF2 chaperonin from Bacillus thuringiensis did not increase PA accumulation or induce folding into cuboidal crystals in transgenic chloroplasts. Trypsin, chymotrypsin and furin proteolytic cleavage sites present in PA were protected in transgenic chloroplasts because only full length PA 83 was observed without any degradation products. Both CHAPS and SDS detergents extracted PA with equal efficiency and PA was observed in the soluble fraction. Chloroplast-derived PA was functionally active in lysing mouse macrophages when combined with lethal factor (LF). Crude leaf extracts contained up to 25 μg functional PA/ml. With an average yield of 172 mg of PA per plant using an experimental transgenic cultivar grown in a greenhouse, 400 million doses of vaccine (free of contaminants) could be produced per acre, a yield that could be further enhanced 18-fold using a commercial cultivar in the field.

Solubilization of serotonin1A receptors heterologously expressed in Chinese hamster ovary cells.

1. The serotonin1A (5-HT1A) receptors are members of a superfamily of seven transmembrane domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. 2. We report here, for the first time, the solubilization of 5-HT1A receptors stably expressed in Chinese Hamster Ovary (CHO) cells using the zwitterionic detergent CHAPS in presence of NaCl followed by polyethylene glycol (PEG) precipitation. We show by ligand-binding assay that the 5-HT1A receptor solubilized this way is functionally active. We have optimized the efficiency of solubilization with respect to total protein and NaCl concentration. 3. Our results show that careful control of salt and protein concentration is crucial in optimal solubilization of membrane receptors heterologously expressed in cells in culture. The effective solubilization of important neurotransmitter receptors such as 5-HT1A receptors which are present in very low amounts in the native tissue may represent an important step in characterizing membrane receptors expressed in mammalian cells in culture.

Ito Channels Are Octomeric Complexes with Four Subunits of Each Kv4.2 and K+ Channel-interacting Protein 2

Mammalian voltage-gated K+ channels are assemblies of pore-forming alpha-subunits and modulating beta-subunits. To operate correctly, Kv4 alpha-subunits in the heart and central nervous system require recently identified beta-subunits of the neuronal calcium sensing protein family called K+ channel-interacting proteins (KChIPs). Here, Kv4.2.KChIP2 channels are purified, integrity of isolated complexes confirmed, molar ratio of the subunits determined, and subunit valence established. A complex has 4 subunits of each type, a stoichiometry expected for other channels employing neuronal calcium sensing beta-subunits.

N-Acetylaspartate synthase is bimodally expressed in microsomes and mitochondria of brain

N-Acetylaspartate (NAA) is an abundant amino acid derivative of the central nervous system that is localized primarily in neurons and has found widespread use in clinical NMR spectroscopy (MRS) as a non-invasive indicator of neuronal survival and/or viability. Its function, although still obscure, is thought to reflect its unusual metabolic compartmentalization wherein NAA synthase occurs in the neuron and aspartoacylase, the hydrolytic enzyme that removes the acetyl moiety, occurs in myelin and glia. The NAA synthase enzyme, acetyl-CoA/ l-aspartate N-acetyltransferase (ANAT), was previously shown to function in mitochondria (MIT), although other subcellular fractions were apparently not examined. In this study we confirmed its presence in MIT but also found significant activity in rat brain microsomes (MIC). The reaction mixture, consisting of [ 14C]aspartate plus acetyl-CoA in Na-phosphate buffer (pH 7), gave rise to [ 14C]NAA that was separated and quantified by TLC. Reaction rates were 29.0±0.46 and 6.27±0.27 nmol/h/mg for MIC and MIT, respectively. K m values and pH optima were similar, and both fractions showed modest enhancement of ANAT activity with the detergents Triton CF-54 and CHAPS. Our tentative conclusion is that ANAT is bimodally targeted to MIT and a component of MIC—likely endoplasmic reticulum. ANAT activity increased in both MIC and MIT between 29 and 60 days of age but differed thereafter in that only MIT ANAT showed a decrease after 1 year.

Characterization of plasma membrane proteins from ovarian cancer cells using mass spectrometry.

To determine how the repertoire of plasma membrane proteins change with disease state, specifically related to cancer, several methods for preparation of plasma membrane proteins were evaluated. Cultured cells derived from stage IV ovarian tumors were grown to 90% confluence and harvested in buffer containing CHAPS detergent. This preparation was centrifuged at low speed to remove insoluble cellular debris resulting in a crude homogenate. Glycosylated proteins in the crude homogenate were selectively enriched using lectin affinity chromatography. The crude homogenate and the lectin purified sample were prepared for mass spectrometric evaluation. The general procedure for protein identification began with trypsin digestion of protein fractions followed by separation by reversed phase liquid chromatography that was coupled directly to a conventional tandem mass spectrometer (i.e. LCQ ion trap). Mass and fragmentation data for the peptides were searched against a human proteome data base using the informatics program SEQUEST. Using this procedure 398 proteins were identified with high confidence, including receptors, membrane-associated ligands, proteases, phosphatases, as well as structural and adhesion proteins. Results indicate that lectin chromatography provides a select subset of proteins and that the number and quality of the identifications improve as does the confidence of the protein identifications for this subset. These results represent the first step in development of methods to separate and successfully identify plasma membrane proteins from advanced ovarian cancer cells. Further characterization of plasma membrane proteins will contribute to our understanding of the mechanisms underlying progression of this deadly disease and may lead to new targeted interventions as well as new biomarkers for diagnosis.

Rpe65 Is a Retinyl Ester Binding Protein That Presents Insoluble Substrate to the Isomerase in Retinal Pigment Epithelial Cells

Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the all-trans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of beta-carotene dioxygenase, appears to play a role in this pathway. Rpe65-/- knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Leber's congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds all-trans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65-/- mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65.

Crystallization and preliminary X-ray analysis of 5′-methylthioribose kinase fromBacillus subtilisandArabidopsis thaliana

Recombinant Bacillus subtilis 5'-methylthioribose (MTR) kinase has been expressed, purified and subsequently crystallized using the hanging-drop vapor-diffusion technique. With PEG 2000MME as the precipitant, two different crystal forms have been grown in the absence and presence of the detergent CHAPS. These crystals belong to space groups P2(1)2(1)2(1) (unit-cell parameters a = 193.7, b = 83.2, c = 51.6 A) and P2(1)2(1)2 (unit-cell parameters a = 213.8, b = 83.2, c = 51.5 A), respectively. The crystals grown in the presence of CHAPS diffract to 2.2 A resolution at Station X8C, National Synchrotron Light Source (NSLS). For both crystal forms, the presence of two monomers per asymmetric unit is predicted (Matthews coefficient V(M) = 2.29 and 2.52 A(3) Da(-1), respectively). Recombinant C-terminally histidine-tagged Arabidopsis thaliana MTR kinase has also been expressed, purified and refolded into its active form. Rod-shaped crystals of this protein were grown from PEG 8000 using the hanging-drop vapor-diffusion technique. These crystals exhibit the symmetry of space group C2 (unit-cell parameters a = 162.3, b = 83.3, c = 91.0 A, beta = 117.8 degrees ) and diffract to 1.9 A resolution at Station X8C, NSLS. Two monomers are estimated to be present in the asymmetric unit (V(M) = 2.82 A(3) Da(-1)).